Ovis dalli dalli) are important herbivores in the mountainous ecosystems of northwestern North America, and recent declines in some populations have sparked concern. Our aim was to improve capabilities for fecal metabarcoding diet analysis of Dall’s sheep and other herbivores by contributing new sequence data for arctic and alpine plants. This expanded reference library will provide critical reference sequence data that will facilitate metabarcoding diet analysis of Dall’s sheep and thus improve understanding of plant-animal interactions in a region undergoing rapid climate change.Dall’s sheep (rbcL gene of 16 arctic-alpine vascular plant species that are known to comprise the diet of Dall’s sheep. These sequences contribute to a growing reference library that can be used in diet studies of arctic herbivores.We provide sequences for the chloroplast The online version contains supplementary material available at 10.1186/s13104-021-05590-z. Ovis dalli dalli) are endemic to alpine ecosystems of northwestern North America, and their populations have been declining in recent decades [Dall’s sheep  were paired upon import, then quality trimmed with BBDuk trimmer . Sequences were normalized, then aligned and assembled using the de novo assembly tool (Geneious Prime). Assembled contigs were uploaded and annotated using BankIt, then submitted to GenBank  Table 11.Table 1We sequenced one DNA extraction from each plant species.The sequencing project was funded through a grant to train new users on Illumina Nextera sequencing.The following are limitations for these data files:Additional file 1. Table of information about the plant specimens used for rbcl sequencing.


Genome-wide association studies have identified thousands of genetic susceptibility loci associated with cancer as well as other traits and diseases. Mapping germline variation in identified genetic susceptibility regions to alterations in nearby gene expression nominates candidate genes potentially related to disease risk for further functional investigation. We developed LDexpress as an online resource that integrates population-specific linkage disequilibrium data from the 1000 Genomes (1000G) project and tissue-specific expression data from the Genotype-Tissue Expression project to better study regional germline variation impacting gene expression. LDexpress is a publicly available web tool designed to be easy to use, flexible to conduct a wide range of variant queries, and quick to efficiently investigate dozens of query variants across multiple tissue types. We demonstrate the utility of LDexpress using example genomic queries and anticipate this tool will accelerate understanding of disease etiology by uncovering associations of regional germline variation to nearby gene expression. Genome-wide association studies (GWAS) have made tremendous advances in identifying germline genetic variation associated with disease risk . IdentifA common approach for selecting nearby candidate genes for functional investigation is by performing an expression quantitative trait locus (eQTL) analysis. eQTL analyses identify germline variants associated with local gene expression by testing for a relationship between variant genotype and gene expression levels –4. Whilehttps://ldlink.nci.nih.gov/?tab=ldexpress) is a new open access web-based tool that seamlessly integrates LD patterns estimated from the 1000G project with gene expression data from GTEx v8 to investigate correlations between genotype and regional gene expression.GTEx is an excellent resource for eQTL information but lacks information on local linkage disequilibrium patterns. Linkage disequilibrium (LD) is the nonrandom distribution of alleles at nearby loci in a defined population . Germlinhttps://ldlink.nci.nih.gov/?tab=ldexpress) asks the user to input information for the desired query including a list of RefSeq (RS) numbers or genomic positions of query variants, the 1000G population of interest, the GTEx tissue or tissues of interest, threshold options for LD metrics , GTEx eQTL p-value, and genomic window of interest. The calculate button submits the form data to the LDexpress Python flask server which pulls phased VCF data  for the 1000G population of interest and calculates LD measures for sequenced variants in the selected window surrounding the query variants. LD measures are calculated as previously described in LDlink [https://storage.googleapis.com/gtex_analysis_v8/single_tissue_qtl_data/GTEx_Analysis_v8_eQTL.tar, accessed 8/19/2020) indexed by variant position for rapid retrieval. eQTL associations that match the user defined GTEx tissue of interest and meet the user provided p-value significance value are returned to the LDexpress web interface. Most variant queries take less than 10 seconds to complete.LDexpress is designed to be accessible to a wide range of genomic investigators interested in disengangling tissue- and population-specific eQTL associations. The landing page for accessing LDexpress (n LDlink . Varianthttps://ldlink.nci.nih.gov/?tab=apiaccess) using the options described above to allow batch queries.LDexpress organizes query results into a menu of clickable links for each query variant that has an eQTL association in LD within a selected GTEx tissue. The variant links will direct the user to a searchable and sortable table that provides details on variants in LD that have eQTL associations in GTEx. The table displays information on variant RS number, position and LD measures with the query variant as well as provides details on the eQTL association including gene symbol, Gencode ID, tissue type, effect size and p-value. Custom links are also generated to dbSNP, NCBI Gene, Ensembl and GTEx where further details and plots on the variants, genes and eQTL association are provided. Variants with no eQTL associations in LD in GTEx will be displayed as warnings. All LDexpress returned results can also be downloaded in a tab-delimited text file for further investigation. LDexpress web content is programmed in HTML5 for cross platform compatibility. The LDexpress web tool is available and fully supported for two full years following publication and does not require mandatory registration to use the resource. LDexpress can also be accessed through the LDlink API  that were associated with altered expression of SF3A3, INPP5B, and FHL3 in transverse and sigmoid colon tissues. The second example query is from a recent cutaneous melanoma GWAS [ITGB3BP gene in skin tissue , a tumor suppressor gene. CSR1 binding leads to migration of SF3A3 to cytoplasm and reduces splicing efficiency of epidermal growth factor receptor and platelet-derived growth factor receptor [http://www.proteinatlas.org) also suggested that the expression of SF3A3 protein might be a prognostic factor in liver cancer [INPP5B