PKHD1 gene, 8 variants in the TMEM67 gene, 4 variants in the PKD1 gene, 2 variants in the HNF1B gene and 2 variants in BBS1 and NPHP1 genes, respectively. NGS is a valuable tool in the diagnostics of various forms of cystic kidney diseases. Its results changed the clinically based diagnoses in 16% (n = 5) of the children.Cystic kidney diseases are a very heterogeneous group of chronic kidney diseases. The diagnosis is usually based on clinical and ultrasound characteristics and the final diagnosis is often difficult to be made. Next-generation sequencing (NGS) may help the clinicians to find the correct final diagnosis. The aim of our study was to test the diagnostic yield of NGS and its ability to improve the diagnosis precision in a heterogeneous group of children with cystic kidney diseases. Next-generation sequencing of genes responsible for the formation of cystic kidneys was performed in 31 unrelated patients with various clinically diagnosed cystic kidney diseases gathered at the Department of Pediatrics of Motol University Hospital in Prague between 2013 and 2018. The underlying pathogenic variants were detected in 71% of patients (n = 22), no or only one  pathogenic variant was found in 29% of patients (n = 9). The result of NGS correlated with the clinical diagnosis made before the NGS in 55% of patients (n = 17), in the remaining 14 children (45%) the result of NGS revealed another type of cystic kidney disease that was suspected clinically before or did not find causal mutation in suspected genes. The most common unexpected findings were variants in nephronophthisis (NPHP) genes in children with clinically suspected autosomal recessive polycystic kidney disease . Overall, 24 pathogenic or probably pathogenic variants were detected in the  Cystic kidney diseases (CKD) are a very heterogeneous group of chronic kidney diseases that in pediatric patients comprise mostly inherited kidney diseases, such as autosomal recessive polycystic kidney disease (ARPKD), nephronophthisis (NPHP), renal cysts and diabetes syndrome (RCAD) or autosomal dominant polycystic kidney disease (ADPKD) . In contIn the last decade, next-generation sequencing becomes the method of choice, allowing simultaneous mutational analysis of panel of genes that can be especially useful in genetic diagnosis of young patients with unclear or doubtful phenotype, in etiologically complex syndromes, or in differential diagnosis of phenotypically overlapping syndromes. In very rare cases a combination of mutations in two genes (digenic disease), can be found in children with severe neonatal clinical phenotype . TherefoThe aim of our study was to test the diagnostic yield of NGS and its ability to improve the diagnosis precision in a heterogeneous group of children with clinically diagnosed cystic kidney diseases.Altogether, there were 31 patients with clinical diagnosis of different cystic kidney diseases–ARPKD, ADPKD, NPHP, RCAD syndrome and Bardet-Biedl syndrome. The group of patients was gathered at the Department of Pediatrics of Motol University Hospital in Prague between 2013 and 2018. The characterization of the group  is summarized in Clinical diagnosis of ARPKD was based on the fulfillment of established clinical criteria of ARPKD including 1) typical kidney involvement on ultrasound , with or without 2) liver involvement  and 3) normal renal US of both parents consistent with autosomal-recessive inheritance [Clinical diagnosis of ADPKD was based on the presence of one or more kidney cysts in one/both kidneys with or without positive family history of ADPKD and was mostly based on expert opinion as no consensus diagnostic criteria were stated for children under the age of 15. Retrospective evaluation shows that diagnosis of ADPKD in our patients followed the international consensus statement for radiological criteria for children and young people published in 2019 ,5, whereThe clinical diagnosis of nephronophthisis was based on the clinical finding of 1) polyuria/ polydipsia or 2) renal concentrating defect or 3) biopsy findings of chronic interstitial nephritis or US findings of normal sized hyperechogenic kidneys, all together with compatible autosomal recessive inheritance . NephronHNF1B nephropathy was mainly based on the presence of cystic kidney dysplasia with/without MODY and/or hypomagnesemia and/or pancreatic malformation, together with presence of other distinctive features described in detail in [Clinical diagnosis of renal cysts and diabetes syndrome (RCAD)–etail in .Clinical diagnosis of Bardet-Biedl syndrome was in our group of patients (patient n.25) based on the presence of cystic kidney disease, obesity, polydactyly and cognitive impairment as described among other features in .th percentile [Hypertension was defined as blood pressure ≥95rcentile .All children with severe cystic kidney disease or systematic disease with presence of cysts treated in the Department of Pediatrics of Motol University Hospital between 2013 and 2018 were included in our group of patients. Cases of typical ADPKD phenotype with mild manifestation and positive family history of the disease, as well as patients whose parents/legal guardians did not give informed consent, were excluded from the study.Detailed clinical, laboratory and ultrasound information of patients are summarized in The study was approved by the Ethics Committee of General University Hospital in Prague and all patients/legal guardians were consulted by geneticist prior to molecular-genetic analysis and gave written informed consent for genetic testing.PKD1, library preparation from long-range PCR products was chosen for PKD1 library preparation  has been chosen. The full list of genes contained in the analyzed panel is in ribed by , PrimeSTThe bioinformatic analysis was provided by in-house bioinformatic pipeline. The analysis included detection of single-nucleotide polymorphisms (SNPs), small insertions and deletions and, in the case of panel sequencing, copy-number variants (CNV).PKD1 sequencing) and quality check of the reads and their trimming was provided by Trimmomatic 0.36 [https://github.com/lh3/bwa/tree/master/bwakit). The hs38DH genome provided by bwa.kit was used. Mapped reads were cleaned up and sorted using samtools fixmate and samtools sort, respectively . The PCR duplicates were then marked with picard MarkDuplicates 2.9.2-SNAPSHOT (Picard Toolkit by Broad Institute) and mapped reads indexed with samtools index (v1.6). For variant calling, freebayes v1.1.0-9-g09d4ecf was used [https://www.ncbi.nlm.nih.gov/snp/) and dbNSFP 3.2a (https://sites.google.com/site/jpopgen/dbNSFP).First, the quality of the FASTQ files created by MiSeq Reporter was evaluated using FastQC v0.11.5 (Babraham Institute). In the second step, cutting off the adapter (TruSeq3-PE-2) sequences (and PCR primers in case of tic 0.36 . The triwas used . The varwas used  and SnpSwas used  compilinThe variant prioritization was composed of automatic filtration (point 1 and 2) followed by evaluation by molecular geneticist in cooperation with nephrologist (points 3–5):The variant causes change on protein level (i.e. nonsynonymous sequence changes in exons) or the variant is in the intronic region in the donor  or acceptor  splite site.The variant has a population frequency (of European descend) lower than 0.1. 1000 Genome Project EUR population and Exome Aggregation Consortium (ExAC) was used for the assessment.in silico prediction by programs: SIFT [http://www.mutationtaster.org/), MutationAssessor [Evaluation of the variant based on ms: SIFT , PolyPhems: SIFT , LRT [18Assessor , FATHMM Assessor , PROVEANAssessor , CADD [2Assessor , ensemblAssessor , and conhttp://www.humgen.rwth-aachen.de/) and Autosomal Dominant Polycystic Kidney Disease Mutation Database: PKDB (https://pkdb.mayo.edu/).The consultation of variant with VarSome search engine  containiThe consultation with attending nephrologist regarding the possible pathogenic effect of detected variants with uncertain significance on patient phenotype.trans localization in the patient.If available, analysis of variant segregation in family members. In case of recessive inheritance, analysis of variation in proband’s parents to determine The copy-number variant calling was done using CNVkit 0.8.6.dev0  in all sPKHD1 gene done within our previous project. These patients were either clinically suspected to have ARPKD or had neonatal/infantile onset of the disease. The sequencing method, data analysis and variant classification used in this project were in detail described previously [PKHD1 gene) of patients 3–5 and 7 were already published and discussed in [PKHD1 gene alone  have not been analyzed by panel or PKD1 sequencing . Patient 24 was analyzed in commercial laboratory. The complete list of patients and their respective genetic analysis are summarized in Patients 1–24 had had mutational analysis of the eviously . The defussed in . The patPKHD1 and HNF1B followed the genetic analysis of the PKHD1 gene in patients in whom no/one mutation in PKHD1 was found. The kits used for MLPA analysis were SALSA MLPA P341 PKHD1 mix 1, P342-PKHD1 mix 2 and SALSA MLPA P241 MODY Mix 1 (MRC Holland). The generated data were analyzed by Coffalyser.Net (MRC Holland).MLPA (Multiplex Ligation-dependent Probe Amplification) analysis was used for detection of large genome rearrangements. MLPA of NPHP1 gene found by panel sequencing. The kit used for the analysis was SALSA MLPA P387 NPHP1.The MLPA analysis was also applied in patient 29 to confirm the complete deletion of both alleles of PKHD1 confirmed the diagnosis in 10 (50%) of them. In the other 10 patients, the sequencing of PKD1 and panel of genes yielded the diagnosis in additional 5 of them . Four children harbored only one pathogenic mutation in the recessive PKHD1 