To evaluate the follicular dynamics, superovulatory response, and embryo recovery following superstimulatory treatment initiated at estradiol-17β induced follicular wave emergence and its comparison with conventional superstimulatory protocol in buffaloes.th day of the estrous cycle with same FSH dose regimen and similar timings for PGF2α injections. In both groups, half of the buffaloes were treated with luteinizing hormone (LH) 25 mg and other half with 100 ug buserelin; gonadotrophin releasing hormone (GnRH) analog at 12 h after the end of FSH treatment. All buffaloes in both protocols were inseminated twice at 12 and 24 h of LH/GnRH treatment. Daily ultrasonography was performed to record the size and number of follicles and superovulatory response.Six normal cycling pluriparous buffaloes, lactating, 90-180 days post-partum, and weighing between 500 and 660 kg were superstimulated twice with a withdrawal period of 35 days in between two treatments. In superstimulation protocol-1 (estradiol group) buffaloes were administered estradiol-17β  and eazibreed controlled internal drug release (CIDR) was inserted intravaginally (day=0) at the random stage of the estrous cycle. On the day 4, buffaloes were superstimulated using follicle stimulating hormone (FSH) 400 mg, divided into 10 tapering doses given at 12 hourly intervals. Prostaglandin F2α analogs (PGF2α) was administered at day 7.5 and day 8, and CIDR was removed with the second PGF2α injection. In superstimulation protocol - 2  buffaloes were superstimulated on the 10Significantly higher number of small follicles (<8 mm) was present at the time of initiation of superstimulatory treatment in the estradiol group compared to the conventional group , however, the number of ovulatory size follicles (≥8 mm) did not differ significantly between the respective groups . Total embryos and transferable embryos recovered were non-significantly higher in the estradiol group compared to the conventional group . The significant higher proportion of transferable embryos were recovered in buffaloes treated with LH compared to GnRH .The average number of ovulatory size follicles (>8 mm), corpora lutea, and transferable embryos was higher in buffaloes superstimulated at estradiol-induced follicular wave compared to the conventional protocol: Further the percentage of transferable embryos was significantly higher in buffaloes administered with LH compared to GnRH. Howevern cattle ,3. In bun cattle ,5. Similn cattle  reportedth day), to coincide with the approximate time of emergence of the second follicular wave. It has been observed that asynchrony of even 1 day between wave emergence and initiation of superstimulatory treatment may significantly reduce the superovulatory response [et al. [So far conventional superstimulatory protocols have been used in buffaloes involving the initiation of superstimulatory treatment during mid-cycle  or hormonal approaches (reviewed in ). In bufThe present study being a part of a larger study for doctorate thesis, approval had been obtained from the Institutional Animal Ethics Committee. The embryo collection was done as per standard procedure without harming the animals.Six normal cycling healthy buffaloes, with average body weight 500 - 660 kg, 90-180 days post-partum, superstimulated twice with a withdrawal period of 35 days in between two treatments were used for the study.th day (day of follicular wave emergence) using follicle stimulating hormone (FSH) 400 mg NIH-FSH-P1, given daily intramuscularly in divided, tapering doses , over a period of 5 days. All the buffaloes were given two injections of prostaglandin F2α analog (PGF2α) cloprostenol 500 μg at 84 and 96 h after the start of superstimulatory treatment and CIDR was removed at the time of second prostaglandin injection.In Group I : Buffaloes were administered estradiol-17β  along with insertion of eazibreed controlled internal drug release (CIDR) (designated as day=0), the superstimulatory treatment was initiated on the 4th day of induced estrus using same dose regimen of FSH as above. Similarly, buffaloes were given two injections of PGF2α  at 84 and 96 h after the start of superstimulatory treatment.Group II conventional superstimulation protocol: Buffaloes (n=6) were superstimulated as per the conventional protocol by initiating the superstimulatory treatment on the 10In both Groups I and II, half of the buffaloes were administered 25 mg luteinizing hormone (LH), and half were administered 100 ug buserelin (gonadotrophin releasing hormone (GnRH) analog) at 12 h after the end of superstimulatory treatment. The fixed time insemination was done at 12 and 24 h of LH/GnRH treatment.Ultrasonography was carried out daily using ultrasound machine  equipped with B-mode linear array trans-rectal probe (7.5 MHz) from the start of experiment till the day of embryo recovery to record the size and the number of follicles followed with number of ovulations and luteal dynamics. Embryos were collected by flushing the uterus of the donor animals non-surgically on day 5.5 using two-way Worrlien catheter using flushing media - Dulbecoo’s phosphate buffered saline supplemented with 0.4% bovine serum albumin and antibiotics at standard rates. Embryos were searched and evaluated morphologically using zoom stereomicroscope and were graded as per the manual of International Embryo Transfer Society .t-test (two-tailed) was applied to compare mean values of treatments in SPSS-16 Statistical program. A p=0.05 was considered as significant.The data are presented as means and standard errors for all variables. After confirming the normality of data and homogeneity of variance, Student’s All the buffaloes responded to superstimulatory treatment by developing multiple corpora lutea (CLs) on the ovaries. The results are presented in No significant differences were observed in percent ovulation , the number of CLs on day 5 post artificial insemination (AI) , embryo recovery rate (47.95% vs. 45.16%), and total number of recovered embryos  in the estradiol group compared to the conventional group, respectively. Further, a mean number of transferable embryos recovered were non-significantly higher in the estradiol group compared to the conventional group .The administration of LH or GnRH did not have any significant effect on ovulation rate and a number of embryos recovered in buffaloes . HoweverIn vivo embryo production could be a viable option in buffaloes on the identification of good donors. However, the detection of estrus is more difficult in buffaloes than cattle which made implementation of the conventional superstimulatory protocols more difficult in buffaloes resulting in poor success rates. The initiation of superstimulatory treatment following exogenous control of follicular wave emergence using estradiol-17β could eliminate the need for estrus detection and avoid the unnecessary waiting period until mid-cycle to initiate the conventional protocol. Therefore, the present work was undertaken to study the follicular dynamics, superovulatory response, embryo recovery following superstimulation protocol initiated at the beginning of estradiol-17β-induced follicular wave emergence and its comparison with the conventional protocol in buffaloes.et al. [et al. [et al. [et al. [et al. [The number of small follicles (≤8 mm) present at the time of initiation of superstimulatory treatment was significantly higher in the estradiol group compared to the conventional group . Similaret al.  and Carv [et al.  who show [et al. . However [et al. ,16. Howe [et al.  and Camp [et al.  reportedThe reason for higher number of follicles reaching ovulatory size (≥8 mm) in the estradiol group compared to the conventional group in the present study could be emergence of a new follicular wave 4 days later following administration of estradiol-17β  which leet al. [et al. [et al. [In general, the both groups showed higher ovulation rate (74% and 84%) in the present study. The similar high ovulation rate in superstimulated buffaloes has been reported by Lipinski et al. . The mod [et al. . However [et al. ,21. Pate [et al.  also repth day post AI in both groups was higher than reported by Misra and Joshi [et al. [The superovulatory response in terms of number of CLs on the 5nd Joshi  and Agar [et al. . Misra [ [et al.  in a stuth day post AI could be due to the higher number of follicles reaching ovulatory size (≥8 mm) in the estradiol group compared to the conventional group. The presence of higher number of dominant follicles at the initiation of superstimulatory protocol in the conventional group could be another reason. The decreased superovulatory response due to the presence of dominant follicle has also been reported by others [et al. [The comparatively higher number of CLs in the estradiol group than the conventional group on the 5y others . Li et a [et al.  reportedet al. [et al. [The moderate embryo recovery rate of ~45-48 was observed in the present study. The recovery rate achieved in the present study was higher than (~34%) reported by Baruselli et al.  and Carv [et al.  but lowe [et al.  also rep [et al. , failure [et al. , fragile [et al.  higher n [et al. ,28, howe [et al.  was unsu [et al. . The exaet al. [et al. [et al. [The average number of embryos recovered in the present study was higher in the estradiol group than the conventional group. The two studies in the literature have reported the recovery of similar or higher number of embryos. Misra et al.  have rec [et al.  reported [et al.  reported [et al.  and admi [et al.  has alsoet al. [et al. [et al. [The average number of transferable embryos recorded in the estradiol group was non-significantly higher than the conventional group. Misra et al.  reportedet al. , >4 tranet al.  and 7 tret al.  has alsoet al. ,20,21. T [et al. , Heleil  [et al.  and lowe [et al.  achievedet al. [Differences in superovulatory response, embryo recovery and a number of transferable embryos were non-significant between buffaloes administered LH or GnRH in the present study . Howeveret al.  reportedet al. .et al. [et al. [et al. [et al. [Techakumphu et al.  reported [et al.  did not  [et al.  reported [et al.  observedSignificantly higher number of growing follicles was present in the estradiol group at the initiation of superstimulatory treatment compared to the conventional group. Superovulatory response in terms of number of CLs, total, and transferable embryos tend to be higher in the estradiol group compared to the conventional protocol indicating that the estradiol based protocol could be successfully used for superstimulation in buffaloes. In addition to this, the administration of LH in buffaloes subjected to fix timed AI following superstimulation produced a significantly higher percentage of transferable embryos compared to administration of GnRH. Therefore, the results of the present study indicated that initiation of superstimulatory treatment subsequent to the synchronization of follicular wave emergence by estradiol-17β and fixed timed AI could be successfully used for embryo production in buffaloes.The present study was part of NS’s PhD dissertation. The work was designed by GSD and PSB. The execution of experimentation protocol and performing ultrasound scanning was done by NS, VSM, and MH. The lab work was performed by NS, SS, and DD. Statistical analysis and drafting of the manuscript were done by NS, GSD, and DD. All authors read and approved the final manuscript.