is a member of inositol polyphosphate-5-phosphatase family which regulates calcium signaling. The expression of INPP5B protein is prognostic for renal, head and neck, and pancreatic cancers in The Human Protein Atlas. Finally, FHL3 has been shown to modulate SOX4 expression leading to abrogation of self-renewal and epithelial-mesenchymal transition in cancer cells [We showcase the utility of LDexpress by identifying eQTLs with receptor . The Humr cancer . INPP5B er cells , 14.ITGB3BP) suggest this transcriptional coregulator that binds to and enhances the activity of members of the nuclear receptor families could be important for melanoma risk at 1p31.3. Functional studies in breast cancer cells indicates ITGB3BP induces apoptosis through a caspase 2-mediated signaling pathway.In the melanoma example, LDexpress demonstrated ancestry and tissue-specific identification of eQTLs by finding variants in linkage disequilibrium at 1p31.3 with the tagging rs670318 variant. The ancestry-specific population groups in the 1000 Genome Project sequencing data used by LDexpress enables cancer researchers to further disentangle potential differences in cancer risk based on genetic ancestry. The identification of an eQTL with integrin subunit beta 3 binding protein  were White. Previously, population-biased eQTL (pb-eQTL) were examined in GTEx across 31 tissues and discovered only 178 pb-eQTL suggesting that ancestry likely accounts for a limited proportion of all eQTLs . DespiteOur example LDexpress queries showcase the convenience and potential etiologic insights LDexpress can add to post-GWAS analyses that is not easily accomplished with simple GTEx queries, which are unable to perform comprehensive population-specific searches for proxy variants in LD. We anticipate LDexpress to be a useful tool for researchers that assists with identifying potential genes relevant for disease risk for future functional investigation.


Liver cancer is considered one of the deadliest diseases with one of the highest disease burdens worldwide. Among the different types of liver cancer, hepatocellular carcinoma is considered to be the most common type. Multiple conventional approaches are being used in treating hepatocellular carcinoma. Focusing on drug treatment, regular agents in conventional forms fail to achieve the intended clinical outcomes. In order to improve the treatment outcomes, utilizing nanoparticles—specifically lipid based nanoparticles—are considered to be one of the most promising approaches being set in motion. Multiple forms of lipid based nanoparticles exist including liposomes, solid lipid nanoparticles, nanostructured lipid carriers, microemulsion, nanoemulsion, phytosomes, lipid coated nanoparticles, and nanoassemblies. Multiple approaches are used to enhance the tumor uptake as well tumor specificity such as intratumoral injection, passive targeting, active targeting, and stimuli responsive nanoparticles. In this review, the effect of utilizing lipidic nanoparticles is being discussed as well as the different tumor uptake enhancement techniques used. Liver cancer is the sixth highest cancer in terms of incidence rate and the third highest cancer in mortality rates. The world health organization (WHO) estimates the occurrence of 905,677 new cases in the year 2020 with 830,180 new deaths during the same year  score, and HKLC (Hong Kong Liver Cancer) staging . HoweverOther classification systems have been employed based on the molecular signature of HCC. These classification systems include G1-G6 (gene-signature based classification), iHCC 1–3 (metabolic classification), immune-high, immune-mid and immune-low  and poorly polyploid and highly polyploid  .Current conventional HCC therapies include liver resection, liver transplantation, local ablative therapy, transarterial therapy, and systemic therapies (tyrosine kinase inhibitors). However, regular chemotherapeutic agents such as doxorubicin (DOX) and gemcitabine show limited success due to the high ability of HCC to possess drug resistance both intrinsic and acquired .Liver resection is the process by which part of liver is removed. The extend of partial removal is governed by the tumor extension as well as the histological change of the underlying parenchyma. However, liver transplantation possesses the ability to completely remove both detectable, un-detectable nodules, preneoplastic lesions as well as treating the underlying cirrhosis . ChoosinLocal ablative therapy is considered to be the go-to therapy for early stage, unresectable tumors . LocallyTransarterial therapy can be classified into transarterial embolization, transarterial chemoembolization (TACE), and transarterial radioembolization (TARE) . TransarGenetic mutations in cancer setting result in dysregulating tyrosine kinases . TyrosinDespite multiple approved drugs, drug treatment of unresectable, advanced HCC still does not meet the intended outcomes. These insufficient outcomes can be attributed to multiple reasons including low drug bioavailability, and nonspecific drug delivery which lead to high risk of side effects with low drug concentration in the target tissue . One of Nanoparticles are defined recently by the British Standards Institution as 3D nano-objects with 3 external dimensions in the nano range with the nano range extending between 1 and 1000 nm . NanoparLipidic nanoparticles are biocompatible, non-toxic, and well tolerated nanoparticles formulated mainly of physiological lipids . Using lThis review article is constructed with the aim of elucidating the huge potential of using lipidic nanoparticles Fig.  to delivLipidic nanoparticles possess the ability to improve the encapsulated drugs bioavailability and permeability, as well as enhance the payload stability through providing protection against physiological barriers . These eLiposomes are considered to be one of the most studied nanoparticles . LiposomLiposomes have been extensively studied as an approach to HCC treatment. Yang et al. prepared liposomal formulation entrapping docetaxel. The prepared liposomes composed of soy phosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). HepG2 and SMMC-7721 cells were used to assess the efficacy of drug encapsulation in liposomal formulation as well as the safety of different liposomes forming materials. In both cells, soy phosphatidylcholine was the safest liposome forming material at both 24 and 48 h except for SMMC-7721 cells at 48 h, DMPC was the safest option. In HepG2 cells, drug loaded DOPC liposomes exhibited the highest cytotoxicity. While in SMMC-7721, drug loaded soy phosphatidylcholine liposomes had the highest cytotoxicity. It is also worth mentioning that in both cell lines, combining free drug with blank liposomes demonstrated higher cytotoxicity than drug encapsulated liposomes. Drug