gene, and the final diagnosis remained unknown.The group of 31 patients formed by patients from Motol University Hospital gathered between years 2013 and 2018 was analyzed within our project. The underlying pathogenic variants were detected in 71% of patients (n = 22), no or only one  pathogenic variant was found in 29% of patients (n = 9) . The resPKHD1 and those with childhood onset, without a clinical suspicion of ARPKD, a panel sequencing and sequencing of the PKD1 gene yielded the diagnosis in 57% patients (12 of 21). The most common unexpected findings were changes in nephronophthisis (NPHP) genes in children with clinically suspected ARPKD (n = 4). The most common incorrect clinical diagnosis was ARPKD (n = 10) with four patients having genetically proven NPHP, one ADPKD de novo and further five patients having no definitive genetic diagnosis.In patients without a yield by sequencing of PKHD1 gene (14 patients), 8 variants in the TMEM67 gene (4 patients), 4 variants in the PKD1 gene (4 patients), 1 variant in HNF1B (1 patient) and 2 variants in BBS1 (1 patient). Moreover, whole gene deletion was detected in heterozygous (HNF1B gene) and homozygous (NPHP1 gene) state in two patients.Overall, 24 pathogenic or probably pathogenic SNP variants were detected in the The molecular genetic data of patients are summarized in HFN1B and NPHP1 was detected. In both genes, whole gene deletions are the most common variants described in literature [Sequencing analysis performed in 31 pediatric patients with suspected cystic kidney diseases detected causal variant in 71% (n = 22) of patients. This roughly correlates with results presented by Bullich et al.  where taterature .TMEM67 variant), which was already described in 2009 by Otto et al. [NGS confirmed the clinical diagnosis in 55% (n = 17) patients and did not confirm or changed the clinical diagnosis in 45% (n = 14) of them. The most common clinical diagnosis that had to be changed thanks to the NGS results was ARPKD. Four children  with a clinical diagnosis of ARPKD was diagnosed as having genetically NPHP11 and one patient (28) de novo ADPKD. The clinical and ultrasound picture of ARPKD and NPHP can be similar making the differential diagnosis difficult mainly in children with non-neonatal manifestation in ARPKD and in absence of extra-renal/hepatic manifestation such as eyes or central nervous system abnormalities present in NPHP. The less severe NPHP phenotype in our patients was caused by the fact that all children carried the allele p.Cys615Arg  or had another clinical diagnosis discovered during follow-up . In three children (15%) with clinically diagnosed ARPKD only one pathogenic variant in the n et al. . Even thn et al.  suggestsNo or partial genetic findings in some patients could be caused by several reasons:DZIP1L [CEP83 [Mutations in other ciliopathic genes which were not in our NGS ciliopathy panel  covered by genetic analysis, e.g. regulatory regions in deep intronic sequences, untranslated regions of genes, first exons of genes or even promotor regions, that are especially important for genes coding transcription factors whose function strongly relates to expression level. Also, the causing variant could have been filtered out as variant of unknown significance (VUS) as its function is yet unknown or could have been missed by bioinformatic processing.Moreover, a combination of heterozygous mutations in two different ciliopathic recessive (or dominant) genes could theoretically cause a severe cystic kidney disease phenotype as an oligogenic inheritance where variants in different genes modify final phenotype  was already described in ciliopathies (reviewed in ), as welAnother possibility for the discrepancy between clinical and genetic diagnosis is that the cystic kidney disease in a given child is non-inherited  which is however very rare in pediatric patients especially if manifested in neonatal/infant age.Extra-renal manifestations, such as eye or CNS abnormalities were the best clues for the correct clinical diagnosis of patients with non-ADPKD cystic kidney diseases . NystagmThe strengths of our study are a very well clinical and ultrasound characterization of the patients, the long-term follow-up and the high number of ciliopathy genes in our panel. The genetic diagnosis consisted of several methods of sequencing and CNV detection and thus provided reliable results. We are aware of the limitations of our study—the low number of patients, single-center study, lack of some non-ciliopathy genes in our panel and lack of whole-exome sequencing in the genetically unresolved patients.In conclusions, our study demonstrated that the next-generation sequencing method facilitates molecular analysis of different types of cystic kidney diseases and enables precise genetic diagnosis. Its results changed the clinical diagnosis in number of the children. The correct final genetic diagnoses enable better management of children with different forms of cystic kidney diseases and provide efficient genetic counseling.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table+, yes; -, no.(PDF)Click here for additional data file.


We conducted a genome-wide association study of 1317 centenarians from the New England Centenarian Study and 2885 controls using >9M genetic variants. The most significantly associated variants were correlated to 4131 serum proteins in 224 study participants. The genetic and protein associations were replicated in a genome-wide association study of 480 centenarians and ~800 controls of Ashkenazy Jewish descent and a proteomic scan of approximately 1000 participants of the same study. The analysis replicated a protein signature associated with APOE genotypes and confirmed strong overexpression of BIRC2 (p < 5E-16) and underexpression of APOB in carriers of the APOE2 allele (p< 0.05). The analysis also discovered and replicated associations between longevity variants and slower changes of protein biomarkers of aging, including a novel protein signature of rs2184061 (CDKN2a/CDKN2B in chromosome 9). The analyses show that longevity variants correlate with proteome signatures that could be manipulated to discover healthy aging targets.


Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory tract illness in infants and affects the elderly and the immune-compromised. RSV infection can cause pulmonary obstruction and disease, but the mechanisms contributing to these outcomes remain elusive. Despite the major health burden caused by infection, there is currently no licensed RSV vaccine and treatments are limited. The Special Issue on “Pathobiology of Respiratory Syncytial Virus (RSV)” covers the attributes of disease pathogenesis, immunity, and vaccine development. In an article by Pedro Piedra, “Antibody Response to the Furin Cleavable Twenty-Seven Amino Acid Peptide p27) of the Fusion Protein in Respiratory Syncytial Virus (RSV) Infected Adult Hematopoietic Cell Transplant (HCT) Recipients” , the ant7 of the


The purpose of this study was to explore the relationship between nursing home volunteer programming and quality measures and increase the knowledge base of nursing home volunteer programming in various settings. Fifty-two nursing homes were surveyed using electronic surveys and personal interviews. Questions focused on the organization of the nursing home, characteristics of the volunteer program and volunteer activities. Of the 52 facilities surveyed, 19 were not part of a chain, 37 were in urban settings, and 24 were for-profit entities. Volunteers were used in 46 nursing homes with a mean number of volunteers of 51.7 onsite an average of 4.9 days per week. Bivariate analysis revealed statistically significant correlations (p<.05) between organizational and volunteer programming characteristics and six quality measures . Statistically significant (p<.05) inverse relationships were found between volunteers providing individualized activities  and the incidence of urinary tract infections and the use of psychotropic drugs. Multiple regression analysis revealed a statistically significant (p<.05) inverse relationship between personal volunteer services such as combing hair and doing nails and the use of hypnotic drugs and antipsychotic drugs. This study indicates a significant correlation between volunteer programming and quality measure scores. A larger study of these relationships is indicated.


Semen samples were then cryopreserved in TEST-yolk buffer and stored in liquid nitrogen at -196°C.The identification of the differentially expressed proteins (DEPs) between the control and NSTC groups was con- ducted via Scaffold software. The criteria for the selection of DEPs for validation by Western blot (WB) included: 1) proteins involved in reproductive system development and function; 2) proteins involved in the top canonical pathways; 3) proteins with a higher difference of abundance between the experimental groups; 4) proteins with a well-described function in the literatureThe authos identified 189 differentially expressed proteins (DEPs) in the dataset, from which five DEPs related to sperm function and fertilization were selected for validation by Western blot. And they indentified that underexpression of the mitochondrial complex subunits NADH:Ubiquinone Oxidoreductase Core Subunit S1 (NDUFS1) and ubiquinol-cytochrome C reductase core protein 2 (UQCRC2), as well as the underexpression of the testis-specific sodium/potassium-transporting ATPase subunit alpha-4 (ATP1A4) in the NSTC group.The results indicate that sperm mitochondrial dysfunction may explain the observed decrease in sperm concentration, total sperm count and total motile count in NSTC patients. The identified DEPs may serve as potential biomarkers for the pathophysiology of subfertility/infertility in patients with NSTC.