Essential hypertension (EH) is a complex, polygenic condition with no single causative agent. Despite advances in our understanding of the pathophysiology of EH, hypertension remains one of the world’s leading public health problems. Furthermore, there is increasing evidence that epigenetic modifications are as important as genetic predisposition in the development of EH. Indeed, a complex and interactive genetic and environmental system exists to determine an individual’s risk of EH. Epigenetics refers to all heritable changes to the regulation of gene expression as well as chromatin remodelling, without involvement of nucleotide sequence changes. Epigenetic modification is recognized as an essential process in biology, but is now being investigated for its role in the development of specific pathologic conditions, including EH. Epigenetic research will provide insights into the pathogenesis of blood pressure regulation that cannot be explained by classic Mendelian inheritance. This review concentrates on epigenetic modifications to DNA structure, including the influence of non-coding RNAs on hypertension development. Hypertension (HT) affects more than 1 billion people globally and is a major risk factor for stroke, chronic kidney disease, and myocardial infarction . The pubEpigenetic modification is recognised as essential in biological processes and in recent years there has been studies investigating its role in the development of specific pathologic conditions. Research into epigenetic mechanisms will provide insights into the pathogenesis of blood pressure regulation that cannot be explained by classic Mendelian inheritance. Previous reviews in this field report on the various epigenetic mechanisms impacting CVD ,5,6 or tEpigenetics refers to all heritable changes to the regulation of gene activity, without changes to the DNA sequence itself . In otheThe launch of international initiatives, the Human Epigenome Project , which fS-adenosyl-l-methionine is bound to position 5 of the cytosine ring, forming 5-methyl-cytosine (5mC) .,19.10,19et al. [IGFBP3, KCNK3, PDE3A, PRDM6) and renal function  [The most robust data on the involvement of methylation in blood pressure regulation comes from the study by Kato et al. . Kato anet al. . An inveet al. , suggest7, TBX2) . InvestiHSD11B2 gene is hypermethylated [et al. [et al. [HSD11B2 promoter methylation was associated with EH, with parallel disruptions to the THF/THE ratio.Initial DNA methylation research centred on correlating EH with the global level of 5mC , but morthylated ,28. The thylated ,28, in a [et al. . Friso e [et al.  also fouPRCP gene encodes a lysosomal prolylcarbopeptidase protein, which is implicated in cleavage of c-terminal amino acids linked to proline in peptides such as angiotensin II and III [PRCP gene was hypomethylated in EH subjects [Atgr1a) were analysed in both spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar Kyoto rat (WKY), expression of Atgr1a was significantly increased by week 20 in the SHR. Bisulfite sequencing revealed that the Atgr1a promoter from endothelial cells in the aorta and mesenteric arteries of the SHR rats became progressively hypomethylated with age compared to their WKY counterparts [Atgr1a expression in the SHR was related to the hypomethylation of the Atgr1a promoter and may have a role in the maintenance of high blood pressure. Somatic ACE (sACE) converts angiotensin I to the active form, angiotensin II and is, therefore, a key regulator of blood pressure [Due to the well-known involvement of the RAAS system on arterial pressure regulation, any changes in the activation status of RAAS-regulated genes have a pronounced effect on an individual’s potential to develop HT. This effect has been extensively tested in animal models of HT . Hypomet and III . Methylasubjects . Similarterparts , suggestpressure . Bisulfipressure .+-K+-2Cl− cotransporter 1 (NKCC1), participate directly with fluid and electrolyte loss and therefore arterial pressure regulation [Sic2a2 gene, mediates the transport of sodium, potassium and chloride into and out of the cells [+, K+, Rb+, and Cl− in hypertensive vascular smooth muscle cells [Sic2a2 gene promoter in the SHR aorta and heart, result in increased expression of NKCC1 and is positively correlated with HT [Membrane transporters such as Nagulation . NKCC1, he cells . Changesle cells . Hypomet with HT .N-methyltransferase (PNMT), which can act similarly to methyl CpG binding protein 2, has been shown to exacerbate the decrease in norepinephrine uptake, thereby enhancing the local and systemic catecholaminergic effect [Methyl CpG binding protein 2 methylates the norepinephrine transporter gene, silencing its expression . Hypermec effect .DNA is packaged into the dynamic protein structure of chromatin, whose basic unit is the nucleosome. A nucleosome comprises of two copies each of the histone proteins H2A, H2B, H3, and H4 . Post-trEpigenetic histone modification occurs when the N-terminal tail is subjected to a variety of post-translational modifications . Up to 6et al. [et al. [et al. [et al. [Histone modification affecting arterial pressure levels has been documented in a variety of human and animal tissues, including vascular smooth muscle. Vascular oxidative stress can contribute to endothelial dysfunction—a hallmark of HT—and the development of HT. A study by Bhatt et al.  document [et al. . Epigene [et al. , possibl [et al. . eNOS ac [et al.  found th [et al.  observed [et al. .et al. [et al. [et al. [Ace1 promoter regions of the SHR tissues were more enriched with H3Ac and H3K4me3, and concluded that Ace1 is locally up-regulated in SHR tissues via histone code modifications [The study by Riviere et al.  noting t [et al.  further  [et al.  also fouications .et al. [et al. [Nkcc1 mRNA and protein in the aortas of Sprague–Dawley (SD) rats were significantly increased after administration of an angiotensin II infusion. Cho et al. [Nkcc1 during HT development.In line with observations by Lee et al. , Cho et  [et al.  found tho et al.  found tho et al. , suggestDisruptor of telomeric silencing-1 (DOT 1), a methyl-transferase, enhances methylation of lysine 79 residue of histone H3 (H3K79) . DOT1-me2AR) leading to cyclic AMP (cAMP) production, and increased activity of renal epithelium sodium channels (ENaC) [et al. [et al. [WNK4 promoter region that contains a negative glucocorticoid-responsive element [et al. found WNK4 expression was inhibited via these pathways leading to overexpression of the sodium chloride co-transporter (NCC) and the onset of HT [Activation of the renal sympathetic nervous system has long been thought to play a crucial role in the development of salt-reactive HT . Overacts (ENaC) . WNK4 sis (ENaC) ,48. Mu e [et al.  found th [et al. . Mu et a [et al.  also not element . Mu et aet of HT .et al. [Non-coding RNAs (ncRNA) are implicated in several epigenetic processes, notably small RNAs that can influence histone modifications and cytosine methylation which are connected with gene expression regulation . NcRNA met al. . Here weMiRNAs are the most commonly studied small ncRNA; currently there is no research available investigating the involvement of the other types of small and mid-sized ncRNAs in EH. There are more than 1800 miRNAs in the genome, each approximately 22 nucleotides in length . miRNAs et al. [AGTR1 mRNA. This site has a Mendelian C (minor) and an A (major) allele. The minor-C allele is associated with EH [AGTR1 mRNA in EH individuals who inherit this allele. Individuals with the C allele did not have reduced AGTR1 mRNA and exhibited a greater pressor effect in response to angiotensin II [AGTR1 expression in umbilical vein endothelial cells [DOT1 gene [In regards to the RAAS-regulated genes, it appears that miRNAs may also have a role modulating ACE mRNA transcription . MiRNA het al.  to targe with EH . Has-miRensin II . Has-miRal cells . A recenal cells . Two miRal cells . In refeal cells  (mentionOT1 gene . FurtherOT1 gene . AngioteOT1 gene . siRNA tOT1 gene .Long non-coding RNAs (lncRNAs), a heterogeneous group of non-coding transcripts longer than 200 nucleotides, regulate their targets by influencing epigenetic control, mRNA processing, translation or chromatin structure. LncRNAs have been implicated in several biological processes, including transcriptional regulation by epigenetic mechanisms . An exemet al. [ADD3, NPPA, ATP1A1, NPR2, CYP17A1, ACSM3, and SLC14A2 were connected with cis-lncRNA transcripts [NPPA) gene (present in cardiac hypertrophy and heart failure) and, therefore, has potential CVD involvement [Large intergenic non-coding RNAs (lincRNAs) have been identified both as modulators of health development ,65,66 anet al.  have deset al. . The proet al. , suggestet al. . LncRNAsnscripts . Of thesolvement .We are entering a new era of understanding how the genome interacts with the environment to affect disease pathogenesis. There is now emerging evidence that epigenetic, as well as genetic, factors are key players in regulating and maintaining blood pressure, and strong evidence for a complex interaction of genetic and environmental factors that influence the risk of HT in each individual . Many ep


Nanotechnology has offered a wide range of opportunities for the development and application of structures, materials, or systems with new properties in the food industry in recent years. The developed nanomaterials could greatly improve not only food quality and safety but also the foods’ health benefits. In this special issue, different nano-sized vehicles  are reported as efficient bioactives delivery systems and sensitive detection materials.w/v lecithin). Additionally, the naringenin-loaded nanoliposomes still maintained good stability during 31 days of storage at 4 °C. Zhou et al. [To resolve the low chemical instability, poor water solubility, and intestinal efflux limitations and challenges of bioactive ingredients, constructing an effective delivery vehicle using food-grade polymers is supposed to be a novel and feasible strategy. For example, Chen et al.  encapsulu et al.  fabricatu et al.  reportedu et al.  fabricatu et al. . Two typu et al.  systemat2+ in water. This probe exhibited an extremely low limit of detection of 0.22 nM. Meanwhile, a possible fluorescence-quenching mechanism was proposed in this study. In another study, to achieve the rapid detection of Listeria monocytogenes, Zhu et al. [3) was used as a photosensitive material, which was modified with gold nanoparticles to immobilize complementary DNA, and amplified the signal by means of the sensitization effect of CdTe quantum dots and the shearing effect of exonuclease I (Exo I) to achieve high-sensitivity detection. This strategy had a detection limit of 45 CFU/mL in the concentration range of 1.3 × 101–1.3 × 107 CFU/mL, providing a new way to detect Listeria monocytogenes.Another focus of the published articles in this special issue is the sensitive detection of various contaminants  ions, pathogenic bacterium) associated with food safety through different nano-techniques. In the study by Feng et al. , a novelu et al.  used aptOverall, these articles extend the knowledge on the application of nanomaterials in food nutrition and safety, promoting the development of nanotechnologies in food industry.Finally, we would like to thank all of the authors for their submissions, and all of the referees for their valuable suggestions for improving the manuscripts.