loaded soy phosphatidylcholine liposomes were able to decrease lactate dehydrogenase leakage, alleviate drug induced intracellular malondialdehyde (MDA) production, while increasing superoxide dismutase activity compared to free drug in HepG2 cells .1/2 compared to chitosan coated PLGA nanoparticles and free drug. Nanoerythrocytes coated chitosan coated PLGA nanoparticles also had higher drug accumulation in the liver [Chitosan can be applied as a coat to increase the liposomes stability and minimize its aggregation . Quagliahe liver .50 compared to blank nanoparticles and free drug. Pharmacokinetic testing demonstrated that drug loaded liposomes were able to accumulate more in the plasma and liver compared to free drug while the free drug and drug loaded liposomes were both not toxic to the N-nitrosodiethylamine rats’ vital organs. Drug loaded liposomes therapeutic and protective effects were assessed in which the prepared liposomes exhibited the highest therapeutic and cancer preventive effect in comparison to free drug. Resveratrol loaded cationic liposomes also showed lower number of nodules as well as lower liver marker enzymes [Due to conventional chemotherapeutics agents’ drawbacks such as toxicity and cancer recurrence, there is an ongoing search for new compounds with anti-cancer activity. One of the most explored options is the usage of phytochemicals . Phytoch enzymes .50 respectively, which the authors attributed to the easier availability of the drugs to the cancerous cells. Yet, the authors also stated that these results may indicate longer action time of the prepared liposomes resulting in higher anti-proliferative effect. In vivo testing was done on H22 tumor bearing mice on which the prepared formulation showed the lowest tumor volume as well as the lowest tumor weight, while decreasing SOR exposure to normal tissue [Combination therapy has been extensively used in cancer therapy. Combination therapy utilizes the pharmacological actions of two or more pharmaceutical agents in either an additive or a synergistic manner in order to combat the heterogenic nature of cancers . Employil tissue . Wang etl tissue . Sarfrazl tissue Another approach for combination therapy is combining a chemotherapeutic agent with other forms of therapy such as microwave ablation and radiotherapy. Wu et al. prepared DOX loaded liposomes and utilized it in combination with microwave ablation. The liposomal formulation consisted of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and DSPE-PEG2000. Drug loaded liposomes exhibited higher cellular uptake and cytotoxicity in HepG2 and Huh7 cells compared to free drug. Applying microwave ablation enhanced the cytotoxic efficacy compared to microwave ablation alone or drug loaded liposomes alone. Combining microwave ablation with drug loaded liposomes showed the highest reduction in tumor volume and weight in HepG2 tumor bearing mice compared to the free drug, microwave ablation alone and a combination of both. Microwave ablation also possessed the ability to enhance the cellular uptake of both drug loaded liposomes and free drug both in vitro and in vivo with drug loaded liposomes showing higher uptake . Shin etRSG), and remote loading with hypertonic loading (LRHG)-. The liposomal formulations were composed of DPPC, cholesterol, and DSPE-PEG2000. Utilizing the different combination of loading techniques significantly enhanced the entrapment efficiency of gemcitabine compared to passive loading technique. Incorporating gemcitabine in both liposomal formulations enhanced the cellular uptake in Huh7 cells resulting in lower IC50 compared to free drug. LRSG and liposomal DOX were chosen to assess the in vivo efficacy in Huh7 tumor bearing mice. LRSG combined with liposomal DOX significantly delayed tumor growth time while enhancing the animals’ survival time compared to free drug combination and control group [Tamam et al. explored a new loading approach to increase gemcitabine entrapment entitled hypertonic loading. The new loading approach depends on admitting a high osmotic pressure which forces the water molecules including the drug through lipid bilayer. The authors prepared two gemcitabine loaded liposomes combining different loading techniques—remote loading with small volume loading  and did not cause cell lysis in hemolysis assay. The authors also stated that both formulations increased the bioavailability of SOR. Multiple cell lines  were used to assess the cytotoxicity effect of the prepared NLCs. Drug loaded NLCs containing Captex® 355 EP/NF showed higher cytotoxicity than the free drug. Drug loaded Miglyol® 812 NLCs showed dose dependent cytotoxic effect in HepG2, Huh7 and PLC/PRF/5 cells, and no cytotoxic effects in Hep3B cells . The samVarshosaz et al. prepared ASGPR targeted NLCs through chemically binding lactobionic acid to stearyl amine. The prepared NLCs composed of glyceryl monostearate, lecithin, oleic acid or Labrafac®, and tween 80 or Solutol® HS15. The prepared NLCs were used to entrap 5-fluorouracil. The optimized targeted, drug loaded formula which contained oleic acid and Solutol® HS15 was assessed in vitro using HepG2 cells. The results showed that the prepared NLCs had higher cytotoxicity as well as higher cellular uptake than untargeted NLCs .As mentioned earlier, HCC is considered to be one of the most resistant cancers to treatment. Liu et al. explored the ability of NLCs to overcome drug resistance through the utilization of 10-hydroxycamptothecin resistant HepG2 cells. The authors entrapped 10-hydroxycamptothecin in NLCs composed of soya oil, tween 80 and used xyloglucan as a coat to target ASGPR. The prepared NLCs were compared against microemulsion containing 10-hydroxycamptothecin and the free drug. NLCs showed the highest cytotoxicity and the highest cellular uptake. Also, in in vivo drug resistant xenograft model, NLCs showed longer residence time in the blood, higher accumulation in the liver, improved drug safety by increasing the lethal dose in comparison to free drug, as well as higher tumor inhibition rate . Zhao et50 when tested on HepG2 cells in comparison to the free drug as and other NLCs containing other liquid lipids. NLCs containing coix seed oil also showed the highest anti-tumor effect and inhibition rate in HepG2 tumor bearing mice [The process of formulating NLCs -as well as other drug delivery systems- requires the addition of pharmaceutical excipients alongside the active pharmaceutical ingredient. Excipients are defined by the International Pharmaceutical Excipients Council as “substance(s) other than the active pharmaceutical ingredient that are included in a drug delivery system to enhance the attributes of the overall safety and effectiveness of the drug delivery system during storage or use.” . Excipieing mice .50 