Conventionally, (p)ppGpp collectively refers to two nucleotides, ppGpp, and pppGpp in the literature. Initially, (p)ppGpp has been discovered as a transcription regulatory molecule as it binds to RNA polymerase and regulates transcriptional gene regulation. During the past decade, several other target proteins of (p)ppGpp have been discovered and as of now, more than 30 proteins have been reported to be regulated by the binding of these two signaling nucleotides. The regulation of diverse biochemical activities by (p)ppGpp requires fine-tuned molecular interactions with various classes of proteins so that it can moderate varied functions. Here we report a structural dynamics of (p)ppGpp in the unbound state using well-defined computational tools and its interactions with target proteins to understand the differential regulation by (p)ppGpp at the molecular level. We carried out replica exchange molecular dynamics simulation studies to enhance sampling of conformations during (p)ppGpp simulation. The detailed comparative analysis of torsion angle conformation of ribose sugar of unbound (p)ppGpp and bound states of (p)ppGpp was carried out. The structural dynamics shows that two linear phosphate chains provide plasticity to (p)ppGpp nucleotides for the binding to diverse proteins. Moreover, the intermolecular interactions between (p)ppGpp and target proteins were characterized through various physicochemical parameters including, hydrogen bonds, van der Waal’s interactions, aromatic stacking, and side chains of interacting residues of proteins. Surprisingly, we observed that interactions of (p)ppGpp to target protein have a consensus binding pattern for a particular functional class of enzymes. For example, the binding of (p)ppGpp to RNA polymerase is significantly different from the binding of (p)ppGpp to the proteins involved in the ribosome biogenesis pathway. Whereas, (p)ppGpp binding to enzymes involved in nucleotide metabolism facilitates the functional regulation through oligomerization. Analysis of these datasets revealed that guanine base-specific contacts are key determinants to discriminate functional class of protein. Altogether, our studies provide significant information to understand the differential interaction pattern of (p)ppGpp to its target and this information may be useful to design antibacterial compounds based on (p)ppGpp analogs. Escherichia coli culture. The concentration of these nucleotides was enhanced drastically in E coli cells during amino acid starvation and these nucleotides were inhibiting rRNA synthesis ppGpp is used for two nucleotides, guanosine 5′-diphosphate-3′-diphosphate (ppGpp) and guanosine 5′-triphosphate-3′-diphosphate (pppGpp). These nucleotides are synthesized by (p)ppGpp synthetase by transferring pyrophosphate groups from ATP to GDP/GTP to form ppGpp and pppGpp, respectively . There aThe molecular dynamics simulation studies provide structural dynamic information in the solution state of a molecule. Although, glycosidic bond and ribose sugar in the nucleotide structure exhibit conformational flexibility and classical molecular dynamics simulation on nucleotides is challenging because of energy barriers and sampling of conformations are limited. Therefore, Replica Exchange Molecular Dynamics (REMD) simulation was carried out on ppGpp and pppGpp nucleotides in solution states. In REMD, several identical replicas run in parallel at different temperatures and these allow enhanced sampling of high energy conformations. Moreover, these replicas are allowed to swap their states based on Boltzmann-weighted probability at neighboring temperature state. This process is repeated iteratively during the simulation and subsequently enhanced sampling of conformations is achieved at various temperatures.3 for both molecules and upon addition of buffer, the box volume was expanded to 145262 and 150766 Å3 for ppGpp and pppGpp, respectively. Negative charges, due to phosphates groups of (p)ppGpp molecule, were neutralized by the addition of 20 mM Mg2+ in the simulation system. The Optimized Potential for Liquid Simulations 3 enhanced (OPLS3e) force field was selected for the simulation ppGpp-protein complexes. The structural alignment was carried out in PyMol using an atom alignment algorithm. The distribution frequency of glycosidic bonds in unbound states of ppGpp and pppGpp were plotted at 300 K simulation pose in Schrodinger. The torsion angle and phase angle values were calculated from Pseudo-Rotational Online Service and Interactive Tool (PROSIT) , 2005.The structures of (p)ppGpp-protein complexes were downloaded from Protein Data Bank (PDB). The PDB ligand code G4P for ppGpp, 0o2, and C1Z for pppGpp was used as a search term to obtain structures of ppGpp-protein and pppGpp-protein complexes, respectively. As shown in Molecular dynamics simulation is one of the most commonly used approaches to understand structural dynamics of biomolecules in solution state . It has 2+ ions during the simulation, yet higher flexibility of phosphate atoms was observed in the RMSF plot. In contrast to (p)ppGpp, the phosphate groups in cyclic messenger nucleotides show lesser flexibility due to the unavailability of free phosphate chains (2 group of guanine ring of (p)ppGpp in REMD simulation. The detailed analysis of molecular properties shows the overall quality and conformational dynamics of (p)ppGpp during the simulation ppGpp were obtained by energy minimization in Maestro. The structural coordinates of bound (p)ppGpp were extracted from structures of (p)ppGpp-protein complexes. The structural alignment of unbound state and bound state of (p)ppGpp shows substantial similarity in the guanine ring region with RMSD of atoms less than one. However, the phosphate chain shows divergence upon structural alignment which is in agreement with our simulation RMSF plot.syn conformation, however, some occurrences were observed in anti conformation. Our results on the frequency distribution of glycosidic conformation in unbound states are similar to those observed in the bound states in the crystal structure. As most of the structures of (p)ppGpp-protein complexes have anti conformation of glycosidic bond except in few cases it was found in syn conformation such as nucleosidase (6GFM)  and maximum puckering amplitude (νmax) which show that ribose sugar adopts north conformation in both states ((p)ppGpp nucleotides exhibit a substantial extent of conformational flexibility because of various rotatable torsion angles present in the structure particularly a ribose ring . Therefoe (6GFM) , lysine e (6GFM) . The sugormation . Whereasd pppGpp . The fivse sugar . The ribse sugar . Additioh states . The com2+ ions. As seen by the hydrogen bonds histogram, the major interatomic polar interactions were observed in guanine ring and phosphate group atoms. The aromatic ring of guanine hadπ-π base stacking interactions with the side chain of tyrosine in the structures of (p)ppGpp synthetases and nucleotide metabolic enzymes. The ribose moiety of (p)ppGpp makes comparatively fewer interactions with protein atoms which may be the consequence of the structural restrain of the ribose ring. Similarly, van der Waal’s interactions were found majorly in the region of the guanine ring of (p)ppGpp ppGpp . The avaBacillus subtilis ppGpp synthetases are apparent (p)ppGpp binding proteins as these enzymes synthesize ppGpp and pppGpp from ATP and GDP/GTP, respectively. However, most of the structures determined so far are in complexes with substrate analogs as these complexes explain the catalytic mechanism. A brief structural review on (p)ppGpp synthetase proteins has been described previously  hence cusubtilis ; small as aureus  and one mophiles . The (p)2+ ions. Therefore, these interactions are considered as ionic interactions. The observed binding mode of (p)ppGpp to the nucleotide enzymes is similar due to an analogy of substrate nucleotide structure to the (p)ppGpp. As shown in the graph, the N1, N2, N7, and O6 atoms of the aromatic guanine ring of (p)ppGpp is a major contributor to hydrogen bond interaction with protein residues.There are six crystal structures of (p)ppGpp and nucleotide metabolic enzymes complexes available in the Protein Data Bank which includes Xanthine phosphoribosyltransferase (XPRT) , PyrimidThere are five crystal structures of GTP binding GTPase proteins in complex with (p)ppGpp available till now. These include Obg GTP binding protein , GTPase RNA polymerase was the first protein complex reported to regulate its activity by binding of (p)ppGpp . There aThere are several crystal structures of various other functional class of (p)ppGpp binding proteins reported including translation peptide chain release factor 3 (PDB: 3VR1) , acetylt2+) plays a significant role in the structural stability of nucleic acid and nucleotides by neutralizing highly negative charged phosphate groups (2+ ions assist in the specific interaction of (p)ppGpp to their target protein. As shown in the graph ppGpp synthetase, RelTt, it binds to only one chain of phosphate ppGpp but it also mediates interactions between (p)ppGpp and protein side chains. Each phosphate chain of (p)ppGpp is making interactions with one Mg2+ ion in the complex of RNA polymerase-(p)ppGpp. Altogether, we have observed Mg2+ ions in several other complexes of (p)ppGpp-protein structures except for few complexes which may be the limitation of electron density interpretation as discussed earlier ppGpp have linear phosphate chains that provide additional flexibility to adapt various conformations according to the stereochemistry of the binding site of a respective target protein. Overall, our results support the hypothesis that the conformation flexibility of glycosidic bond, ribose sugar puckering, and phosphate groups provide structural plasticity to the (p)ppGpp for binding to the various functional class of proteins. The structures of unbound ppGpp and pppGpp in solution states obtained by MD simulation are similar to that observed in the structures in bound form, hence, these structures resemble biologically active conformations of (p)ppGpp. The gyration conformation profile of glycosidic bond is in agreement with the conformation of (p)ppGpp observed in the bound state. The analysis of (p)ppGpp-protein interactions reveals that the binding pattern of these nucleotides governs the regulation for a particular class of target proteins.The original contributions presented in the study are included in the article/GK and NB conceptualized the study. GK and AP performed the study. All authors analyzed the data, wrote and edited the manuscript, and agreed on the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sarothrura ayresi was sequenced using next generation sequencing technology. The size of the genome is reported as 16,767 bp and comprises 13 protein-coding genes, 2 rRNAs and 22 tRNAs. The organization of the genome is comparable to that of other bird species. A phylogenetic comparison mapped the relative relationship of Sarothrura ayresi with respect to other species in the order Gruiformes.The mitochondrial genome of  The Critically Endangered white-winged flufftail  forms part of the genus Sarothrura IUCN . The speTM – Tissue MiniPrep Kit (Zymo Research Corporation), library preparation and Illumina high-throughput sequencing . Sequences were assembled using the CLC Genomics platform . The mitogenome was assembled under the de novo option, using the standard parameters. Various kmer sizes were used during optimisation and the full mitochondrial genome for the WWFT was assembled using kmer size 31. The mitochondrial assembly constituted 8597 reads out of the 18,567,330 that remained after quality trimming and removal of contaminating adapters, using the Trimmomatic software  in January 2014 was selected for mitochondrial analysis. This was performed using standard extraction using the ZR Genomic DNAGallus gallus as reference  was performed using MITOS  and Heliornithidae (Heliornis fulica) than with members of the Gruidae (Anthropoides paradiseus) and Otidae (Otis tarda).The WWFT mitogenome constitutes the first whole mitochondrial genome entry within the Sarothruridae family. In order to evaluate its relative position within the order Gruiformes a phylogenetic tree (maximum-likelihood) was constructed to place the WWFT among species representing the Rallidae, Gruidae, Heliornithidae and Otididae Families using MEGA version 6 (Tamura et al.