Kibria et al. was incorrectly included as reference 28. As a result, all subsequent references are misnumbered. References 29–50 should be references 28–49. All occurrences of Kibria et al. should refer to Migneco et al. .As a result of the inclusion of the wrong reference there is an error in There is an error in There are errors in the Supporting Information files S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


The authors of “Revision Total Knee Arthroplasty Using Robotic Arm Technology”  would liWe have reviewed the article by Steelman et al  and openMatthew Bullock is a paid presenter for Smith & Nephew; is an unpaid consultant for Osso VR; has stock in Stryker; received educational support from Stryker, Smith & Nephew, Depuy, and Zimmer/Biomet; is a part of Editorial Board Arthroplasty Today; is a board member of AAKHS Patient Education Committee and West Virginia Orthopaedic Society Education Committee. The other authors declare no potential conflicts of interest.https://doi.org/10.1016/j.artd.2022.101091.For full disclosure statements refer to


Journal of Clinical Medicine with great interest. This systematic review and meta-analysis on the effects of assisted reproductive technologies (ARTs) on DNA methylation in human offspring could be of major importance for the health of children conceived through these techniques. Unfortunately, there are many limitations and confusing elements in this study that lead to questioning the veracity of the results.We read the study by Cannarella et al.  recentlyIt is first surprising that 50 articles were selected for quantitative synthesis  (as shown in the first figure of the paper (page 4)), but only 12 were finally included. The methodology used in this systematic review to select studies and how they were distributed into the different analyses must be clarified.The way the meta-analysis was conducted and its motivations are very confusing. The meta-analysis is greatly similar to the one published by Barberet et al. , but theH19 CTCF3 region in ART newborns compared to controls. However, a duplication error was made in the analysis for buccal smears, and results from Puumala et al. [n = 29, mean = 43.09, SD = 7.82). It is also hardly understandable that results from Choux et al. [The main result of this study suggests a hypomethylation in the a et al.  are wronx et al.  are consH19 CTCF6, the sample size is wrong for Sakian et al. [KCNQ1OT1, the sample size for Gomes et al. [KvDMR1 gene”, which is an imprinting region, or when referring to “H19 methylation”, which does not allow us to know if the authors are discussing CTCF3, CTCF6, or H19/IGF2 DMRs regions in each corresponding study.There are also other figures in this study that seem to be wrong and may compromise the overall results for other genes. For n et al.  and Sakis et al.  is highes et al. : “In addWe hope that these items can be corrected or justified and will no longer confound the results of Barberet et al.  who eval


More than 10 studies have confirmed the association of antibiotic overuse with colorectal cancer. The exact cause is unknown, but most authors hypothesize that disturbance of colon microbiota is the main culprit. In this commentary, an evolutionary explanation is proposed. It is well known that antibiotics can induce antibiotic resistance in bacteria through selection of mutators—DNA mismatch repair deficient (dMMR) strains. Mutators have an increased survival potential due to their high mutagenesis rate. Antibiotics can also cause stress in human cells. Selection of dMMR colon cells may be advantageous under this stress, mimicking selection of bacterial mutators. Concomitantly, mismatch repair deficiency is a common cause of cancer, this may explain the increased cancer risk after multiple cycles of oral antibiotics. This proposed rationale is described in detail, along with supporting evidence from the peer-reviewed literature and suggestions for testing hypothesis validity. Treatment schemes could be re-evaluated, considering toxicity and somatic selection mechanisms.The association of antibiotics with colon cancer is well established but of unknown cause. Under an evolutionary framework, antibiotics may select for stress-resistant cancerous cells that lack mechanisms for DNA mismatch repair (MMR). This mimics the selection of antibiotic resistant ‘mutators’—MMR-deficient micro-organisms—highly adaptive due to their increased mutagenesis rate. Multiple studies have found a positive association between overuse of antibiotics and risk for colorectal cancer, though the exact cause remains unknown. In this article, a connection with DNA mismatch repair (MMR) genes is speculated, under the view of somatic selection. Below, a detailed description of the most significant studies that found the antibiotics–cancer association is included. Studies are summarized in et al. [et al. [et al. [et al. [P < 0.001). Along the same lines, Armstrong et al. [P < 0.001). Simin et al. [et al. [et al., [et al. [P < 0.001) or duration of antibiotic exposure . The most recent studies were performed by Lu et al. [et al. [et al. [et al. [Kilkkinen et al. , using a [et al.  surveyed [et al.  studied  [et al.  found ang et al.  found a n et al.  in a hug [et al.  in a met[et al., , more th [et al.  searchedu et al.  and Anek [et al. . In the  [et al. , 40 545  [et al.  was a meet al. [et al. [The association of antibiotics use and cancer is not restricted to colorectal tumors. Indicatively, two studies are referred here. The study of Kilkkinen et al.  found an [et al.  reportedAccording to this vast amount of published data, there can be little doubt that antibiotics overuse is associated with increased colorectal cancer risk, often in a dose- or time-dependent manner. Antibiotic type was usually not found to be a significant parameter of this association; however, some studies agree on penicillin and anti-anaerobic antibiotics. Association of antibiotics with other cancer types probably needs further investigation. Is colorectal cancer–antibiotics a causal relationship? The obvious explanation is the disturbance of colon microbiota. This is the explanation that most authors give for their results. In this perspective article, an alternative evolutionary explanation will be discussed, related with the selection of DNA MMR-deficient cells under antibiotic stress. I would like to state here that I do not neglect the probable significance of microbiota to cancer. DNA MMR deficiency is probably a part of a complicate equation that drives to cancer.DNA MMR is considered one of the most important mechanisms of DNA damage repair and one of the most conserved molecular mechanisms in all living organisms . MMR proMLH1, MLH3, MSH2, MSH3, MSH6, PMS1 and PMS2) have been identified. For some of them, the exact function is not clear. Deficiencies in the MMR pathway are a frequent cause of carcinogenesis.In humans, seven DNA MMR genes/proteins  [MLH1-associated tumors is not a gene mutation but hypermethylation of the promoter. Promoter hypermethylation of MLH1 is found in at least nine more cancer sites including gastric cancer (21.6%) [Most cancer cases are associated with somatic mutations in oncogenes and tumor suppressor genes. MMR genes are considered as tumor suppressor genes. Inherited neoplasias represent ∼ 5–10% of all cancer cases and usually follow an autosomal dominant model of inheritance. Mutations in the MMR genes are responsible for hereditary nonpolyposis colorectal cancer/Lynch syndrome (HNPCC/LS), and other cancer-predisposing Lynch variant syndromes. The majority of mutations in HNPCC/LS occur in and PMS2 . Additioand PMS2 . Specifi1 (9.8%) , 17. Fre (21.6%)  and oral (21.6%) .MSH2 and MLH1 genes [Microsatellite instability (MSI) is considered the classical method for detecting MMR pathway deficiency in colorectal or other tumors. Microsatellites are short tandem repeats (STRs) that are found throughout the genome. The most common ones in the human genome are the dinucleotide repeats, especially (AC)n. In case of a deficient MMR pathway, genetic instability is detected as presence of multiple alleles (instead of two) per each analyzed STR in tumors’ DNA . The NatH1 genes . TreatmeH1 genes , despiteH1 genes . ImmunotH1 genes .MMR gene mutations are observed in monocellular as well as multicellular organisms. In multicellular organisms, these mutations can cause cancer. In monocellular organisms, these mutations can offer an adaptive advantage through the ‘mutator’ phenomenon. Eucaryotic somatic cells with MMR gene mutations may have also increased fitness under the concept of ‘mutator’ cells. By virtue of the MMR mutation that may increase their fitness, mutator cells are also potentially cancerous cells.Escherichia coli mutators were among the first that were studied [The term ‘mutator’ is used for cells that have increased mutagenesis rate, which contributes to their survival under demanding or hostile environments. Most of the knowledge we have of this phenomenon comes from antibiotic-resistant bacteria or other drug-resistant microorganisms. Commonly, mutator microorganisms’ strains have a defective MMR pathway , 28. Esc studied . In thesPseudomonas aeruginosa is antibiotic-resistant and has increased virulence [P.aeruginosa lung infections are a life-threatening condition for these patients. Generally, mutator multidrug-resistant bacterial strains are common in chronic infections, like cystic fibrosis or urinary tract infections. Patients in these cases receive multiple antibiotic cycles and bacteria are continuously under positive selection for antibiotic resistance [Salmonella strains have also been identified with mutations in MMR genes [Cryptococcus, Candida and Aspergillus genus, all of which are characterized by increased mutagenesis rates and rapid adaptation to antifungal drugs [There are several examples of mutator strains. Studies show that MMR-deficient irulence . This hasistance , 33. AntMR genes , 34. Funal drugs , 35.et al. [NOTCH1 and TP53 genes, the most frequently mutated ones in esophageal cancer. NOTCH1 mutations in normal esophagus were several times higher than in esophageal cancers. Similar results were published soon after for many other healthy human tissues like endometrial, colorectal and liver [Recent advances in genomic analysis of somatic tissues challenge the standard knowledge that somatic mutations in oncogenes and tumor suppressor genes are always pathogenic. Mutations in oncogenes and tumor suppressor genes can lead to clonal expansions and adaptation in cells harboring these mutated genes. Martincorena et al.  found thnd liver . Many ‘dIntestinal epithelial cells with mutator capabilities have an adaptive advantage under stressful conditions, e.g. anticancer therapy . MMR-defet al. [Under repeated antibiotic courses, MMR-deficient colon mucosa cells can be selected as in the case of bacteria. These evolutionary pressures probably affect colon crypt stem cells, which are small clonal units occupying intestinal spaces referred to as crypts. Mutations in non-stem cells usually do not accumulate since they have limited life spans, while the stem cells are responsible for cell proliferation of the crypt. Despite stem cells being quite resistant to mutagenesis, inevitably mutations appear during ageing . Crypt set al.  showed tStudies have reported a gut microbiome imbalance (dysbiosis) in patients with colorectal cancer, showing an increase of the population of ‘bad’ microbes compared to a decrease of ‘good’ microbes . In lighThe MMR genes’ hypothesis could be tested by multiple ways. A population-based study would be ideal, by arranging prospective cohorts of patients treated frequently with antibiotics. Steps: (i) Patients undergo once a year colonoscopy examination, checking for any alterations in their colon mucosa, (ii) biopsies must be taken from any abnormal forms of tissue, like polyps or cancer-like malformations, (iii) DNA from those tissues will be tested for MSI, (iv) exome sequencing can be performed in polyp DNA or tumor DNA, looking for mutations in MMR genes or other implicated genes, (v) groups of cancer patients with an already MSI-tested biopsy, can be examined for a previous history of multiple antibiotic treatments, comparing the MSI-positive and the MSI-negative ones.The weakness of testing this hypothesis in humans is the need of colonoscopy. Colonoscopy is considered an invasive method, and this may be problematic under a research protocol. Additionally, biopsy testing cannot differentiate between direct and indirect effects of antibiotics. An alternative way to test this hypothesis is the use of animal models. Mice and zebrafish can be used as well. Steps: (i) Antibiotics can be administered in mice or zebrafish for a prolonged time, (ii) After some months (multiple time points can be used), DNA from multiple cell types, including intestinal cells, could be checked for any MMR gene pathogenic mutations, (iii) Results must be compared with antibiotic-free animals of the same age. Experiments can be designed to be more complicated, e.g. by performing comparisons between microbiota-free animals vs normal microbiota animals. Additionally, cancer incidence must be estimated, between treated and non-treated animals.Similar experiments can be performed in cell cultures, preferably colon cell tissue cultures. Cultured cells treated for a prolonged time with antibiotics and antibiotic-free cells can be tested for MMR gene mutations. More advanced technology can be used like organ-on-a-chip models as well. Microfluidic organ-on-a-chip models of human intestine are available . Chip exThe above suggestions can confirm or reject the hypothesis of MMR-deficient mutator cell selection. In addition, it would be important to consider whether extensive use of antibiotics by cancer patients could be risky as MSI-negative tumors can be transformed to MSI-positive after exposure to a harsh micro-environment. These tumors are more aggressive than the previous ones. It is probably wise for cancer patients to carefully consider antibiotic treatments or generally drugs that can increase death resistance of their cells.In conclusion, an evolutionary explanation is proposed for the association of antibiotics with colorectal cancer, which has been revealed in multiple large-scale population-based studies. Testing this hypothesis is feasible, especially in national cancer reference centers, where large cohorts of patients exist. Somatic selection is the key for the understanding of many conditions related with human disease.