when assessed on HepG2, Huh7, and human colorectal carcinoma (HCT-116) [Another new advancement in designing drug delivery systems is the utilization of ion pair amphiphile (IPA). IPAs are cheap, lipid like alternative to phospholipids . They arHCT-116) .Microemulsions are bi-continuous dispersions of oil droplets in water that are thermodynamically stable, optically isotropic. These droplets exist in a diameter larger than swollen micelles .Ma et al. prepared and assessed the effectiveness of microemulsions in entrapping tanshinone  and tansTrepanier et al. incorporated CRV431  in self-microemulsifying drug delivery system. The prepared formulation consisted of vitamin E, Maisine® CC, propylene glycol, Transcutol®, and Cremophor® RH40. The prepared drug loaded nanoparticles exhibited high drug accumulation in the liver in healthy rats. The prepared drug loaded nanoparticles also exhibited higher Cmax and significantly larger AUC in healthy human subjects compared to Neoral®  .Nanoemulsions consist of oil, water and an emulsifier that form an emulsion with droplet size ranging from 20 to 500 nm . They diTabassum et al. incorporated the extract from five-day sprout of nigella sativa in nanoemulsion. Sefsol® 228, tween 80, and ethanol were used for the nanoemulsion formulation. The prepared drug loaded nanoemulsion was able to induce cytotoxicity in HepG2 while being non-toxic to normal human liver (WRL-68) cells. Treated HepG2 cells with drug loaded nanoemulsion showed increased levels of internal reactive oxygen species (ROS) which is hypothesized to be the utilized pathway for apoptosis by the prepared drug loaded nanoemulsion . NigellaSweed et al. formulated self-nanoemulsifying drug delivery system incorporating rosuvastatin calcium. Peceol®, tween 80, and Transcutol® P were used to construct the self-nanoemulsifying drug delivery system. Blank nanoparticles exhibited almost no toxicity in HepG2 cells indicating the safety of the prepared formulation. Drug loaded nanoparticles enhanced the cytotoxic effect of free drug in HepG2 cell as well as increased the apoptotic cells percentage .Ahmad et al. prepared nanoemulsion formulation composed of Sefsol® 218, Kolliphor® RH40, and PEG400 that was used to encapsulated silymarin. Drug loaded nanoemulsion exhibited cytotoxic effect as well as apoptotic signs in HepG2 cells compared to untreated control. The prepared drug loaded nanoemulsion also promoted intracellular release of ROS to which the authors attributed the generated apoptosis. Pharmacokinetic study in healthy rats demonstrated the ability of encapsulating the drug in nanoemulsion in improving the pharmacokinetic parameters of the free drug. The prepared drug loaded nanoemulsion exhibited higher Cmax, shorter Tmax, and larger AUC compared to free drug .Despite the phytochemicals robust pharmacological actions and their success in vitro. Their clinical use is limited due to their poor in vivo absorption. One of the suggested strategies to deliver phytochemicals is the usage of phytosomes . PhytosoFreag et al. assessed phytosomes ability to enhance the intestinal absorption of diosmin. The research group prepared lyophilized complex of soy phosphatidylcholine and diosmin with the aim of the in vitro formation of phytosomes upon exposure to the gastrointestinal tract aqueous media. The pure drug was not able to penetrate intestinal membrane in vitro. However, drug loaded phytosomes were able to penetrate intestinal membrane and allow the drug presence in significant amount in the dissolution medium .Komeil et al. prepared genistein loaded phytosomes using three different phospholipids namely Lipiod® S100, Phosal® 53 MCT, and Phosal®75 SA. The prepared phytosomes  exhibited the ability to enhance the drug accumulation in the liver, blood, and intestinal serum lipoproteins in healthy rats. These results indicate the ability of the phytosomal preparation to enhance lymphatic uptake as well as protecting the drug from intestinal degradation. The prepared drug loaded phytosomes  exhibited lower cytotoxic effect than free drug initially then had higher cytotoxic effect later on in HepG2 cells with Lipiod® S100 phytosomes exhibiting higher cytotoxic than Phosal®75 SA phytosomes. The authors attributed these results to the longer time necessary for the phytosomal preparation to exert their cytotoxic effect due to the complex structure between the drug and phospholipid. In DEN model in rats, both drug loaded Lipiod® S100 and Phosal®75 SA phytosomes exhibited higher caspase-8 expression with lower VEGF expression compared to free drug and untreated control. Drug loaded Lipiod® S100 phytosomes exhibited the highest improvement in alanine aminotransferase and aspartate aminotransferase. Drug loaded Phosal®75 SA phytosomes exhibited the highest AIF, and caspase-3 expression as well as lower MMP9 expression . LoadingCoating nanoparticles can be defined as the introduction of a material on the surface of nanoparticles . Shao etKhan et al. prepared lipid coated, pH sensitive calcium carbonate nanoparticles entrapping cisplatin and oleanolic acid. The lipids used in the coating layer are HSPC, cholesterol, and DSPE-PEG2000. The prepared nanoparticles exhibited higher in vitro release in pH 5.5 compared to pH 7.4. Co-loaded nanoparticles exhibited higher cytotoxic effect compared to single drug loaded nanoparticles in HepG2 cells. Co-loaded nanoparticles were also able to ameliorate cisplatin’s induced liver toxicity resulting in higher hepatoprotection . Liu et Nanoassemblies are core–shell structure nanoparticles that form due to the presence of a hydrophobic and hydrophilic portion co-existing together . If one Hanafy et al. prepared bromopyruvic loaded nanoassemblies composed of oleic acid conjugated chitosan. The prepared nanoassemblies were coated with folic acid conjugated bovine serum albumin in order to target folic acid receptor. Drug loaded nanoparticles exhibited higher cytotoxic effect on HLF cells compared to free drug with minimal cytotoxic effect caused by blank nanoparticles . MonajatIn order to increase the treatment efficacy, prodrug nanoassemblies have been developed. Prodrug nanoassemblies utilize both prodrug and nanoparticles through modifying the active constituent structure with another moiety rendering the structure amphiphilic . Zuo et Nanoparticles face multiple biological barriers that limit their ability to greatly accumulate in the tumor tissue . Upon reChemotherapeutic agents lack the ability to solely affect cancerous cells. This inability to discriminate between normal and cancerous cells significantly lowers the treatment efficiency . To addrRen et al. evaluated the effect of intratumoral injection of free and liposomal DOX on the pharmacokinetics, biodistribution, and anti-tumor effect in H22 