Recent studies have reported ERp57 overexpression in various human cancers, and altered expression and aberrant functionality of ERp57 are associated with cancer growth and progression and changes in the chemosensitivity of cancers. ERp57 may become a potential biomarker and therapeutic target to combat cancer development and chemoresistance. Here, we summarize the available knowledge of the role of ERp57 in cancer and the underlying mechanisms.Endoplasmic reticulum resident protein 57 (ERp57) has a molecular weight of 57 kDa, belongs to the protein disulfide-isomerase (PDI) family, and is primarily located in the endoplasmic reticulum (ER). ERp57 functions in the quality control of nascent synthesized glycoproteins, participates in major histocompatibility complex (MHC) class I molecule assembly, regulates immune responses, maintains immunogenic cell death (ICD), regulates the unfolded protein response (UPR), functions as a 1,25-dihydroxy vitamin D With cancer incidence and mortality rates increasing rapidly, cancer has become a global problem and is expected to become the leading cause of death worldwide in the 21st century according to the Global Cancer Statistics 2018 report The PDI family includes more than 20 members that catalyze cysteine-based redox reactions and play critical roles in productive protein folding in vitro analyses and clinical trials support that ERp57 is overexpressed in a variety of cancers. ERp57 expression is significantly different between tumor tissues and normal tissues (P < 0.05) in a wide variety of cancers, including stomach adenocarcinoma, colon adenocarcinoma, liver hepatocellular carcinoma (HCC), breast invasive carcinoma, and prostate adenocarcinoma, as shown in Figure http://ualcan.path.uab.edu/analysis.html) Recent research from In vitro, knockdown of ERp57 in HeLa cells inhibits cancer invasiveness and metastasis Some studies have demonstrated that ERp57 is a primary cellular target that inhibits cancer cell proliferation + T cells is enhanced when ERp57 is knocked out in human CD8+ T cells because ERp57 knockout in CD8+ T cells regulates the expression of multiple immune regulators and effectors on the cell surface + T cells need to be explored in detail to expand the utility of ERp57 in cancer immunotherapy strategies.Calnexin (CNX) and calreticulin (CRT) predominantly act as lectins that specifically interact with nascently synthesized glycoproteins in the ER, and ERp57 forms complexes with these proteins to accomplish efficient folding and proper formation of intramolecular disulfide bonds in glycoproteins in vivo because CRT translocation is inhibited and immunogenic cell death is abolished Immunogenic cell death (ICD), a type of regulated cell death (RCD), induces adaptive immune responses to kill cells directly Due to its disulfide isomerase activity, ERp57 forms a complex with CNX and CRT to specifically modulate the folding of newly synthesized glycoproteins by facilitating the formation of native disulfide bonds Recent data suggest that ERp57 plays an important role in the DNA repair process. ERp57, accompanied by high mobility group proteins 1 and 2 (HMGB1 and HMGB2), acts as a component of a complex that detects damaged DNA, inactivates the mismatch repair system and decreases the sensitivity of cells to thiopurine treatment Taken together, the evidence indicates that ERp57 has a protective effect on the survival of tumor cells due to its contribution to the DNA repair process, which regulates the sensitivity of the tumor to DNA-modifying chemotherapy. Thus, it may become a biomarker to predict therapeutic failure and success. However, further studies are required to confirm these findings and identify potential antitumor targets.2D3 is the active form of vitamin D, and its analogs exert inhibitory effects on the proliferation and progression of multiple cancers, including melanoma 3-membrane-associated, rapid response to steroid binding  receptor, mainly located on the plasma membrane and Golgi/ER 2D3 to form a ligand-receptor complex to mediate several rapid signal transduction cascades; for example, it regulates STAT3 2+ release from lysosomes to activate autolysosomal degradation In vitro studies have revealed that knockdown of ERp57 inhibits HCC cell proliferation 1,25(OH)Mitomycin C (MMC) exerts anticancer effects by inducing DNA cross-linking in cancer cells Affymetrix microarray results suggest that cytoskeletal remodeling and the Wnt signaling pathway are the two of the top pathways disrupted by ERp57 silencing in HeLa cells 3-MARRS receptor ERp57 plays an important role in the natural history of cancer and the therapeutic process. As an ER chaperone, ERp57 is associated with CRT and CNX, participates in MHC class I molecule assembly


Cervical cancer is a common gynecologic malignancy worldwide. It is the fourth for both incidence and mortality. For cervical cancer, imaging and pathology assessments are incorporated in the revised 2018 Federation of Gynecology and Obstetrics (FIGO) staging system. Uses of imaging techniques for the pre-treatment work-up of cervical cancer have been increasing. Among imaging techniques for the evaluation of cervical cancer, ultrasound is cheaper, faster and widely available than other imaging techniques such as computed tomography (CT) or magnetic resonance imaging (MRI). Advanced technique in ultrasound, such as three-dimension (3D) ultrasound and color Doppler, have improved the clinical application of ultrasound in cervical cancer. Ultrasound may provide highly accurate information on detecting tumor presence and evaluating local tumor extent if performed by ultrasound-trained gynecologists; the experience of readers is also critical for correct pretreatment staging and assessment of response to treatment. Sonographic images could be useful to predict response of neoadjuvant chemotherapy, radiotherapy, chemotherapy and concurrent chemoradiotherapy in patients with cervical cancer. This review article attempted to present the most updated specific applications of ultrasound in cervical cancer. Cervical cancer is a common gynecologic malignancy among women. It is the fourth for both incidence and mortality in Global cancer statistics 2018 Patient age, performance status, para-aortic lymph node status, tumor size, and pelvic lymph node status are significantly associated with progression-free interval Cervical cancer can spread by lymphatic or hematogenous dissemination or direct extension. Staging cervical cancer is critical for approaching the most effective treatment strategy and predicting the outcomes. Stage IA cancer can be treated with cervical conization procedure or simple hysterectomy, while later stages may require neoadjuvant chemotherapy, radical surgery, radiation, chemotherapy or a combination of therapies. It is valuable to evaluate the response to treatment in cervical cancer women.For cervical cancer, imaging and pathology assessments are incorporated in the revised 2018 Fédération internationale de gynécologie et d'obstétrique (FIGO) staging system Transvaginal and transrectal ultrasound (TVUS and TRUS) are typically performed by the gynecologist with the advantage of low cost and wide availability A study enrolled 52 patients, 13 were advanced-stage disease (stage ≥IIb) and 39 were early-stage disease (stage ≤IIa), to evaluate the combined use of transvaginal elastography (TVES) and high-resolution TVUS in comparison with the diagnostic performance of magnetic resonance imaging (MRI) in detecting parametrial invasion in cervical cancer. It could be promising and economic for pre-operative work-up of cervical cancer by a dedicated gynecologic radiologist using TVES combined with TVUS In the cases with negative paraaortic lymph nodes on computed tomography (CT) or positron emission tomography (PET-CT), it could be necessary to perform dissection of the paraaortic lymph node in surgical staging. Obtaining an ultrasound or CT-guided tru-cut biopsy in equivocal extrauterine lesion is recommended to reduce false positive findings by imaging methods Hence, uses of imaging techniques for the pre-treatment work-up of cervical cancer have been increasing Among imaging techniques for the evaluation of cervical cancer, ultrasound is faster, radiation-free, cheaper, noninvasive, no contrast medium required and more widely available than other imaging techniques The characteristics of the cervix blood flow in normal women and in women with precancerous lesions or invasive cancers were reported by using three-dimensional power Doppler ultrasound (3D PDU) indices, including the intensity of flow at the time of volume acquisition (FI), the number of vessels within the volume of interest (VI), and both blood flow and vascularization (VFI) TVCD combined with colposcopy was used to diagnose precancerous lesions and early stage cervical cancer A thorough evaluation to determine the extent of cervical cancer should be performed if the diagnosis has been identified. There were several studies involving detection of the extension of cervical cancer lesion Table .The role of ultrasound for staging cervical cancer was reported in early 90s The accuracies of staging with TRUS and clinical staging were 83% and 78%, respectively. The sensitivity, specificity and accuracy of TRUS for extent of parametrial involvement were 78%, 