MINFLUX is purported as the next revolutionary fluorescence microscopy technique claiming a spatial resolution in the range of 1–3 nm in fixed and living cells. Though the claim of molecular resolution is attractive, I am concerned whether true 1 nm resolution has been attained. Here, I compare the performance with other super-resolution methods focusing particularly on spatial resolution claims, subjective filtering of localizations, detection versus labelling efficiency and the possible limitations when imaging biological samples containing densely labelled structures. I hope the analysis and evaluation parameters presented here are not only useful for future research directions for single-molecule techniques but also microscope users, developers and core facility managers when deciding on an investment for the next ‘state-of-the-art’ instrument.This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 2)’. The significant milestones have been confocal laser scanning microscopy , 2-photoThe major advantage of the direct combination is in resolution enhancement by increasing the information per photon. For readers interested in the technical details of these methods, following are some excellent recent reviews on this topic –19. MINF 1.New biological insights through molecular resolution 2.A priori structural information and subjective event filtering 3.Multi-colour, three-dimensional, live imaging and the caveats when imaging ideal, well-defined structures 4.Spatial resolution versus localization precision, densityMINFLUX is presently the most photon efficient method to localize molecules and the aim of this article is not to argue otherwise. Here, I evaluate MINFLUX on the following four broad categories:I hope this detailed categorization helps scientists to evaluate whether MINFLUX is the right microscopy technique for their research.. 2et al. [The primary highlight of the paper by Gwosch et al.  is the a (a)NPCs are symmetrical structures of eight subunits arranged in an octagonal geometry with an outer diameter of approximately 120 nm (see  (b)et al. [et al. [et al. [It is worth noting that no independent validation of molecular copies or subunits of NPCs was done in Gwosch et al.  using ei [et al.  used the [et al.   and single-molecule localization microscopy (SMLM) roughly 50 and 10 years ago, respectively ,22. For  (c)et al. [et al. [et al. [Gwosch et al.  used Nup [et al. . As a ge [et al. . Moreove (d)et al. [et al. [Gwosch et al.  state th [et al.  MINFLUX  [et al.  created  [et al. , please . 3For years, in electron and single-molecule localization microscopy, filtering of imaging data has been done to enhance contrast and optimize visualization. In this section, I highlight how event filtering can be used to attain higher localization precision, purport new biological structures not present in the raw data and the need for blind samples to standardize the resolution claims. (a)a priori information to achieve molecular resolution, I use nuclear pore data from [www.ebi.ac.uk/biostudies/BioImages/studies/S-BIAD8. For downstream analysis at individual nuclear pore level, I zoomed-in at an image section with 4831 localizations  was chosen to provide close to four copies per subunit. In this regard, we question the choice of pixel and kernel size used in Gwosch et al. [4a and 6e but a kernel of 2 nm and a pixel size of 0.2 nm for figure 4a, f in Gwosch et al. [A single nuclear pore (yellow box) is further highlighted to show the impact of visualization and image rendering. Note how the individual components of the eight subunits of NPCs cannot be resolved with image zoom but can be resolved with a manually chosen pixel size (0.5 nm) and Gaussian kernel (8 nm). The choice of pixel size and Gaussian kernels were done with h et al. . For exah et al. . (b)a priori structural information. For structures with prior information like NPCs, the manual filtering can lead to additional biases for live imaging, see figure 6b. For structures with little prior information, the reduction in the number of molecules would lead to under-sampled structure (figure 6a). Furthermore, it raises questions on the authenticity of the structures if an independent validation via electron microscopy or other super-resolution methods is not provided.The MINFLUX filtering is done at various levels based on photon counts, targeted coordinate pattern to account for true emission and those considered as background events. For a fair comparison, both raw and filtered data should be presented to demonstrate that any such filtering is not biased by et al. [Lastly, it is not clear if the molecular components of NPCs will still be visible in raw/unfiltered data as the increased density of signals will tend to overlap like in the two-colour images . It is wet al. ) leads t (c)DNA origami is commonly used to measure the spatial resolution for different microscope setups. DNA origami has a pre-defined arrangement and blind samples with no knowledge on the prior arrangement are needed for proper calibration. This will make the photon count-based filtering more accountable as origami samples, unlike biological samples, have almost zero background see . As signResults obtained by imaging and analysing DNA origami will likely be a poor predictor of performance for real biological samples where problems of out-of-focus blur, non-specific background, light scattering and other sample aberrations exist. Thus, the resolution claimed based on DNA origami needs to be thoroughly investigated in this context and efforts made not to mislead the researchers interested in real biology.In fact, a new quality metrics for microscopy techniques of the relative deviation between nominal and real life resolution in different biological situations needs to be introduced. After all, conventional fluorescence microscopy rarely achieves its theoretical limit. However, the relative deviation is much smaller than in MINFLUX. (d)For the lateral spatial resolution, so far, MINFLUX imaging has been done on NPCs , microtuFor the biological targets with no underlying structure, an independent validation with another super-resolution method or a relative comparison under identical imaging conditions and length scales is essential. For such studies, a minimum signal density and labelling efficiency should be considered as a minimum requirement.For the biological targets with an underlying organization such as NPCs, the filtering of data can be misused to attain a certain resolution . Further. 4et al. [MINFLUX was originally published in 2017  with nan (a)et al. [et al. [The individual proteins of the eight NPC subunits (roughly 40 nm apart) that can be observed in single-colour images, vanish in two-colour images , puttinget al.  and Thev [et al. , respect (b)et al. [et al. [Regarding the three-dimensional imaging of well-defined structures like NPCs, the rendered MINFLUX data appears to be highly clustered, under-sampled and unevenly distributed  and Nup96 (SNAP-Alexa Fluor 647) again are barely comparable with standard three-dimensional SMLM images. The ring distribution of WGA is unresolved and instead appears like a random distribution of points. The Nup96 octamer is also hardly visible, both laterally and axially . (c)a (this paper) or figure 3A and Movie S6 (http://movie-usa.glencoesoftware.com/video/10.1073/pnas.2009364117/video-6) of Pape et al. [MINFLUX has been used to probe the sub-mitochondrial localization of the core MICOS proteins . The autfigure 5e et al. . As no iThe microtubules: Historically, FWHM of microtubules (MTs) has been the benchmark for resolution demonstration in STED microscopy. MTs are continuous and have a denser tubular organization (25 nm) when compared to the nuclear pores. Hence, any subjective filtering would lead to an under-sampled image. The hollow tube structure of MTs has been resolved with SMLM and DNA-PAINT setups. For well resolved three-dimensional cylinders of microtubules with SMLM, see Huang et al. [et al. [et al. [g et al. , Li et a [et al. . On the  [et al.  claim 15In summary, MINFLUX performance on continuous structures with a well-defined organization, such as MTs or those for which prior information is not available, remains to be proven. For MICOS, post-synaptic proteins, etc. which do not have an underlying structure, any resolution and shape can be claimed but is it valid? Thick samples where background is high will be another big challenge for MINFLUX. (d)b). The observed uneven distribution of nuclear pores is possibly due to heavy filtering of localizations (greater than MINFLUX imaging of nuclear pores (Nup96–mMaple) in living U2OS cells shows a highly under-sampled configuration with no data on cell viability or photo-bleaching b. The obh & Curd  for a deRegarding excitation powers for live imaging, the authors used powers old less . For str. 5MINFLUX nanoscopy claims to provide a ‘resolution in the range of 1–3 nm for structures in fixed and living cells’. It is important to remember that 1–3 nm range here refers to localization precision, and more specifically of fluorophore and not that of the target protein . As argu (a)For years, localization precision and spatial resolution have been interchangeably used by physicists given their inherent dependence on photons. Resolution, i.e. the ability to resolve close-by structures or target molecules, primarily depends on how densely a biological structure is labelled while localization precision implies how well these labels are detected, see For single-molecule studies, localization precision follows an inverse square-root relationship with the total number of detected photons, whereas MINFLUX has an inverse quadratic relationship with detected photons under ideal conditions with zero background. For real-life biological applications, MINFLUX is severely limited by the background photons as this compromises the positioning of the donut zero. In general, MINFLUX is likely to achieve better precision numbers than single-molecule imaging but so far, due to multiple reasons stated in this article, the spatial resolution of the method has been much poorer than SMLM methods . (b)et al. [et al. [Gwosch et al.  applied The second resolution assessment is made by subtracting the mean localized position from all localizations. This process is similar to the first approach and will be biased by the filtering of the data. The third approach is based on Fourier ring correlation (FRC) and it is not clear whether filtered or raw data was used for FRC analysis. FRC measures correlation between subset of localizations and will again be affected by filtering of the data.Prakash & Curd  estimate (c)A resolution cannot be better than the localization precision of the detected fluorophore. As a general rule, a more conservative upper limit for localization precision or full-width at half-maximum should be used to account for the linkage errors. From a biochemistry point of view, how reasonable to have a resolution in the range of 1–3 nm when the SNAP-tag itself is about 3–3.5 nm in size? Given the resulting uncertainty of relative position of the dye molecule, the stated nanometer resolution  can only be claimed for the fluorophore, but not the biological target. In this context, I urge the readers to take careful note when authors state the precision of the fluorophore and that of the target protein. (d)Generally speaking, the resolution is the fundamental ability to determine a structure. From a physicist perspective, it is satisfying that MINFLUX can achieve 1-nm localization precision under ideal conditions (based on photon statistics), whether it materializes in resolving real life biology or not. From a biologist perspective, for a 1-nm resolution claim, MINFLUX is off by a factor of 5 for synthetic samples like DNA origami and a factor of 40 for biological samples like NPCs.For a true 10 nm structural resolution, a general expectation would be to resolve a well-established 10 nm bead-on-string structure of nucleosomes or a turn of DNA helix which is about 3.4 nm (for 1-nm claim). From Nyquist viewpoint to achieve 10 nm structural resolution, one would need a dye every 4 nm, which for many biological structures is hard to achieve due to steric hindrance or low copy number/binding sites. Alternatively, for structural biology, with true 1-nm resolution claim, I wonder if one can compare MINFLUX with X-ray crystallography, which has a broad range of resolution (anything between 1.5 and 10 Å) depending on the crystal quality.. 6 (a)The spatial resolution in light microscopy has become something of a numbers game where scientists cite an arbitrary FWHM or an FRC resolution or filter localizations to achieve any desired resolution. Given the large number of potential biological reference structures in the range of 1–100 nm size scale, for example, DNA (2 nm), nucleosomes (10 nm), microtubules (20 nm), NPCs (40 nm), synaptonemal complexes (60–150 nm), I recommend using these well-defined structures to support spatial resolution claims. For DNA origami researchers, I encourage usage of blind samples where the origami arrangement or the distances are not known in advance. This will make the photon count-based filtering more accountable. (b)et al. [et al. [Gwosch et al.  and Balz [et al.  have mad (c)MINFLUX papers have shown several loop-holes in current scientific reporting and standards. A clear distinction needs to be made about experimental precision, spatial resolution, cross-validation of new structures by independent methods, extent of sample variability versus measurement uncertainty, hardware adaptability, image quality/standards , availability of raw data/codes, reproducibility of the analysis pipeline, automated versus manual components of data analysis (like filtering) and proper procedures to report them .From a historical perspective, 4Pi, STED and now MINFLUX have been closed systems with little focus on making the hardware adaptable or raw data/codes available. Several units of highly priced 4Pi microscopes were sold around 2000s as the next generation microscope, however, not many biological findings resulted. This was followed with the launch of STED microscopes with very few novel biological results.Science is about reproducible measurements. Improvement in precision leads to refined measurements. MINFLUX improves the precision with which fluorophores can be localized but by not making the hardware adaptable, raw data and codes available, it is hindering reproducibility, open science efforts and overall progress of the field. (d)One of the important aspects of science is to promote cutting-edge ideas even if they offer few gains in the short run. MINFLUX research provides an important conceptual advancement and leads to overall progress in the field. The aim of this article is not to discourage MINFLUX research but to highlight that the technology is still underdeveloped and needs further validation from other independent labs before it is made commercially available, especially considering the high price tag of the commercial system.As research grants mostly come from tight public funds and are a zero-sum game, I hope this critique provides the scientists, microscope users, developers and core facility managers with an alternative viewpoint when deciding about an investment in the next ‘state-of-the-art’ microscope instrument.