tumor bearing mice. The prepared drug loaded liposomes were able to decrease the drug concentration in the plasma and healthy organs (except for liver and spleen) while exhibiting lower toxicity and lower mortality rates. Drug loaded liposomes also significantly enhanced the tumor tissue concentration compared to the free drug. The free drug showed lower anti-tumor effect compared to drug loaded liposomes .Emulsomes are a type of nanoparticles which consist of a phospholipid layer surrounding a solid lipid core . Xu et aFu et al. prepared paclitaxel and DOX co-loaded liposomes. The prepared liposomes were loaded into thermoresponsive nanocomposite gel for intratumoral delivery. The prepared gel exhibited sol phase at 4 °C and gel phase at 37 °C with limited corrosion when incubated in phosphate buffer solution at 37 °C. The prepared liposomes showed no toxicity on three different cell lines  while exhibiting enhanced cellular uptake and internalization in SMMC-7221 cells. Intratumoral injection of the prepared liposomal gel significantly enhanced the accumulation in the tumor site while minimizing accumulation to other organs in SMMC-7721 tumor bearing mice. In vivo results also demonstrated the possible ability of the prepared gel to delay the payload release .Irreversible electroporation is an emerging cancerous tissue ablation technique. It depends on the delivery of strong, short pulses of electric field that result in killing tumor cells as well as creating membrane defects . It was For the tumor cells to grow, excessive supply of oxygen and nutrients must be attained by the tumor cells. This need for oxygen and nutrients promote the tumor angiogenesis process through which new blood vessels supplying the tumor with its needs are formed . The newZhao et al. prepared Fasudil loaded pegylated liposomes using DSPE-mPEG2000. The prepared liposomes showed significantly higher accumulation in the tumor tissue as well as in the liver, spleen, stomach, and kidney in Hep3B xenograft tumor model when compared to free Fasudil . Huang em which resulted in lower drug leakage and slower drug release [Another approach that can be used to enhance targeting is altering the composition of nanoparticles . Li et a release .As previously mentioned, passively targeted nanoparticles possess higher blood circulation time. However, one common limitation of this targeting approach is the possible premature release of the active payload . NanoparpH responsive nanoparticles respond to changes in pH through two main pathways: protonation/ionization of functional groups and removal of acid-labile bonds . In protGuo et al. prepared liposomal formulation containing DOX and MDM2 inhibitor MI-773. The liposomal formulation was coated with carboxymethyl chitosan as a pH sensitive coat. The prepared coated nanoparticles exhibited negative charge in pH 7.4 which converted into positive charge at lower pH indicating the dissociation of the carboxymethyl chitosan coat. Coated liposomes also exhibited higher in vitro drug release at pH 6.5 compared to pH 7.4. Cellular uptake in HepG2 cells was assessed in pH 6.5 and 7.4. Coated liposomes showed improved uptake in pH 6.5 compared to pH 7.4 due to the charge reversal from negative to positive .Duan et al. synthesized N-acetylgalactosamine modified and pH sensitive DOX prodrug. This prodrug was co-encapsulated with SOR in lipid nanoparticles for the treatment of HCC. The prodrug gained pH sensitive capabilities due to the presence of hydrazine functional group. pH sensitive modified nanoparticles exhibited higher in vitro drug release in pH 5.5 compared to pH 7.4. The authors this increase in DOX release to the cleavage of hydrazone bonds in lower pH. They also stated that these results indicate that DOX will be released more easily in the tumor lower pH environment .The tumor redox microenvironment is controlled by nicotinamide adenine dinucleotide phosphate (NADP) -NADPH/NADP +-and glutathione. It was reported that glutathione levels in tumor tissue is significantly higher (4 folds) than in normal tissue. Glutathione exerts its reducing effects mainly through fragmenting disulfide bonds . DisulfiZhou et al. encapsulated DOX in redox responsive liposomal nanohybrid cerasomes through the incorporation of disulfide bond. Redox responsive nanoparticles exhibited higher in vitro release in glutathione rich release medium compared to glutathione deprived release medium. While glutathione addition showed no impact on regular nanoparticles. Redox responsive nanoparticles exhibited cytotoxic effects on SMMC-7721 cells and human breast cancer cells (MCF-7) while showing minimal effect on human embryonic kidney cells (293) .Utilizing ultrasound as a trigger for payload release is coupled with the usage of ultrasound contrast agents micro/nanobubbles. Micro/nanobubbles compose of gas-filled inner core (such as fluorocarbon) that allows the particles to oscillate at high speed when exposed to ultrasound field resulting in its destruction . EmployiZhu et al. prepared liposomal microbubble entrapping DOX using perfluoropropane as the gaseous core. Drug loaded liposomal microbubbles had a significant cytotoxic effect on H22 cells in the presence of ultrasound. However, drug loaded liposomal microbubbles had limited cytotoxicity in the absence of ultrasound. Drug loaded liposomal microbubbles enhanced the tumor ultrasound signals with high nanoparticles signals around the tumor in H22 tumor bearing mice. Tumor blood flow also increased after nanoparticles destruction indicating liposomal microbubbles’ ability to enhance blood flow inside the tumor .Guo et al. used perflenapent as the gaseous core in formulating arginine-glycine-aspartic acid (RGD) modified liposomal nanobubbles loaded with fingolimod and superparamagnetic iron oxide nanoparticles. In vitro release of drug increased from the prepared liposomal nanobubbles when ultrasound was applied. The enhancement in drug releases was directly proportional to the ultrasound power applied. Cellular uptake of the prepared nanoparticles in HepG2 cells improved when ultrasound was applied compared with no ultrasound application. This improvement was attributed by the authors to the cavitation process .For nanoparticles to gain responsiveness to magnetic field, materials possessing magnetic behavior must be included. One of the most commonly used materials is iron oxide nanoparticles . UtiliziGrillone et al. prepared SOR and superparamagnetic iron oxide nanoparticles loaded solid lipid nanoparticles. The prepared solid lipid nanoparticles enhanced cellular uptake in HepG2 cells when magnetic field was applied. Magnetic field application was able to focus the cytotoxic effect in the desired tumor tissue as a result of magnetically driven tumor accumulation .Chen et