89% and 87%. TRUS could be helpful applied to routine pretreatment assessment of patients with cervical carcinoma In the assessment of parametrial infiltration in cervical cancer, a study included 29 patients using MRI and 2D and 3D ultrasound examination before treatment. The result showed that 2D and 3D ultrasound had similar moderate agreement with MRI. However, 2D and 3D ultrasound examinations are more available at low cost than MRI. Ultrasound could be considered as a method for preoperative work-up of cervical cancer The agreement of clinical examination, 3D and 2D sonography with MRI for local staging of cervical carcinoma was evaluated For evaluating the tumor's volume and intra-tumoral vascularization, the study enrolled 27 patients, with cervical cancer and stage 1B1 disease, who have an initial 2D ultrasound examination and followed by 3D volumes of the cervix. The Virtual Organ Computer Aided Analysis (VOCAL) program was used to calculate the tumor volume and vascularization A recent study reported the local staging of cervical cancer for 46 patients who underwent MRI of the pelvis and high‐resolution TVUS A previous prospect study, included 38 cervical cancer patients, involved the TRUS in the evaluation of cervical carcinoma and comparing with spiral computed tomography (SCT) and MRI For locally advanced cervical cancer, 3-dimensional transvaginal ultrasound (3D-TVUS) was used to diagnose the extent of invasive cervical cancer and compare to that of Magnetic resonance imaging For cervical primary tumor confined to the origin, the sonographic detection rate is high; transrectal or TVUS is a highly accurate modality in classifying early-stage tumors The diagnostic performance of TVUS and MRI for assessment of presence and extent of cervical cancer was investigated by a prospective study The diagnostic performance of TVUS examination with regard to local extent and tumor size of the disease of early stage cervical cancer was investigated Assessment of the extension of the lesion has been researched Furthermore, angiogenesis, the formation of new vessels in a specific area, is required for tumor growth and progression Transvaginal Color Doppler in cervical cancer allows non-invasive assessment of tumor angiogenesis Color Doppler findings associated with risk factors was reported by a study involving neoangiogenesis measured in early cervical cancer cases Angiogenic parameters, including subjective assessment of pulsatility index (PI) and the amount of vessels within the tumor (scanty-moderate or abundant), were evaluated by transvaginal color Doppler ultrasound for 27 early stage cervical cancer patients. The risk factors  were recorded. Tumors with abundant vasculization were significantly associated with parametrial involvement, lymph-vascular space involvement, pelvic lymph node metastases etc. Postoperative treatment was significantly more prevalent in patients with profuse vascularization The p53 protein expression in cervical cancer after RT correlated with transvaginal color Doppler ultrasound findings was reported by a recent study Several studies have evaluated the role of ultrasound for predicting the response to varied treatment in women with cervical cancer.The role of TVUS in early prediction of pathological response in a large series of locally advanced cervical cancer patients arranged neoadjuvant treatment followed by radical surgery was investigated by using 3D PDU Using Power Doppler vascularity index (PDVI) to predict the response to neoadjuvant chemotherapy (NACT) in bulky early stage cervical carcinoma was reported A prospective study involved 42 women with locally advanced cervical cancer referred for NACT Transvaginal Color Doppler sonography used to predict the response to chemotherapy in patients with advanced cervical cancer was reported The residual tumor in locally advanced cervical cancer patients receiving chemoradiation and radical surgery was assessed by 3D power Doppler, 2D ultrasound parameters, or contrast-enhanced indices Ttransvaginal color Doppler ultrasonography (TCD) used to predict response to preoperative chemoradiation for locally advanced cervical carcinoma patients was investigated Alterations in cervical intra-tumoral vascularization during and after radiotherapy investigated by 3D PDU was reported The evaluation of radiotherapy response of cervical cancer by suing gray scale and color Doppler ultrasonography was report It was reported that Transvaginal color Doppler sonography could be useful in predicting clinical response to concurrent chemoradiation for locally advanced cervical carcinoma The investigation of early response of locally advanced cervical cancer patients undergoing concurrent chemo-radiotherapy (CCRT) by ultrasound was reported The role of intraoperative ultrasound guidance in intracavitary brachytherapy of cervical cancer was investigated in a recent retrospectively study In a recent systematic review and meta-analysis, a decrease of uterine perforations by using ultrasound image-guided applicator insertion for cervical carcinoma patients underwent intracavitary brachytherapy was revealed TRUS guided high dose rate (HDR) interstitial brachytherapy for patients of cervical cancer was investigated For the ability of evaluating morphology and intensity of blood flow, ultrasound technique might be used to improve cervical cancer screening, and this need more studies to confirm the applications. Imaging during the primary diagnostic work-up is essential for accurately assessment of tumor extent and selecting the best therapeutic option for cervical cancer patients. Ultrasound may be a useful modality to evaluate local extent of disease in cervical cancer. This technique is limited for evaluating lymph node status. Tumor vascularization by Doppler ultrasound could be useful for monitoring and predict response to therapy.


The Medication-Related Osteonecrosis of Jaws (MRONJ) diagnosis process and its prevention play a role of great and rising importance, not only on the Quality of Life (QoL) of patients, but also on the decision-making process by the majority of dentists and oral surgeons involved in MRONJ prevention (primary and secondary). The present paper reports the update of the conclusions from the Consensus Conference—held at the Symposium of the Italian Society of Oral Pathology and Medicine (SIPMO) —after the newest recommendations (2020) on MRONJ were published by two scientific societies , written on the inputs of the experts of the Italian Allied Committee on ONJ (IAC-ONJ). The conference focused on the topic of MRONJ, and in particular on the common practices at risk of inappropriateness in MRONJ diagnosis and therapy, as well as on MRONJ prevention and the dental management of patients at risk of MRONJ. It is a matter of cancer and osteometabolic patients that are at risk since being exposed to several drugs with antiresorptive  or, more recently, antiangiogenic activities. At the same time, the Conference traced for dentists and oral surgeons some easy applicable indications and procedures to reduce MRONJ onset risk and to diagnose it early. Continuous updating on these issues, so important for the patient community, is recommended. Medication-Related Osteonecrosis of Jaws (MRONJ) and its prevention play a role of great and rising importance, not only on the Quality of Life (QoL) of patients, but also on the decision-making process of the majority of dentists and oral surgeons, involved every day in MRONJ prevention (primary and secondary). The present report illustrates the update of conclusions adopted from the Consensus Conference held at the Symposium of the Italian Society of Oral Pathology and Medicine (SIPMO) on 20 October 2018, in Ancona (IT), when opinions were discussed and synthesized by 24 Italian experts. The Conference was the first national consensus to address the MRONJ disease and specific interventions , it sharThe present report was considered by the experts as convenient and worthwhile in the light of the new findings ,12,13 anThe updated Consensus Conference focused on the common practices still at risk of inappropriateness in MRONJ diagnosis and prevention, and on the dental management of patients exposed to ONJ. It is matter for the cancer  and osteometabolic patients, since they are exposed to an increasing number of drugs with antiresorptive  or antiangiogenic activities  ,16,17,18The experts focused on the topic of MRONJ using the following 6 main subcategories (issues): (1) clinical diagnosis; (2) radiologic diagnosis; (3) primary prevention (before and during drug intake); (4) dental management; (5) drug holiday (temporary suspension vs. therapeutic suspension); and (6) therapy.The IAC-ONJ planned that a recommended practice equivalent will be advised for any inappropriate practice, within these 6 issues.Assume that the Italian Consensus adopted the previously described definition of MRONJ ,5, as be(#1) Restricting the anamnestic interview only to the assumption of bisphosphonates for diagnosing MRONJ.(#2) Carrying out routine bone biopsies when suspecting an MRONJ.(#3) Considering the presence of exposed necrotic bone, in the oral cavity, as an essential (conditio sine qua non) sign to diagnose MRONJ.(#4) Judging the presence of pain as an essential symptom for diagnosing MRONJ.