There is a growing trend in complementary and alternative medicine (CAM) usage among the population with medical conditions. However, there is hesitancy for medical practitioners to integrate its application with the current treatment modality, despite governance by the authority. Hence, our objective is to systematically evaluate the healthcare perception towards integrating CAM in their practices. We systematically searched three large and renowned databases i.e., Scopus, Web of Science and PubMed, regarding “Perception on Integrating CAM Usage in Patient's Treatment among Healthcare Practitioners” from 2016 until 2020. At least two independent reviewers comprehensively screened and extracted the data from the accepted articles. A total of 15 studies were included in the final qualitative synthesis following a strict and rigorous assessment checked using MMAT 2018 checklist. The studies included providing the richness of information due to the qualitative nature of the study design. There were three main domains extracted i.e. knowledge, attitude, and perspective of the healthcare practitioner towards CAM integration. Limited knowledge of CAM among healthcare providers may be the possible main reason for non-supportive attitude and negative perspective on CAM. However, those who showed an inclination towards CAM were found to be more open and ready to learn about CAM if it provides benefits to the patients. There is a heterogeneity of perception towards CAM integration from healthcare providers' point of view. A proactive and systematic CAM literacy awareness program may help to improve their understanding and possibly gain more trust in its application. The world population is consistently facing health disease burden either from communicable or non-communicable disease. The practice of conventional medicine is the mainstream health system in most countries in treating diseases. However, in this current era, the treatment modalities are accessible within the spectrum of conventional medicine to complementary and alternative medicine (CAM). CAM is a general term referring to a broad field of medical “therapies” that is different from the conventional medical treatment practice in hospitals. According to the National Centre for Complementary and Integrative Health (NCCIH), there are five main groups of CAM, namely alternative medical system, mind-body interventions, biologically therapies, manipulative and body-based methods as well as energy therapies . The NCCAs reported in a systematic review, there was substantial CAM use (9.8% - 76%) among the general population in 15 countries surveyed . MeanwhiAt the same time, evidence had shown that conventional medicine has been steadily reducing morbidity and mortality as well increase the quality of life for the past decades in managing most of the disease. However, CAM practised were perceived to be more effective compared to conventional medicine based on the population survey conducted among elderly patients in Malaysia. Half of the respondents (55.1%) agreed, from a total of 256 respondents in the study . On the At the time being, there is a scarce study on CAM among healthcare practitioners. Therefore, this review aims to determine the perception of integrating CAM in patient's treatment among healthcare practitioners.Search strategy: this systematic review was conducted using three large and renowned databases, i.e. PubMed, Web of Science, and Scopus, regarding the “Perception on Integrating CAM Usage in Patient's Treatment among Healthcare Practitioners” from the year 2016 until 2020. This search was conducted in accordance with the Preferred Reporting Items for a Systematic Review and Meta-Analysis (PRISMA) checklist [Annex 1 and PRISMA Checklist as Annex 2. The keywords used were as below and the search strategies as Annex 3: “Complementary medicine” OR “traditional medicine” OR “alternative medicine” AND “Belief” OR “perception” OR “perspective” OR “attitude” AND “Healthcare practitioner” OR “healthcare worker” OR “healthcare professional” OR healthcare personnel” OR “doctor” OR “physician” OR “medical assistant” OR “nurse” OR “pharmacist”.hecklist . A tableInclusion and exclusion criteria: the target population of this search was any healthcare practitioner. The inclusion criteria from the database searches were (a) Original article , (b) All medical disease or advice, and (c) Availability of full-text article. The exclusion criteria in this search were based on (a) Quantitative type in design, (b) Systematic and narrative review paper article, and (c) non-English article. The articles were then identified through the titles and abstracts screening process according to the eligibility criteria. Full-text articles obtained were subsequently included in the qualitative synthesis. The flow of the article search is described in Data extraction tool: all included articles were extracted by two independent authors and in case of inconsistency, a third author was consulted. The data was customized into (a) Number; (b) Year; (c) Author and Country; (d) Titles (e) Study Design; (f) Type of methods and analysis; (g) Result-themes generated; and (h) Conclusion.Operational definition: the definition stated by NCCIH on CAM were simply as a group of diverse medical and healthcare interventions, practices, products, or disciplines that are not generally considered as part of conventional medicine. The healthcare providers include all practising professionals in the medical field who may have direct or indirect contact with the patient either by giving treatment or medical advice. They may include doctors, physicians, surgeons, nurses, medical assistance, occupational therapist, physiotherapist, pharmacist etc.Annex 4.A total of 511 articles were initially obtained for the title and abstract screening, while only 50 articles were left for full-text screening. The common reason for omitting the other 35 articles upon full-text review was due to the study design, different objectives, not the target population, and not related to CAM. Two reviewers assessed the quality independently using the mixed method assessment tool Mixed Method Assessment Tool (MMAT) version 2018. Articles were only selected if both reviewers agreed with the quality. Any disagreement between the assigned reviewers will employ a third independent reviewer. All the included studies answered “yes” for all the questions of the respective domains of MMAT checklists which are risk of bias assessment that is present as The distribution of the articles varies with four articles from Asia ; three articles each from the USA and Australia; two articles each from the United Kingdom and Germany; and one paper from Ghana.The findings can be broadly categorized into three main domains which are knowledge, perspective, and attitude towards CAM by the healthcare provider. Due to the richness of the data from qualitative type of research, some of the themes overlap with one another. Therefore, two of our domains which are perspective and attitude were further broken down into more sub-themes and categories. Factors affecting the practitioner's perception in integrating CAM are described knowledge):Domain ( overall, studies that looked into this domain reported the lack of knowledge in CAM among medical practitioners [et al. [et al. [itioners -13. Acco [et al. , almost  [et al.  were the [et al.  suggesteperspectiveDomain: Positive perspective: there are two sub-themes derived in this domain, which are positive perspective and negative perceptive. The integration of CAM with conventional treatment have received two types of fates, either being positively accepted or the idea clearly being denied by the medical practitioner. To put in highlight, the reason for positive acceptance by the healthcare worker was the probable good additional impact that CAM could contribute to the treatment. This is especially true in pain management as most of the practitioners who have a positive perspective on CAM echoing its usage as an additional modality. A study by Sharp et al. [et al. [et al. [p et al.  on patie [et al.  that uti [et al.  claimed  [et al.  stated t [et al.  describeNegative perspective: the opposite sub-theme that emerged was the negative perspective on CAM integration. One of main the reason for the negative perspective towards CAM was the skepticism towards it despite patients claiming of having a good result after using it. According to Christina et al. [et al. [et al. [et al. [a et al. , nurses a et al.  found th [et al.  also fou [et al.  explaine [et al. . Becker  [et al.  on the oOther than being skeptical, another reason for the negative perspective on CAM was the modality that has some link with cultural and religious belief. For example, Corina, Christine and Klein  in theirattitudeDomain: Supportive attitude: the sub-theme of supporting attitude towards CAM can be further subdivided into three categories. One category that supports the integration was based on its true definition of being a complementary or supporting treatment. Anheyer et al. [et al. [et al. [et al. [r et al.  found th [et al.  also add [et al.  showed a [et al.  supportiet al. [et al. [The positive attitude towards CAM was also shown in the sub-theme of displaying interest in the intervention. Tagharrobi, Mohammadkhan Kermanshahi and Mohammadi  showed tet al.  where alet al. ; Christi [et al.  showed tet al. [Another reason for the supportive attitude on CAM by medical practitioners was due to the respect of patient's choice. Liem  highlighet al.  that demet al.  extendedNon-supportive attitude: on the other hand, the sub-theme of non-supportive attitude towards CAM can be categorized into two; due to concern of implication and from the lack of support in healthcare services. Anheyer et al. [et al. [r et al.  raised t [et al.  and Liem [et al.  highligh [et al.  were conet al. [et al. [et al. [Another category, which is lack of support in healthcare services was found to be a non-supportive attitude towards CAM. This reason was mentioned in studies by Kretchy et al.  and Beck [et al. . Apart f [et al. , the lacet al. [The interest in CAM has dramatically increased over the past years. In the UK, there was a high prevalence of herbal medicinal products that were bought over the counter, mostly self-prescribed. Based on our findings of the positive perspective subdomain, several articles reported the use of complementary and alternative medicine, such as chiropractic and acupuncture due to the reasoning of having an additional impact on the treatment and whole-person healing ,20. Thiset al.  that higet al. .With the above-mentioned positive perspective of benefit, it is not surprising for some medical practitioners to have a supportive attitude towards CAM. A study in India found that more than half of doctors working in tertiary hospital utilized CAM therapies and the most commonly utilized therapy was Homeopathy . The docAnother noted reason for medical practitioners to have a supportive attitude towards CAM was respect for patient's choice. Respecting patient's choice is one of the arts in treating disease. It shows effective doctor-patient communication, especially in giving support to patients to find hopes in curing their diseases. According to Kelak, Cheah and Safii , doctor'Although our subdomain religion showed a negative perspective among medical practitioners towards CAM, many other studies found otherwise. A review by Alrowais and Alyousefi  noted reWe also found that medical practitioners have negative a perception towards CAM is because of their skepticism towards it. In a study conducted among the oncologist in Brazil, some of the participants had negative views on CAM due to the limitation of the resources in the healthcare system, thus, they need more evidence-based medicine for practice . On the Our systematic review showed a mixture of perception among healthcare practitioners in the integration of CAM in patients' management that were presented into three main domains and respective subdomains. Many factors were highlighted, ranging from personal experience until the concern of implication if CAM superseded conventional treatments. Nevertheless, knowledge on CAM still remains low among healthcare providers. More awareness program, targeting medical professional, is needed, to successfully integrate CAM in patients care.CAM among healthcare practitioners is a new body of knowledge although it is widely practice in some population;Some patients used CAM as a replacement modality to treat their chronic non-communicable diseases;The integration of CAM together with conventional medicine is limited due to lack of safety and efficacy data.The study has highlighted a low level of knowledge about CAM and its limited application among the healthcare practitioners;The heterogeneity of perception regarding integration of CAM modality with conventional treatment hinders its application by the healthcare practitioners;Healthcare practitioners' attitude towards CAM can skewed towards acceptance as a result of environment and patient factors.