al. encapsulated DOX with superparamagnetic iron oxide nanoparticles in PEG stabilized liposomes. The in vitro release of the entrapped drug significantly increased upon the application of magnetic field. Drug loaded nanoparticles cytotoxicity significantly improved upon applying magnetic field compared to its absence in Huh7 cells .Liu et al. formulated DOX loaded thermosensitive liposomes. Radiofrequency responsive manganese and zinc doped superparamagnetic iron oxide nanoparticles were used in mixture with the liposomal formulation. The combination was used to induce hyperthermia allowing the release of the encapsulated payload. The authors utilized a strong static gating field containing a sharp zero point superimposed on the radiofrequency field. This modification allowed radiofrequency waves to affect only nanoparticles at zero or near zero point. Thus, increasing the resolution of the targeted release. In vitro release of DOX increased with increasing the temperature of the dissolution medium. DOX in vitro release also increased with the presence of magnetic nanoparticles. Liposomal formulation in combination with magnetic nanoparticles exhibited higher cytotoxic effect in Huh7 cells in the presence of magnetic field compared to its absence .Increasing the tumor tissue temperature to 40–43 °C is termed hyperthermia . As mentDirect tumor killing using microwave ablation by heat generation was explored by Zhou et al.. Sodium chloride that is used as a thermo-seed was encapsulated in liposomal formulation. Liposomes loaded with sodium chloride exhibited better heat conversion than blank liposomes and free sodium chloride. Sodium chloride loaded liposomes exhibited the highest cytotoxic effect on HepG2 cells while showing hemocompatibility on the application of microwave. The in vivo efficacy of the prepared liposomes was assessed in both HepG2 xenograft animal model and MHHC97H orthotropic animal model. In both models, the prepared nanoparticles exhibited the highest anti-tumor effect while applying microwave compared to the usage of free sodium chloride, microwave ablation alone, and the prepared nanoparticles alone without applying microwave .Guo et al. prepared icaritin loaded microemulsion with coix seed oil as an unconventional liquid phase. The prepared microemulsion was entrapped in thermosensitive liposomes. In vitro release of the entrapped drug was slow at 37 °C. However, in vitro release was significantly higher at 42 °C. The prepared microemulsion and thermosensitive liposomes exhibited enhanced cellular uptake in HepG2 cells compared to free drug using clathrin-mediated internalization pathway. Applying mild hyperthermia enhanced the prepared nanoparticles cytotoxicity. The prepared liposomes enhanced tumor uptake and exhibited higher tumor penetration in HepG2 + LX-2 desmoplastic 3D tumor spheroids before hyperthermia application. Hyperthermia application enhanced the in vivo anti-tumor effect of the thermosensitive liposomes compared to the anti-tumor effect in the absence of hyperthermia in HepG2 + LX-2 tumor bearing mice .Zhu et al. modified iron oxide nanoparticles with oleic acid. These modified magnetic particles were encapsulated alongside hydroxycamptothecin to obtain thermosensitive magnetic liposomes. Drug in vitro release was enhanced when the release media was heated to 42 °C compared 37 °C confirming the thermosensitive characters of the prepared nanoparticles. The prepared drug loaded nanoparticles enhanced tumor treatment in Huh7 tumor bearing mice when magnetic strips and external heating source (42 °C water bath) were applied compared to the anti-tumor effect in their absence .Multiple clinical trials were conducted validating the efficacy of DOX loaded thermosensitive liposomes as an adjuvant therapy to radiofrequency ablation , 211.1O2) upon activating a photosensitizer. The generated 1O2 is responsible for killing cancer cells [Phototherapy is described as the utilization of light (preferably near-infrared light) through activating phototherapeutic agents. These agents should possess low toxicity in the dark as well as the ability to kill cancer cells under light activation without affecting normal cells . Photother cells . Payloader cells .Youssef et al. encapsulated hypericin in solid lipid nanoparticles. The encapsulated photosensitizer exhibited lower degradation when compared to free photosensitizer. Both free and encapsulated photosensitizer exhibited no significant effect on HepG2 cells in the dark. However, upon light exposure, both free and encapsulated photosensitizer exhibited significant cytotoxicity. The free photosensitizer exhibited higher cytotoxic effect. These results were attributed by the authors to the possible quenching deactivation of the photosensitizer as a result of the solid lipid nanoparticles compact and thick structure .3Na)4ZnPc)- was synthesized by Abdel Fadeel et al.. The prepared photosensitizer was evaluated for its photodynamic efficacy after being loaded into liposomes and transferosomes [A new photosensitizer -thiophenyl sulfonated zinc phthalocyanine  in thermosensitive liposomes. Encapsulating the photosensitive agent in liposomal formulation enhanced its photostability compared to free agent. The drug in vitro release from the prepared thermosensitive liposomes increases significantly upon light application. The prepared nanoparticles were able to attain more cellular uptake in Hep3B cells upon light activation. This enhancement in cellular uptake was attributed by the authors to the production of ROS and the generation of thermal effect which promoted cellular uptake. The cytotoxic effect of the prepared nanoparticles on Hep3B cells significantly improved upon light activation compared to the cytotoxic effect in absence of light. The prepared nanoparticles also had higher anti-tumor effect in Hep3B tumor bearing mice upon light activation compared to its absence .One of the potential risks of phototherapy is the induction of severe hypoxia as a result of oxygen consumption in generating ROS. This may result in a rise in cancer metastatic risk . Yang etHigher understating of disease progression and molecular targets has allowed the development of nanoparticles that actively target the tumor tissue on a cellular level limiting nanoparticles unspecific binding to healthy organs and tissues . Active ASGPR is a C-type lectin composed of 48 kDa (ASGPR-1) and 40 kDa subunits (ASGPR-2) that is responsible for clearing glycoproteins that terminate with a galactose or N-acetylgalactosamine moiety . ASGPR iPathak et al. utilized arabinogalactan conjugated to cholesterol as the targeting moiety for DOX loaded liposomes. Non-targeted drug loaded liposomes showed comparable cell inhibition on HepG2, breast cancer (MCF7), colon cancer (HT-29), and lung cancer (A549) cells. While targeted liposomes showed higher uptake in HepG2 cells. In vivo biodistribution was conducted on healthy mice through radiolabeling the formulated nanoparticles. Both liposomal formulations were able to accumulate in the liver and spleen with minor amounts being present in the bladder. These results indicate the ability of both nanoparticles to mitigate DOX mediated nephrotoxicity. In vivo analysis in HepG2 tumor bearing mice exhibited smaller tumor volume when compared to free drug . Bansal In a research done by Ding et al., the effectiveness of three different ligands was assessed in enhancing cellular uptake of 10-Hydroxycamptothecine loaded liposomes. Galactose and di-galactose were bound to stearic acid targeting ASPGR while galactose and biotin were co-bound to stearic acid targeting both ASPGR and biotin receptors. Galactose-biotin liposomes showed the highest cellular uptake in HepG2 cells followed by di-galactose liposomes then galactose liposomes. Cellular uptake studies also exhibited low uptake in A549 (lung cancer cells), Hela , and SGC-7901 (gastric cancer cells). All targeted liposomes also exhibited lower cellular uptake in LO2  than HepG2 cells .Qu et al. synthesized Octanoyl galactose ester as a targeting ligand for HCC which was used to provide active targeting capabilities to formulated microemulsion. Targeted microemulsions showed higher uptake than non-targeted microemulsion in HepG2 cells. Cellular uptake studies also showed that targeted microemulsion uptake decreased significantly when HepG2 cells were pre-treated with galactose. Targeted microemulsion exhibited higher localization in tumor site with longer retention time when compared to non-targeted microemulsion in HepG2 tumor bearing mice. The authors also stated that there was no difference in pharmacokinetics between orally administered targeted and non-targeted microemulsion which the authors attributed to the lack of effect on galactosylation modification on oral absorption .Wei et al. utilized both passive and active targeting approaches in enhancing the delivery of DOX. DSPE-PEG2000 was used to convert the liposomes into pegylated liposomes as well as conjugating the targeting moeity which was Lactoferrin. Targeted liposomes showed higher cellular uptake in ASPGR positive HCC cell lines HepG2, BEL7402, and SMMC7721. However, both targeted and non-targeted liposomes showed no difference in cellular uptake in ASPGR negative mouse embryonic fibroblast cell line NIH 3T3. DOX showed the highest cellular uptake in all cell lines which the authors attributed to DOX ease of diffusion into the cells while nanoparticles accumulate in the cell using receptor mediated and non-receptor mediated endocytosis .1/2. Huh7 orthotopic tumor xenograft model in mice as well showed higher accumulation for targeted liposomes in the tumor area [The possibility to tackle both MDR as well as active targeting to HCC using mitoxantrone loaded liposomes was explored by Zhang et al.. The research group synthesized galactosyl conjugated Pluronic P123 utilizing P123 ability to reverse MDR while targeting ASPGR using the galactosylated moiety. The ability of P123 to reverse MDR was validated using BCRP-overexpressing Madine-Darby Canine kidney cell line. P123 exhibited dose dependent enhancement in the drug accumulation while galactosylated P123 showed comparable effect indicating that galactosylation possesses limited effect on MDR reversal. Targeted liposomes and P123 modified liposomes showed comparable enhancement in mitoxantrone uptake when compared to conventional liposomes. Conventional liposomes also improved mitoxantrone uptake compared to free drug which the authors attributed to the ability of liposomes to bypass efflux transporters. Targeted liposomes showed higher cellular uptake in ASPGR positive Huh7 cells when compared to P123 modified liposomes and conventional liposomes. Liposomal formulations were able to improve free drug pharmacokinetics with targeted liposomes showing the highest AUC and longest Tmor area .A summary of other approaches targeting ASPGR is provided in Table GA is the aglycon derivative of the widely present in licorice roots glycyrrhizic acid . GA is oCellular uptake and clearance mechanism of both free GA and GA modified liposomes in HepG2 cells were evaluated by Sun et al.. Free GA showed both concentration and temperature dependent uptake with higher uptake achieved in higher concentrations and higher temperature. Different proteins showed varying effects on GA uptake. Bovine serum albumin showed no interference in GA uptake. Fetal bovine serum and cytoplasm protein slightly decreased GA uptake while cytomembrane protein showed the highest decrease in GA uptake. GA coupled liposomes showed time dependent uptake with higher uptake achieved with longer incubation time. GA coupled liposomes showed higher dependence on active transport, endocytosis, and caveolae-dependent endocytosis cellular uptake mechanisms while clathrin-dependent endocytosis and micropinocytosis were not significantly utilized by GA coupled liposomes. Free GA clearance best fitted exponential decay kinetics while GA coupled liposomes best fitted second order kinetics . The samChu et al. coupled GA to DSPE-PEG2000 and used it as a targeting moiety for curcumin loaded NLCs. Varying concentrations of GA were used to assess whether cellular uptake increases with increasing the targeting ligand concentration on HepG2 cells. The results showed that with increasing the ligand concentration, the cellular uptake increases until reaching a certain limit then decreases. The authors attributed these results to the overabundance of ligands which may result in stearic hinderance which may result in blocking ligand-receptor interactions. Targeted NLCs with the best ligand concentration exhibited higher cellular uptake in HepG2 in comparison to free drug, conventional NLCs, and pegylated NLCs .Docetaxel loaded liposomes were modified by Li et al. through synthesizing 3-succinyl-30-stearyl GA. Non-targeted liposomes showed no difference in cellular uptake in L02 cells and hepatic nonparenchymal cells (LX-2) while targeted liposomes showed enhanced uptake in L02 cells through receptor mediated endocytosis. Targeted liposomes had enhanced pharmacokinetic profile compared to non-targeted liposomes when assessed in healthy rats. However, the difference was not significant between the two groups. The authors attributed these results to the inability of hydrophilic succinic anhydride to overcome the hydrophobicity of GA. This led to rapid MPS uptake of hydrophobic targeted and non-targeted liposomes .1/2 and higher elimination rate for targeted liposomes which the authors ascribed to the targeted liposomes’ ability to distribute quickly to the tissues as well