(#5) Believing that all MRONJ are preceded by dental invasive procedures.(#1) Evaluate not only the intake of bisphosphonate drugs (current or past), but also further pharmacological therapies  and perform a thorough physical examination and medical history, together with targeted radiologic examinations see  (confirm(#2) Perform jaw bone biopsies only if there is a suspicion of metastases in cancer patients (confirmed).(#3) Take into the account not only the presence of exposed necrotic bone but considering also other clinical signs and first/second-level imaging (confirmed).(#4) Consider that the symptom “pain” may not always be present in MRONJ cases, especially in the early stages (confirmed).(#5) Consider that some cases of MRONJ can arise from the presence of dental-periodontal diseases or spontaneously, without any relation to invasive dental procedures (confirmed).Radiologic imaging is assumed to be a fundamental tool for diagnostic completion, defining the extent of the disease at the skeletal level, staging and the correct therapeutic planning. Even if it is well known that no specific radiological signs of disease can be defined until now, an increasing volume of evidence recognizes some radiological characteristics that, although non-specific, are associated with MRONJ ,23,24,25(#1) Starting therapies at risk of ONJ without a radiologic assessment of dental arches, remarkably in cancer patients.(#2) Omitting the radiologic assessment for those patients at risk of MRONJ in the presence of dental/endo-periodontal diseases.(#3) Prescribing to everybody exposed to therapies at risk of ONJ all imaging techniques to rule out MRONJ.(#4) Requiring radiologic exams without a specific diagnostic hypothesis.(#5) Prescribing radiological exams for MRONJ only after onset of jawbone exposure.(#6) Diagnosing MRONJ only on the basis of the clinical signs, without radiologic assessment.(#7) Prescribing high-resolution imaging for soft tissue (with contrast medium) for diagnosis of MRONJ.(#8) Planning MRONJ treatment without second-level imaging exams for assessing jawbone disease extension and severity.(#9) Monitoring the extension of the MRONJ process only by investigations of first-level exams .(#1) Evaluate clinically and radiologically any local risk factors for MRONJ  for preventive dental screening in patients, remarkably cancer ones, who are candidates for therapies at higher risk of MRONJ; sometimes, MRONJ could manifest like a dental/endo-periodontal disease, or a worsening of a pre-existent disease (updated) .(#2) Perform radiological exams in all cases of dental/endo-periodontal diseases in patients at risk of MRONJ (confirmed).(#3) Reserve the indication for the execution of radiological exams of the second level  to patients at MRONJ risk only in the presence of ascertained clinical or radiologic signs compatible with MRONJ (confirmed) ,9,22.(#4) Specify always the diagnostic hypothesis when prescribing radiological exams of any level  in patients at MRONJ risk (confirmed).(#5) Do not delay in prescribing radiologic exams to investigate any clinical signs of possible MRONJ, independent of the presence of bone exposure or fistulas (updated).(#6) Integrate always the clinical check-up with the appropriate imaging exams for the diagnosis of MRONJ (confirmed).(#7) Prescribe radiologic exams for bone diseases without contrast medium for diagnosis of MRONJ (confirmed).(#8) Set up the MRONJ treatment after defining its extension and severity also by targeted imaging (confirmed).(#9) Use targeted imaging  to monitor MRONJ during follow-up after conservative and/or surgical treatment (updated).Assuming that recently it has been confirmed that MRONJ prevention means applying the correct protocols of (1) primary prevention for both pre-treatment and in-treatment patients, and (2) secondary prevention  , the ConPrimary prevention for MRONJ mainly means elimination/reduction of the oral and dental risk factors ,27,28,29A new emerging and huge aspect is that primary prevention should be performed not only prior to taking the MRONJ-related drugs but also during and after the treatment with antiresorptive agents (AR), in order to eliminate any infective outbreaks of MRONJ. It is the responsibility of the dentist to accurately assess the risk factors leading to the development of MRONJ and suggest a strategy to remove these factors or minimize the risk. The dentist must also stress the importance of maintaining effective dental hygiene , including regular check-ups, for the patient. Both are necessary to maintain oral health, reducing the outbreak of MRONJ and/or detecting possible signs of the early symptoms of this disease.Secondary prevention or early diagnosis is the second pillar in the strategy against MRONJ, since we know that MRONJ diagnosed at an early stage is more likely to be treated successfully. With respect to the diagnostic work-up, the main problem is the possible under-diagnosing of MRONJ cases without bone exposure (or fistula). A recent multicenter study ,30 has d(#1) Keeping teeth with endodontic and/or periodontal diseases at uncertain prognosis only to avoid extractive and invasive procedures, in patients at risk of MRONJ.(#2) Extracting teeth in partial or total inclusion for pure preventive purposes in patients that are candidates for treatment with drugs at risk of inducing MRONJ.(#3) Scheduling in cancer patients the first dental check-up only after diagnosis of bone metastases.(#4) Omitting in osteometabolic patients dental check-ups for oral health status assessment and relative preventive dentistry actions.(#5) Omitting periodic dental check-ups for edentulous patients or wearing removable prostheses, if they are at risk of MRONJ.(#6) Underestimating the usefulness of the imaging performed to evaluate cancer disease extension  with respect to MRONJ early diagnosis (and follow-up).(#1) Extract teeth with endodontic and/or periodontal diseases, in patients at risk of MRONJ, when a conservative approach is not possible and a good tooth prognosis is not guaranteed (confirmed).(#2) Maintain teeth in partial or total inclusion, if no sign and/or symptom is present  (confirmed).(#3) Schedule in cancer patients the first dental check-up as soon as possible, preferably during the tumor staging process (confirmed).(#4) Consider in osteometabolic patients  a potential gradient in MRONJ risk  (updated(a) Administration and duration of treatment with MRONJ-related drugs ,6,37,38;(b) Eventual presence of comorbidities and further medical therapies indirectly associated; assess, during the prescription of the MRONJ-associated drugs, the oral health status even by a questionnaire   39]; sc; sc39]; (#5) Schedule preventive dental check-ups even in patients at risk of MRONJ even if edentulous or wearing removable dentures, in order to intercept and correct any injury on the oral mucosa (confirmed).(#6) Research any sign of jawbone alterations in cancer patients, revising the pictures of the imaging investigations  already performed to stage the cancer disease, in order to potentially detect eventual early signs of MRONJ, before prescribing further specific imaging (updated).Assume that the Consensus Conference has now adopted the new protocols for dental management in patients at risk of MRONJ, published in the recommendations on MRONJ by the two scientific societies SICMF/SIPMO , in whic(#1) Building an implant rehabilitation in cancer patients who have to take or are taking MRONJ-associated drugs due to advanced cancer disease.(#2) Considering as contraindicated, in an absolute sense, implant rehabilitation in osteometabolic patients taking MRONJ-associated drugs (including cancer patients treated with antiresorptive agents for CTIBL).(#3) Prescribing antibiotic prophylaxis before non-invasive dental therapies.(#4) Performing extractive procedures without removing, even if necessary, the alveolar bone, and plan the healing as a second intention .(#1) Avoid in cancer patients implant rehabilitation and plan non-surgical rehabilitation (confirmed).(#2) Plan implant rehabilitation in osteometabolic patients taking into account the cumulative dose of the drug already taken, the periodontal status, lifestyle , comorbidities and other drugs. Share with the patient a hypothetical medium to long-term MRONJ risk due to “implant presence-triggers” (such as peri-implantitis and micro-cracks), beside the lower probable short-term risk due to “implant surgery-triggers” ,41,42,43(#3) Recommend always, in patients at MRONJ risk, antibiotic prophylaxis when surgical procedures involving the dento-alveolar process or maxillary bones are planned  (confirmed).