The shape and load bearing strength of cells are determined by the complex protein network comprising the actin-myosin cytoskeleton. In response to signals received from the external environment, including chemical and mechanical stimuli, the organization of the actin-myosin cytoskeleton may undergo dynamic changes that contribute to the production of physical force necessary for many cellular processes including cell division, endocytosis, intracellular transport and migration. The essential role of the actin-myosin cytoskeleton in so many cellular functions means that aberrant regulation or function can contribute to a variety of human pathological conditions and diseases.Cells includes 11 review articles that present up-to-date perspectives on a range of cytoskeleton-related fields. A prominent theme linking several reviews is the actin–myosin cytoskeleton in neurons. Mikhaylova et al. [This Special Issue of a et al.  also foca et al.  related a et al.  profileda et al.  discusseBlaine and Dylewski  examinedCells also includes four primary research articles. García-Bartolomé et al. [This Special Issue of é et al.  reportedé et al.  demonstré et al.  conditioé et al.  examined


We have read the case report entitled “Clinical presentation vs endoscopy for an early diagnosis of eosinophilic esophagitis: a case report” by Di Stefano et al. (2022). We wouIn the case report , the auOn the other hand, a systematic review by Shaheen et al. (2018), which The authors declare no conflicts of interest.None.


Eichner index is a dental index, which is based on the occlusal contacts between naturally existing teeth in premolar and molar regions. One controversial topic is the association between occlusal status and temporomandibular joint dysfunction (TMD) and its associated degenerative bony changes. Through the use of cone-beam computer tomography (CBCT), the current study sought to ascertain the relationship between the Eichner index and condylar bone alterations in TMD patients. In this retrospective study, the CBCT images of bilateral temporomandibular joints (TMJs) of 107 patients with TMD were evaluated. The patients’ dentition was classified into three groups of A (71%), B (18.7%), and C (10.3%), according to the Eichner index. Radiographic indicators of condylar bone alterations, including as flattening, erosion, osteophytes, marginal sclerosis, subchondral sclerosis, and joint mice, were either present or absent and registered as 1 or 0, respectively. Chi-square test was used to evaluate the link between the condylar bony changes and the Eichner groups.p= 0.00). However, no significant relationship was found between sex and condylar bony changes (p= 0.80).There was a significant relationship between the Eichner index and condylar bony changes (p= 0.05). According to the Eichner index, the most prevalent group was group “A”. The most prevalent radiographic finding was “flattening of the condyles” (58%).Condylar bony changes were found to be statistically related to age ( Patients with greater loss of tooth supporting zones have more condylar bony changes. The temporomandibular joint (TMJ), one of the body's most intricate articulations, has a wide range of anatomical and physiological characteristics  . DifferTMD describes a group of clinical complaints that affect the stomatognathic system, mainly the muscles of mastication  . TMD haDeveloped by Karl Eichner, the Eichner index can be applied for epidemiological studies and it is one of the most widely used dental indices. This index is effective in establishing intermaxillary contacts and extending functional dental invalidity  . Using The relationship between Eichner index and bony changes in the condylar region has only been studied in a few studies  , 29. IIn accordance with IR.SUM.DENTAL.REC.1399.0908, the present study was approved by the Institutional Research Committee. In this study, 107 patients with clinical signs and symptoms of TMD who needed further CBCT investigation were recruited from the archives of a private clinic and an oral and maxillofacial radiology department. There was a wide age range of 16 to 80 years for the patients (39.57±3.31 years). Exclusion criteria were patients with an established history of temporomandibular surgery, acute trauma, congenital abnormalities, musculoskeletal or neurological diseases, and any systemic diseases potentially affecting joint morphology.Based on the occluding pairs in the posterior teeth (two premolars and two molars), the dentition of each patient was divided into four main occlusal supporting zones. All of the four supporting zones are in contact in class A; one supporting zone is missing in class B, or all of the four supporting zones are absent, but theanterior region remains intact; and class C has no occlusal contact between the remaining teeth .This stA New Tom VGi  was used to obtain CBCT images of bilateral TMJs with 110 KVp, 3.05 mA and 3.6 s exposure time in the standard resolution mode (voxel size 0.3). Image fields were 15×15cm. standing upright; the patients were biting their teeth in the maximum intercuspal position. The Frankfort plane was parallel to the floor when their heads were positioned. The NewTom Cone Beam 3D imaging system workstation (NNT Software version 6.2) was employed to prepare the images of TMJ. The reconstructed CBCT scans were assessed using high-resolution monitor (Barco-China) in a dedicated reporting room with appropriate viewing (dimly lit) condition.The raw data were reconstructed primarily for the TMJ. By scrolling the axial images, the system identified the axial view on which the condylar width had the largest mediolateral dimension. The interval and thickness of the image slices were both set at 0.5mm. Afterwards, the corrected coronal and sagittal cross sections of each joint were rectified by drawing a perpendicular and parallel line and reconstructing them to the long axis of the condyle.The criteria used to evaluate condylar bony changes included (1) flattening (loss of convexity of condylar head outlines), (2) surface erosion , (3) marginal bony overgrowth or osteophytes , (4) subcortical erosion or Ely cyst , (5) marginal sclerosis, (6) subchondral sclerosis (Increased radiopacity of thecancellous bone), (7) joint mice (osteophytes that break off and lie free within the joint space) .For each patient, the right and left TMJ areas were evaluated for the presence or absence of one or more of these radiographic changes, and were rated accordingly as 1 or 0.We calculated the left and right TMJ bony changes independently by adding up the scores related to any radiographic finding. For example, when a patient had flattening, erosion, and sclerosis in the condylar bones, the total number of changes was considered "3”. The right and the left scores were then added. Consequently, the association between the condylar bony changes and the Eichner index was examined.The images were evaluated by two dentomaxillofacial radiologists. Each observer evaluated the images independently after a minimum of two weeks. In addition to checking the consistency between the first and the second sets of records produced by each specialist, we also examined the reliability of the inter-examiners for each of the criteria applying κ statistic. Adequate intra-examiner agreement index , as well as strong inter-examiner agreement (κ coefficient: 0.801 to 1) was detected.p= 0.05. A Dunn’s post-hoc test was used to compare the prevalence of overall bone changes between Eichner groups. Statistical calculations were performed using SPSS . To ensure the intra and inter-examiner reliability, κ statistic was used. The chi-square test was used to determine whether there was a correlation between condylar bony changes, age, sex, and the Eichner groups .Significance level was set at Excellent inter-(κ coefficient: 0.801 to 1) and intra-(κ c-oefficient: 0.833 to 1) examiner agreement was observe-d. In the present study, 107 patients wererecruited among which 74 were females with an age range of 17-78 years (mean 39.18+15.20) and 33 were males with the age range of 16-80 years (mean 40.42+17.70). Based on the Eichner index,the most common Eichner group was a (71%) followed by group B (18.7%) and group C (10.3%) .p= 0.00). However, sex did not significantly affect total bony changes (p= 0.80).Bony changes in the condylar region were significantly associated with Eichner index  showed radiographic changes in condylar morphology. Flattening was the most prevalent bony changes (58.9%) followed by erosion (40.2%), marginal sclerosis (20.6%), subchondral sclerosis (14.0%), osteophyte (5.6 %), cyst (3.7%) and joint mice (0.9%).The results showed that total bony changes had a favorable relationship with age (er index .Moreoveer index .et al. [ A common cause of orofacial pain that is not related to dental or infectious conditions is TMD  . Severaet al.  . Many set al.  ; canineet al.  ; discreet al.  , Kennedet al.  , 29 . Since posterior teeth are necessary to maintain uniform occlusal force distribution on TMJ, losing them can have a greater impact than occlusion type. Additionally, due to incisors' inclined planes, entire mandible move backward when posterior teeth are lost. As a result of this movement, the condyles are moved above and behind their normal position in relation to the articular eminence. When a unilateral cause is responsible, only one condyle is affected but when the bite has a closed, both are affected  - 28.et al. [ et al. [ et al. [ et al. [ Only two studies have investigated the relationship between Eichner index and condylar bony changes  , 29. Aet al.   and Jal et al.  , despitet al. [ et al. [ et al. [ et al. [ et al. [ The TMJ is characterized by its ability to remodel when loading forces exceed its normal tolerance. In adults, this adaptive response can alter the condylar bone morphology and articular eminence  . Flatteet al.  , Mathew et al.  , Gharge et al.   also fo et al.  . Unlike et al.   and Tak et al.   found tet al. [ et al. [ In the published literature, there is still controversy over the correlation between condylar bony changes and age. According to several studies, condylar bony changes and aging are positively correlated  - 35, wet al.   and Jal et al.   found n et al.  .et al.'s study [ et al. [ et al. [ et al. [ et al. [ Our study found no significant relationship between sex and total bony changes, which is consistent with Irsan NDH s study  . Alzaha et al.  , Takaya et al.  , and Ja et al.   reporte et al.  , who foVarious radiographic modalities such as plain film radiography  , 29, cThis study aimed to determine whether radiographic bone changes in the condyles are associated with Eichner; however, some factors such as the time between tooth extraction and image time and oral habits were not evaluated in relation to the severity of TMD symptoms and condylar bony changes. To either confirm or refute the results of the present study, further investigation should be conducted with these factors taken into account.The results of this study indicate that condylar bony changes are highly associated with the Eichner index. In addition, CBCT findings were associated with variations in finding condylar bony changes in this study. It is therefore important to consider CBCT as a modality for appropriate diagnosis in clinical practice as well as for patients' therapeutic choices.The authors declare that they have no conflict of interest.