(#4) Practice extractive or surgical procedures planning the healing as the first intention with the clot maintenance (confirmed).Assuming that a drug holiday is a controversial issue, involving both drug prescribers  and oral and dental specialists, it is important to clarify that “Drug holiday” can have at least two meanings in the medical literature when referred to MRONJ. It can mainly mean (1) the temporary and preventative suspension of drug therapy  in a patient at risk of MRONJ, before the necessary dental procedures (tooth extraction or jawbone surgery); or (2) the suspension of drug therapy  due to a diagnosis of MRONJ, hypothetically finalized to stop or to slow the osteonecrosis process (a practice with scarce literature as support). This document will afford only the first meaning of a “drug holiday”.It is well known that suspension of drug therapy can create different cost–benefit balances, according also to the type of molecule in those two patient populations.In cancer patients with advanced disease , suspension of antiresorptive drugs has to be decided on the basis of a difficult balance between a possible positive drug effect  and a potentially higher risk of a side effect anyway present . Clearly that balance could individually change on the basis of the moment along the patient cancer history and along the drug administration history: hypothetically, in the first months of drug administration for bone metastases or myeloma, the suspension is potentially more harmful for the (lack of reduction of) risk of SRE but less dangerous for the risk of triggering MRONJ; vice versa after two years of antiresorptive administration, for example, the suspension appears less dangerous for the risk of SRE (anyway present) whereas the risk of MRONJ (being higher with longer time and higher cumulative drug dose) might receive maximum benefit from the suspension.(a) -BPs have a long half-life and are able to accumulate in the bone tissue, potentially influencing the post-extractive bone tissue repair. Furthermore, for some largely used BPs , a possible antiangiogenic effect was described in the literature, potentially reducing the soft tissue repair process. Consequently, a precautionary and temporary suspension of the drug administration has been claimed by most of authors as potentially useful, even if no definitive data have been published;-®), a monoclonal human anti-RANKL inhibitor, is a drug with a short half-life and does not accumulate in the bone. A possible rebound effect on the bone turnover has been described after drug withdrawal, so that some authors advised against a (long) suspension.Denosumab , receive antiresorptive drugs at lower doses and less frequently . The fracture risk can be determined objectively and correctly by applying the DeFRA79 algorithm validated by AIFA, the Italian version of the FRAX algorithm [(b) lgorithm .-BPs  are synthetic analogues of pyrophosphates, which firmly bind to the hydroxyapatite and reduce bone metabolism/remodeling . The hal-®), a monoclonal human IgG2 antibody that highly binds the receptor activator of nuclear factor-kB ligand (RANK-L), blocks the osteoclast maturation, function and survival. Its half-life is 25–32 days and it does not amass in the bone with a peculiar regimen (1 dose s.c. of 60 mg every six months) [Denosumab (Prolia months) ,47,48. T months) ,50. Of nOn the basis of the two different antiresorptive agents, they are classified as follows:®) without agreeing any temporary suspension with the prescriber (oncologist/hematologist).(#1) Performing dental extraction or oral surgery in patients with active cancer and myeloma bone disease and in therapy with i.v. high-dose bisphosphonates or denosumab (Xgeva(#2) Suspending indiscriminately therapy with antiresorptive drugs  prescribed for osteoporosis (or its prevention), in case of necessity of extraction or programmed oral surgery, without agreeing with the prescribing physician.®) before and after the dental procedure. This combined assessment is mandatory in the absence of univocal data on the efficacy of the suspension of bisphosphonates or denosumab to reduce the risk of “post-extraction” MRONJ, and in consideration of conflicting data about the possible anti-angiogenic effect of zoledronic acid ((#1) Plan, in cancer patients, combined assessment by the prescriber  and by the dentist  to determine whether or not there is the need for a precautionary suspension of i.v. high-dose bisphosphonate or denosumab  (updated). There are two different conditions ((#2) Plan, in osteometabolic patients, a balanced and combined assessment by the prescriber  and by the dentist  to assess whether or not you need precautionary suspension of BPs or postponing the denosumab (Prolianditions .(a) Patient under BPs, a drug suspension may be already considered useful one week before surgical procedures. BP administration can resume once the biological process of healing of the oral tissues is completed (at least 4–6 weeks after surgery) ,14;®); it is still possible to take advantage of the pharmacokinetic of Prolia® in order to define a time interval in those postponable and non-critical dental/periodontal conditions requiring invasive treatment and can theoretically take place without restrictions. This “delayed dosing window” lasts about 2 months, starts ideally 5 months after the last dose of Prolia® and ends at the beginning of the 7th month. If the surgical dental procedure is assessed as urgent and not procrastinable, it is recommended to apply the suspension scheme of denosumab previously described for cancer patients [(b) Patient under denosumab  with respect to therapeutic strategies ,14; it f(#1) Forgetting the adoption of an adequate perioperative antibiotic regimen in case of surgical treatment of MRONJ.(#2) Performing a diagnostic bone biopsy, unless bone metastases are suspected.(#3) Applying of first-level imaging  only to plan surgical treatment.(#4) Awaiting the exfoliation (self-sequestration) of necrotic exposed bone via the use of non-surgical therapies, since this process is unpredictable over time.(#5) Neglecting shaving and smoothing of the bone surfaces as a mainstay of any surgical procedure.(#6) Banishing the surgical option for MRONJ cancer patients based on general statements of residual life expectancy.(#7) Merging BP-related and non-BP-related (anti-RankL and/or targeted therapies) MRONJ patients as a whole when considering temporary interruption of medications for surgical treatment.(#8) Adopting surgical treatment of MRONJ in symptomatic cases only.(#9) Relaying on systemic bone turnover markers to predict the success of surgical therapies in MRONJ patients.(#1) Prescribe a broad-spectrum antibiotic regimen as an integral part of surgical treatment: high-dose amoxicillin/clavulanic acid (1000 mg TID), plus high-dose Metronidazole (500 mg TID), from the day before surgery and up to the 10th post-operative day. Alternatives should be used in case of reported allergy to penicillin (confirmed).(#2) Perform a diagnostic bone biopsy in case of clinical and radiological suspicion of bone metastases to the jaw or myeloma area, in order to distinguish them from site of MRONJ disease, when clinically needed (confirmed).(#3) Use second-level imaging tools, mainly computed tomography (CT), to appropriately plan the extent of jawbone disease before surgery (confirmed).(#4) Anticipate surgical treatment, whenever indicated, to reduce the surgical burden for MRONJ patients and increase the likelihood of long-term healing (confirmed).(#5) Always perform the shaving and smoothing of bone surfaces as a mainstay of any surgical procedure to prevent further bone exposure (confirmed).(#6) Plan the surgical treatment of MRONJ on an individual basis, weighing the impact and the potential benefit of surgery on the general health status of patients (confirmed).(#7) Discuss and plan the temporary interruption of any given medication in agreement with the prescriber and based on its pharmacological properties, before initiating every MRONJ surgical treatment (confirmed).(#8) Adopt the early surgical treatment also in MRONJ asymptomatic patients (confirmed).(#9) Do not rely on systemic bone turnover markers to establish individual treatment algorithms for MRONJ, but carefully examine all potential factors that are likely to influence the long-term success of therapies, including the underlying disease (cancer or non-cancer) and the type of medication used (confirmed).MRONJ is a potentially severe complication of antiresorptive and/or antiangiogenic treatment in patients with skeletal events due to various cancers as well as osteometabolic diseases; MRONJ may lead to a reduced quality of life due to jawbone infections, chronic pain, tooth loss and compromised function. Although notable progress has been made, there remain a number of controversial aspects on MRONJ, especially regarding pathogenesis, diagnosis and treatment.The present paper updated conclusions from the Consensus Conference at the Symposium of Italian Society of Oral Pathology and Medicine (SIPMO) , after the newest recommendations on MRONJ were published in 2020 by two scientific societies , written on the inputs of the experts of Italian Allied Committee on ONJ (IAC-ONJ). Some recommendations were confirmed.We defined six issues of MRONJ and submitted for every issue the current knowledge and the appropriate best practices, in order to improve the management of patients.We highlighted the importance to disclose the appropriate information about MRONJ, and the statements and recommendations presented in this paper might represent a beneficial instrument both at a national and international level to better understand and manage this singular and severe disease.In the absence of clear knowledge on the etiology and given the continuous spread of the application of new target therapies, it will be fundamental to continue the research, to report any cases using new drugs and to clarify by evidence-based medicine all the controversial aspects of MRONJ.


Radiotherapy is an important therapeutic approach to treating malignant tumors of different localization, including brain cancer. Glioblastoma multiforme (GBM) represents the most aggressive brain tumor, which develops relapsed disease during the 1st year after the surgical removal of the primary node, in spite of active adjuvant radiochemotherapy. More and more evidence suggests that the treatment's success might be determined by the balance of expected antitumor effects of the treatment and its non-targeted side effects on the surrounding brain tissue. Radiation-induced damage of the GBM microenvironment might create tumor-susceptible niche facilitating proliferation and invasion of the residual glioma cells and the disease relapse. Understanding of molecular mechanisms of radiation-induced changes in brain ECM might help to reconsider and improve conventional anti-glioblastoma radiotherapy, taking into account the balance between its antitumor and ECM-destructing activities. Although little is currently known about the radiation-induced changes in brain ECM, this review summarizes current knowledge about irradiation effects onto the main components of brain ECM such as proteoglycans, glycosaminoglycans, glycoproteins, and the enzymes responsible for their modification and degradation. Adjuvant chemoradiotherapy with temozolomide is a conventional protocol for the standard treatment for newly diagnosed glioblastoma multiforme (GBM) . Last deRadiotherapy (RT) represents an essential part of this post-surgery GBM treatment, and the current standard usually includes 30 X-ray fractions × 2 Gy, although multiple non-conventional RT sources and regimens are under active investigation .RT aims to destroy the residual GBM cells at the resection border and prevent the disease relapse, but it has wide biological effects on different molecular and physiological parameters in the irradiated brain tissue as well . NegativAlong with that, RT-induced lymphodepletion and subsequent suppression of immune response contribute to the insufficient efficiency of conventional RT and may be limiting the success of GBM treatment . Also, iAt present, more and more evidence is accumulating showing that TME is actively involved in the molecular fate of GBM tumor-initiating cells and tumor development, and significantly modifies the epigenetic landscape of GBM cells with unknown mechanism . IrradiaThe presented data demonstrate that different components of the GBM TME  actively respond to X-ray irradiation, whereas the contribution of extracellular matrix (ECM) to radiation-induced changes in both GBM tumor and normal brain tissue remain much less investigated. The available information on this issue is very scanty and fragmentary, but it may be useful to summarize it in order to outline possible directions for future research in this scientific field.Here, the effects of experimental and clinical irradiation onto key brain ECM components  in normal brain tissue and GBM cells/tumors will be reviewed.Proteoglycans (PGs) are the main components of brain ECM and play important roles in normal brain physiology and gliomagenesis . They reA major component of the brain ECM is chondroitin sulfate proteoglycans (CSPGs), which are actively involved in the organization of GBM TME and glioma invasion . The comThe few available data on radiation effects on CSPGs are quite controversial, although expression of CSPG-coding genes has a tendency to be down-regulated in non-tumor tissues and up-regulated in glioma cells or tissues upon X-ray irradiation . Cranial125I) seed results in an increase of NG2 content in the brain tissue. The U251 cells with activated NG2 expression (U251–NG2) were significantly more resistant to 5 Gy irradiation compared to the NG2-negative U251 cells suggesting NG2 as an important prognostic factor for radiotherapy resistance  activates the expression of CD44 in these cells  is an integral component of any tissue, but it plays a particularly important role in the brain, where it represents a major component of intercellular space .Irradiation effects onto HA remain to be almost uninvestigated. The only study by Yoo et al. demonstrates that irradiation of GBM U87MG cells (2 Gy/day for 3 days) or U87MG orthotopic brain tumors (2.5 Gy/day for 3 days) results in a significant increase of HA content in GBM cell-conditioned medium and experimental tumors, respectively. The increase of HA content in tumor tissue affects the biomechanical tension in the GBM microenvironment and provides pro-invasive extracellular signaling cue due to binding with CD44 receptor and SRC activation sufficient for a mesenchymal shift of GBM cells. These findings suggest an explanation for the frequent brain tumor relapse after radiotherapy .Heparan sulfate proteoglycans (HSPGs) represent one of the main components of the neurogenic niche. Tight involvement of both the polysaccharide heparan sulfate (HS) chains and their degrading enzyme heparanase in the development of the nervous system and growth and invasion of glioma tumors are comprehensively described by Xiong et al. , 45. AmoThere is just fragmentary information on the irradiation effects onto these macromolecules.Experimental irradiation of normal mouse brain (7 Gy in a single dose) did not change expression levels of syndecan-1, glypican-1, and perlecan in short-term period (24–72 h) . HoweverDespite the common extracellular glycoproteins  not being much abundant in brain tissue, they are intrinsic components of brain ECM. Radiation-induced changes in their expression contribute to pathological brain ECM reorganization during radiotherapy.Whole-brain irradiation of experimental animals (a single dose of 10 Gy for rats or 8 fractions × 5 Gy for 4 weeks for mice) decreases collagen IV content in brain tissue by 24 h after irradiation, while no significant changes are shown at 4 or 8 h after irradiation. Simultaneous up-regulation of expression and enzymatic activity of MMP2 and MMP9 seems to be a molecular mechanism for irradiation-induces ECM degradation .Long-term effects (6 months) of irradiation of the whole brain of Rhesus macaques (age 6–11 years), which had received 40 Gy , increased expression of fibronectin 1 (FN1) in brain tissue at mRNA level  and protIrradiation of U87 GBM cells (2 Gy/day for 3 days) does not affect the expression of collagen type-I alpha 1 (COL1A1) and collagen type III alpha 1 (COL3A1) .Brain ECM structure is tightly determined by a complex interplay between the expression of ECM glycoproteins/PGs/GAGs and their proteolytic remodeling by matrix metalloproteinases (MMPs) and GAG-degrading enzymes , 48. RadX-ray irradiation of animal brain results in a quick (4–24 h) up-regulation of expression and enzymatic activity of MMPs: MMP2 and MMP9  and MMP2  . IrradiaIrradiation of U87MG GBM cells at 2–6 Gy doses increases expression and enzymatic activity of MMP2 in these cells and experimental orthotopic U87 tumors obtained from them . This reHeparanase (HPSE) is the main enzyme responsible for the degradation of polysaccharide chains of HS at the cell surface and ECM of all tissues. It involved in normal physiology and pathological reorganization of ECM into TME and cancer progression and metastasis , 45, 49.Gamma-irradiation of brain microvessel endothelial cells results in a significant increase in the release of heparanase, which degrades [35S]-labeled heparan sulfate from the subendothelial matrix. This was most pronounced at the 24 h after irradiation and can affect the interactions of tumor cells with endothelial cells and their microenvironment, which in turn facilitate the formation of metastasis in irradiated tissues . The relAccording to the presented data, X-ray radiation affects all key components of normal brain ECM  in different extent and directions .An interesting observation is that radiation has a much pronounced effect on normal brain tissue than on tumor cells. There is a common tendency to the increased expression of glycoproteins (collagen and fibronectin) and decreased expression of main proteoglycan components of brain ECM  indicating significant changes of normal structure of brain ECM . ReplaceOn the other side, GBM cells and tumors demonstrate more resistant phenotype to X-ray irradiation and completely different pattern of radiation-induced changes. Only three of fifteen ECM components  respond to irradiation by activation of their expression/content . The cooThese radiation-induced changes in brain ECM and the residual GBM cells cooperate to provide a favorable microenvironment for GBM progression. Moreover, multiple studies show that radiotherapy not only serves as a therapeutic mean to eliminate glioma cells but also activates proliferation and invasion of those cells which survived irradiation. This seems to occur due to the selection of radioresistant GBM clones and their active invasion in previously irradiated GBM tumor microenvironment .in vivo, possibly through the imbalance between MMP2 and TIMP2 and ECM degradation (Molecular mechanisms of negative side effects of irradiation are tightly related to GBM TME: subcurative irradiation of primary human GBM tumors in rat brain results in the increased proliferation (3-fold), migration, and invasion of the survived GBM cells associated with increased expression of CD44 and activation of MMP2 expression ; irradiaradation . Besidesradation .Radiation induces also significant changes in immune components of brain microenvironment, which contributes to poor efficacy of anti-GBM radiochemotherapy. Irradiation results in activation of the expression of immune-associated genes  in murine glioma and human glioma U87 cell line, leading to changes in immune microenvironment, glioma cells radioresistance, and treatment failure . Cranialin vivo, where the initial positive effect on inhibition of GBM tumor growth by 14 day is followed by the tumor relapse and higher mortality for the mice with a smaller tumor volume by 21 day (Thus, GBM radiotherapy possesses a double effect—it directly affects brain ECM and induces GBM cells to modulate their microenvironment. The balance between these modalities might be responsible for the ambiguous functional effect y 21 day . OverallEG conceived the study, drafted, and edited the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Accidents, as the third important cause of mortality, are among main challenges of communities due their costs and irreversible damages. This study aimed to investigate the age distribution of people referred to the hospitals affiliated by Iran’s ministry of health because of accidents.In a cross-sectional study, using a CDC checklist, data related to the injured people who referred to the hospitals affiliated by Iran’s ministry of health in a one year period was gathered from hospital records, as well as interview and observation. The collected data was analyzed by descriptive and analytical statistics in SPSS11 software.The commonest age group involved in accidents during the year 1395 (2016) was middle-aged (37%) followed by youth (25%). The commonest accidents in children were: fall (23%), traffic accidents (19%), and burn (13%). In adolescents, the commonest accidents were: traffic accidents (32%), fall (16%), and violence and poisoning (5%). The commonest accidents in youth were: traffic accidents (35%), violence (9%), and poisoning (6%). In middle-aged group, the commonest accidents were: traffic accidents (30%), fall (12%), and violence and poisoning (7%). The commonest accidents in elderly were: fall and traffic accidents (25%), burn (4%), and violence and poisoning (3%).Making plan and policies to prevent accidents and injuries is a health priority and a key tool for improving the safety of the community. Also, informing the all age groups of the community about preventive actions is necessary.Road Traffic Injury, Trauma, Hospital