Gas sensors play an irreplaceable role in industry and life. Different types of gas sensors, including metal-oxide sensors, are developed for different scenarios. Titanium dioxide is widely used in dyes, photocatalysis, and other fields by virtue of its nontoxic and nonhazardous properties, and excellent performance. Additionally, researchers are continuously exploring applications in other fields, such as gas sensors and batteries. The preparation methods include deposition, magnetron sputtering, and electrostatic spinning. As researchers continue to study sensors with the help of modern computers, microcosm simulations have been implemented, opening up new possibilities for research. The combination of simulation and calculation will help us to better grasp the reaction mechanisms, improve the design of gas sensor materials, and better respond to different gas environments. In this paper, the experimental and computational aspects of  Humans cannot live without gas. However, some of these gases are toxic and harmful even though they may be colorless and odorless. When these gases are present in our environment, it is essential to detect them immediately to protect us from their hazards. Research on gas sensors has long been ongoing and of increasing interest due to their role in a wide range of fields, including laboratory health and safety, gas detection, observation, and environmental investigation , with evnt gases . Even th sensors . The resironment . Further of TiO2  with the of TiO2 , the resectivity . The pho, and NO . The quaatalytic , and gasatalytic  and dram explore , and as humidity . It is t testing . In the  testing . Sensiti testing . In part testing . In Tablarticles , orange tum dots , light ycrystals , light be phases , and dar2 fibers ) that weeriments . Since 1material . As the i et al.  showed ti et al. . It was The MOS gas sensor is designed based on the change in conductivity of the sensor material under the action of reducing or oxidizing gas O2 for an n-type semiconductor can be expressed by Equation (5) , in whiction (6) :(5)S=RaiIn Equation (6), Computer technology is also developing rapidly at a time when the level of experimentation is constantly improving. It can be said that a computer’s help is indispensable in every aspect now, including the experimental part. In the same way, the theory of physics has made astonishing progress. The development of quantum mechanics has promoted the rise and application of the density functional theory (DFT). After deducing the famous Kohn–Sham (K-S) equation by local density approximation (LDA), the first-principle calculation based on DFT has become a powerful material development tool . Using cThere are several experimental methods to improve the performance of onductor , which eI41/amd) , and bro) [pbca) , and the) [pbca) ; these s) [pbca) . In addis 600 °C . The ban 2.96 eV . Howeverfficient . There a bandgap . It is fThe performance of the sensor can be improved by comparing the experimental results, as different experimental methods can obtain different results. In the past, in the manufacturing of gas sensors, the sensing material was uniformly covered on the substrate because the electrical conductivity of metal oxides varies with the adsorption and desorption of gas molecules, but it turns out that this may not be good enough . Common In general, the excellent advantages of Various materials have been obtained through different methods , includinditions b,c is giIn addition, unadulterated nanoparticles are used to help improve the performance of gas sensing. Azhar Ali Haidry et al.  used magy’s work  that paro et al.  preparedhe flame . During he flame  used thiElectrostatic spinning is one of the most commonly used methods to produce nanofilaments. Electrostatic spinning technology has also been used in many fields, including in the study of gas-sensitive materials, by being combining with other technologies. Fibers that are -Ah Park . The mixDue to bandwidth gaps formance . On the formance . The maiA d. Zhang  then triu et al.  were abli et al.  doped CuHowever, the results of Abdelilah Lahmar et al.  showed ti et al.  allowed alehifar  preparedL. Aldon et al.  doped Snichphant , a similSingle-layer graphene (SLG) has the purest carbon chemistry. The two-dimensional (2D) atomic structure maximizes the surface-to-volume ratio. The SLG’s tiny resistance value is very friendly to gas detection at a low power consumption and usually enables a significant reduction in operating temperature. If this can compensate for the higher operating temperature of g et al.  used theSimilarly, semiconductor materials are other common doping materials that are doped into TiO2  . The finChunrong Xiong and Kenneth J. Balkus, Jr.  used SnOr et al.  used ther et al.  conducteme ratio .Piotr Nowak et al.  aimed toIn the case of Chachuli . In theiThe same is true of Feng Li et al. , who dopn et al. . From thJinniu Zhang et al.  doped tii et al.  discussei et al.  synthesiIn this section, the authors summarized the papers on U [U + J [Obtaining better results and breakthroughs often requires a lot of research time and the persistence of countless researchers to make gas sensors work better, but there is no doubt that it is often more difficult when we try to explore the mechanism of the reaction. In the last decade, the development of quantum chemical computational molecular simulations and density functional theory has allowed us to take a more microscopic view of the reaction process, which has attracted more and more people to participate in the research. First principles have helped us to take our research to a higher level from a physics perspective. First-principle computing has several attractive features. First, it can obtain the characteristics of a system without any experimental parameters , based oU , DTF + UU [U + J  and the In the microscopic field of theory, physicists use an equation called the wave functions to describe the states of particles, which is generally expressed as Equation (7): as 1927 . The Hoh as 1927  theorem [H=T+U+V , which c[H=T+U+V  equation[H=T+U+V , GGA [87[H=T+U+V , are appFirst-principle calculation can be used to perform many simulations, including some dangerous biological experiments, without any side effects. However, as mentioned earlier, doping is an effective way to improve the performance of semiconductor gas sensors. A doped semiconductor gas sensor has been widely used in first-principle calculation . For exaCompared with STM images, it is found that ang Wang  derived formance . The surn et al. . In Lilin et al.  work, thJuan Liu  and otheaballero  showed tr et al. . The restion (8) ,96. It iIt was found by theoretical calculations that the Oleg Lupan et al.  preparedBharat Sharma et al.  looked aequation :(9)D=0.8ion (10) :(10)ΔE(Tlated as :(11)Eadsessed as :(12)ΔR∝eJoseph Muscat et al.  studied . Garcia  studied  (0 0 1) . This al (0 0 1)  calculatTo verify the accuracy of the results, James A. Quirk et al.  characteMichele Reticcioli et al.  also carBandwidth changes the most quickly after stimulant use because the first principle can show changes before and after doping . The bang et al.  used theisa Ohno  not onlyU or other hybrid functions is proposed. Youngho Kang et al. [U to calculate the electron–phonon interaction and transmission in anatase U (blue solid line). The lower right corner gives the quadrilateral crystal structure with blue atoms of titanium and red atoms of oxygen. To better describe the structural properties, the on-site Coulomb energy In the early stage of the crystal system, the direct use of function calculation results may deviate from the experimental values. Therefore, a solution of DFT + g et al.  used DFTU and Hund’s J corrections with parameters calculated using linear response theory based on an extended first principle, found that this method was able to predict the bandgap more accurately, giving a maximum error of less than Okan K. Orhan and David D. O’Regan , based og et al.  researchg et al.  added thIn addition to the relationship between polarons and electrons, the first-principle calculations can also study the surface or defects of the The surface of anatase ang Wang . The resHarrison  explain It is undeniable that two-dimensional (2D) materials are gaining more and more importance in some fields as researchers continue to refine and develop their research on materials . Additio Hao Sun  performeion (13) :(13)σ∝e−g et al. , who intGraphene, with its unique two-dimensional structure and large specific surface area, has gradually become a hot spot for sensor research . Zhang eIn this section, the authors provided a brief overview of the theoretical basis of DFT calculations for In general, the excellent advantages of


This article (Correspondence) is in response to the recently published article on the role of Pecto-intercostal Fascial Block for cardiac procedures by Zhang et al. in “BMC Anesthesiology”. I greatly appreciate the authors for publishing this study in which Pecto-intercostal Fascial Block, a novel technique for providing pain relief in open cardiac surgical procedures was evaluated. I wish to present my reflections on this article as well as to add a few more points on this topic. Dear Editor,I read the recently published article that has analyzed the effectiveness of Pecto-intercostal Fascial Block (PIFB) for patients undergoing open cardiac surgery  with keeZhang et al.  have staZhang et al.  have alsThe control group of patients had significantly higher levels of blood glucose despite a significant rise in insulin levels, which was attributed as “Insulin resistance”, by Zhang et al. . Also, tZhang et al.  have men


To the editor: We read with interest the article by van Ewijk et al. [k et al.  regardink et al. . In factk et al. . If the k et al. ,5. FurthThere is a previously published report suggesting that policies differentially targeting the vaccinated and unvaccinated would alter VE estimates. A study in New York showed that VE estimates declined simultaneously across different time cohorts after lifting mask mandates exclusively for fully vaccinated individuals, which cannot be explained by waning immunity . AlthougWe previously published a similar study adjusting for high-risk behaviours and mask wearing as well as testing behaviour . We alsoWhen estimating VE, we assume a causal relationship between vaccination and infection/disease  and we r


Saliva is a precious oral fluid that contributes to oral health and when its quantity is diminished, it hampers the quality of life. Individuals suffering from diabetes have a complaint of reduced salivation due to the consumption of xerogenic drugs and autonomic neuropathy. Our study aims to evaluate the effectiveness of the transcutaneous electric nerve stimulation (TENS) device on the salivary flow rate with respect to age and gender in Jaipur population.p value <0.05 was considered to be significant. A descriptive observational study was carried out on individuals in Jaipur at the Department of Oral Medicine and Radiology at Rajasthan Dental College andHospital during a period of 7 months. The study consisted of 200 individuals who were divided into two groups. Unstimulated and stimulated saliva were collectedfor 5 minutes in a graduated beaker. Stimulated saliva was collected after keeping the TENS unit activated at 50Hz. Kolmogorov-Smirnov, Shapiro-Wilks normalitytests and Mann Whitney U test were done. The  The TENS unit was effective in increasing the quantity of stimulated saliva and a highly statistical significance was seen in age groups. TENS was also found to be more effective in increasing saliva in diabetic individuals. The mean unstimulated salivary rate was 1.64ml/5min and the mean stimulated salivary rate was 1.914ml/5min for Group I. The mean unstimulated salivary rate was 1.231ml/5 min and the mean stimulated salivary rate was 1.547ml/5 min for Group II. The p value for Group I and II for unstimulated saliva was 0.01 and for stimulated saliva was 0.03. It seems that TENS has shown positive results in increasing salivary secretions and salivary values may diminish with age; therefore, TENS might be used in aged individuals as well as in diabetic patients to increase the quantity of saliva. Saliva is a precious oral fluid that is often taken for granted but we need to realize that it is very critical for preserving and maintaining oral health  . SalivaHyposalivation has been found to be associated with diabetes mellitus  . XerostApplication of electric impulses to one or more of the three components of the salivary reflex arch should theoretically improve salivary secretion and lessen the various long-term effects of hyposalivation  .Transcutaneous electric nerve stimulation (TENS) is a simple, inexpensive, and non-invasive modality that uses electric current to activate nerves for therapeutic reasons. It is a non-pharmacological method of pain management for which it is widely used  .Earlier, TENS was majorly concentrated on pain, but few clinical trials have been conducted to identify the effect of electrical nerve stimulation on salivary flow  . As theThis descriptive type of observational study was conducted at the Department of Oral Medicine and Radiology at Rajasthan Dental College and Hospital during a period of 7 months (August 2019- February 2020). The ethical certificate was obtained from the college .The total sample size of the study was decided to be200 participants after a discussion with the statistician.Out of the total 200 individuals, 100 healthy individuals  within the age group of 20-40 years were included in Group I. Group II consisted of 100 individuals of age >40 years, out of which 50 were non-diabetics  and the remaining 50 were diabetic individuals . Patients who had any deleterious habits like alcohol or drug consumption and tobacco in any forms, usage of any medications, history of salivary gland pathology that may affect salivary flow, pregnancy, history of head and neck radiotherapy, granulomatous diseases, Sjögren’s syndrome and any systemic diseases except diabetes were excluded from the study as most of these lead to hyposalivation and we wanted our study to be exclusive to notice changes in salivary values due to diabetes alone. Patients were explained about the procedure and informed consents were taken. For the diabetic individuals the random blood sugar (RBS) levels were obtained firstby using a tabletop glucometer (Dr. Morepen GlucoOne blood glucose monitoring system BG 03) and only those individuals who were under medication for at least oneyear were included. The patients were seated comfortably on the dental chair and were asked to refrain from eating or drinking 1 hour prior to saliva collection.The procedure was done during the early morning hours between 9 am to 12 am. Unstimulated saliva was collected for 5 minutes in a graduated beaker by drainingmethod [ p Value of < 0.05 was set as statistical significance. Kolmogorov-Smirnov and Shapiro-Wilks normality tests were used to check whether the variables follow normal distribution. For variables that did not follow normal distribution, Mann Whitney U test was used to compare mean between the groups.The data collected were analyzed by SPSS software, version 20.0. The The mean age for Group I was 28.36±3.1, for Group II non-diabetic individuals was 53.56±7.8 and for Group II diabetic individuals was 52.16±7.4. 85 out of 100individuals experienced an increase in stimulated saliva, 9 had no change and 6 had a decrease in Group I (20-40years), In Group II (>40years) 83patients out of 100 patients had an increase in stimulated saliva, 9 had a decrease and 8 patients had no change in salivary flow. Individuals who did not showany change were the ones who showed no flow initially. Only those individuals who had an initial saliva flow already were the ones who showed a significant increase.For age group 20-40 years, the mean unstimulated salivary rate was 1.64ml/5min and the mean stimulated salivary rate was 1.914ml/5min. In age >40years, themean unstimulated salivary rate was 1.231ml/5min and the mean stimulated salivary rate was 1.547ml/5min. Mann Whitney U test was applied; the p value for both Group I(20-40 years) and Group II (>40 years) for unstimulated salivary flow was 0.01 and for stimulated salivary flow was 0.03, respectively. A significantdifference was seen between unstimulated and stimulated salivary flow while comparing different age groups .For Group I (20-40years), the mean unstimulated salivary rate was 1.64ml/5min and the mean stimulated salivary rate was 1.914ml/5min. For GroupII (>40years), the mean unstimulated salivary rate was 1.231ml/5min and the mean stimulated salivary rate was 1.547ml/ 5min. Mann Whitney U test wasapplied and the p value for unstimulated salivary production and for stimulated salivary production for both males and females was 0.198 and 0.083, respectively.There was no significant difference on salivary production considering gender when the effect of TENS on salivary production wasdone .p value forunstimulated salivary production for both diabetic and non-diabetic individuals was 0.002 and stimulated salivary production was 0.043; a significant difference wasseen (For the diabetic individuals, the mean unstimulated salivary rate was 1.07ml/5min and the mean stimulated salivary rate was 1.44ml/5min. One-sample t-test wasapplied and the p value for unstimulated and for stimulated salivary production was 0.00; a significant difference was seen . The meawasseen .Reduced salivary secretion has been linked to diabetes and not many studies have been done regarding the use of electro-stimulation as a therapeutic modality in treating hypo-function of the salivary gland.et al. [ et al. [ The present study showed that 168 participants of the study had an increase, which may be attributed to the mechanism by which TENS acts on the parotid glands. It causes stimulation of the auriculo-temporal nerve, which controls the secreto-motor drive of the parotid gland. On the application of the TENS device, parasympathetic supply gets stimulated and hence, produces watery and copious saliva. A total of 17 individuals had no change in saliva because these were those individuals who showed no flow initially. A total of 15 individuals had reduced saliva, which may be due to the fact that the brain may have perceived the electrical stimulus to be painful. In addition, for the diabetic individuals our study demonstrated that the mean unstimulated saliva was 1.08ml/5min, which increased to 1.44ml/5min after stimulation. Similar findings were noted in the study conducted by Dhillon et al.   where i et al.   includeet al. [ et al. [ Similar to our studies, where an increase in stimulated saliva was seen, studies carried out by Bhasin et al.   reporte et al.  , it waset al. [ et al. [  In the study by Manoj Kumar et al.  , 62 out et al.  , 65 outet al. [ Bhasin et al.   conductet al. [ Hargitai et al.   conductet al. [ Aggarwal et al.   conductet al. [ et al. [ In the present study, in the age group 20-40 years, the mean unstimulated saliva was 1.64ml/5min, which increased to 1.91ml/5min post-stimulation. This data was consistent with the study conducted by Bhasin et al.   and Sin et al.   where tet al. [ et al. [ In this study, the individuals of the age range of 20-40 years had an increase in the both the mean unstimulated and stimulated salivary values which were 1.64ml/ 5min and 1.91ml/5min, respectively when compared to the individuals of the age >40 years who had mean unstimulated and stimulated salivary values of 1.23ml/ 5min and 1.54ml/5min, respectively. Our study correlates to findings of the study conducted by Manoj Kumar et al.   where m et al.  , there et al. [ et al. [ et al. [ et al. [ The data in our study are also consistent with the studies conducted by Dyasnoor et al.  , Singla et al.  , Aggarw et al.  , and Si et al.   where net al. [ et al. [ et al. [ et al. [ et al. [ et al. [ et al. [ et al. [ et al. [ et al. [ et al. [ et al. [ Our study employed the draining method for saliva collection in which the saliva was collected passively in the floor mouth and was then allowed to drain into the graduated beaker without any forced self-stimulation. The studies conducted by Dhillon et al.  , Aparna et al.   and Har et al.   used th et al.  , Pattip et al.  , Aggarw et al.  , Singh  et al.  , Sakshi et al.  , Lingam et al.  , Manoj  et al.  , Mimans et al.   and Sin et al.   used thet al. [ In this study, the mean value of unstimulated saliva for the diabetics was 1.08ml/5min and for stimulated saliva was 1.44ml/5min. In the study conducted by Jagdhari et al.  , where et al. [ For the diabetic individuals, our study demonstrated that the mean unstimulated saliva was 1.08ml/5min, which increased to 1.44ml/5min. A significant change in saliva was seen post-stimulation. Dyasnoor et al.   conductThe limitation of our study was that the electrode pads were placed on the approximate location on the skin overlying the parotid gland and the exact anatomical measurements were not made. The diabetic staging of the individuals was not done and all individuals who were found to be diabetic were included in the study irrespective of the sugar levels. In addition, the patients could have been re-evaluated after 1 or 2 weeks after the application of the TENS device, to check for its effectiveness.It seems that TENS has shown positive results in increasing salivary secretions in different age groups and in the diabetic individuals. In the future, TENS might beused as an additional treatment modality to manage salivary gland dysfunctions.The authors declare that they have no conflict of interest.