as quick recognition by GAR and ASPGR. Targeted liposomes were able to significantly enhance liver targeting compared to non-targeted liposomes. The results also demonstrated that targeted liposomes accumulated mainly in the liver and kidney with higher amount in the kidney [Zhou et al. explored the potential of synthesizing a single ligand that has the ability to target two receptors simultaneously. The authors synthesized 3-Galactosidase-30-stearyl deoxyglycyrrhetinic acid  which has the ability to target both GAR and ASPGR. The synthesized ligand was used to modify cantharidin loaded liposomes. In vivo testing in healthy rats exhibited lower Te kidney . Double e kidney .Folate Receptor (FR) is a family of proteins that are classified into FRα, FRβ, FRγ and FRδ. They are a group of glycoproteins where FRα, FRβ, and FRδ are glycosyl-phosphatidylinositol anchored cell surface proteins while FRγ is a secreted protein with no anchor . Folate Folic acid was conjugated to DSPE-PEG2000 by Liu et al. and they used this conjugation to modify diacid metabolite of norcantharidin loaded liposomes. Biodistribution study in H22 tumor bearing mice demonstrated high accumulation of targeted and pegylated liposomes in the tumor, liver, and spleen. This accumulation was significantly higher than the free drug accumulation. Targeted liposomes were able to further enhance pegylated liposomes’ ability to target the tumor. However, targeted liposomes showed significantly higher accumulation in the kidney compared to pegylated liposomes. The authors stated that this higher accumulation in the kidney requires further safety evaluation for targeted liposomes containing diacid metabolite of norcantharidin due to higher risk of kidney toxicity . Liu et 1/2 with lower clearance in B16 tumor-bearing mice. Targeted nanosuspensions improved tumor targeting efficiency compared to free drug while pegylated nanosuspensions showed intermediate improvement in targeting efficiency. Both nanosuspensions were accumulated in the liver and spleen more than free drug while decreasing the free drug concentration in the heart, lung, and kindey [Wang et al. prepared docetaxel lipid based nanosuspensions modified using folic acid conjugated DSPE-PEG2000. Targeted and pegylated nanosuspensions showed comparable cytotoxic effect on FR negative HepG2 cells. However, targeted nanosuspensions showed significantly more cytotoxic effect than pegylated nanosuspensions on FR positive B16 cells. Both nanosuspensions improved the cytotoxic effect of free drug on both cell lines. Both targeted and pegylated nanosuspension improved the pharmacokinetic parameters of free drug with higher residence time, and Td kindey .EGFR is a single chain transmembrane glycoprotein . EGFR  to be used as the targeting moiety. EGFR Fab´ conjugated nanoparticles showed higher cellular uptake when compared to non-modified nanoparticles. This improvement in cellular internalization was observed in SMMC-7721 (high expression of EGFR), HepG2 (moderate expression of EGFR) and Huh7 (low expression of EGFR) cells. However, the enhancement in cellular uptake in Huh7 was moderate. EGFR Fab´ conjugated nanoparticles enhanced tumor tissue uptake in SMMC-7721 tumor bearing mice when compared to non-modified nanoparticles. Non-modified nanoparticles exhibited low cellular internalization in the tumor tissue. However, EGFR Fab´ conjugated nanoparticles demonstrated high cellular internalization in a pattern consistent with receptor mediated endocytosis .Low density lipoprotein receptor (LDLR) composes of several domains, most importantly class A repeat and class B repeat. Class A repeat consists of three pairs of cysteines in each seven or eight repeat moieties. Each seven or eight repeat moieties consist of approximately 40 amino acids. Class B repeat contains four-amino-acid sequence of Tyr-Trp-Thr-Asp. Domains containing class B repeat typically consist of both class B repeats and epidermal growth factor repeats. The class B repeats form a structure called β-propeller . Among LApo B was explored as a targeting ligand for LDLR by Wang et al.. The research group utilized Apo B as a targeting ligand for lipid nanoparticles entrapping both SOR and dihydroartemisinin. The prepared nanoparticles were able to enhance the cellular uptake in LDLR + ve HepG2 cells compared to non-targeted nanoparticles. This enhancement in uptake was mediated through receptor mediated endocytosis .Alanazi et al. explored the ability to target LDLR conjugating cholesterol to an active pharmaceutical ingredient. This exploration was based on the theoretical knowledge that this conjugation will result in a cholesterol ester which is a natural component of LDLR. The research group conjugated 5-fluorouracil to cholesterol and incorporated this conjugation into both liposomes and LDL nanoparticles. Drug conjugation to cholesterol was able to enhance drug partitioning into LDL core due to its hydrophobic nature compared to non-conjugated drug. When comparing conjugated and non-conjugated drug loaded liposomes in healthy rats, conjugated drug loaded liposomes significantly enhanced the drug accumulation in liver. Conjugated drug loaded liposomes also were able to accumulate in LDL while non-conjugated drug loaded liposomes failed to accumulate in LDL .LDL nanoparticles packed with docosahexaenoic acid targeting LDLR were explored by both Ou et al.  and YangCD are cell surface glycoprotein that are used to immunophenotype cells. There are more than 350 CD markers that exhibit various functions including cell adhesion, cell activation, and cell inhibition . MultiplCD13  protein is a 150 kDa metalloprotease characterized by the presence of a catalytic domain oriented toward the extracellular matrix. It plays a vital role in multiple processes including cytokines activity regulation through cleaving the present N-terminals as well as regulating inflammatory mediators . Pang etCD90  protein is 25 to 37 kDa glycophosphatidylinositol anchored protein with heavy N-glycosylation on two sites in human and three sites in mouse. CD90 exerts roles in cell–cell and cell–matrix interactions . Yang etd, and t1/2 compared to free drug in healthy rats. Huh7 tumor bearing mice demonstrated higher ability for targeted liposomes to accumulate in the tumor as well as liver and spleen with lower accumulation in heart, lung, and kidney. In vivo evaluation also confirmed the ability of targeted liposomes to enhance tumor uptake compared to non-targeted liposomes and free drug [CD147  protein is a type-1 transmembrane glycoprotein that exerts functions in intercellular recognition. CD147 can be present in either a 27 kDa unglycosylated form or a 43 to 66 kDa glycosylated form , 273. Waree drug .Peptides are low molecular weight protein fragments that compose of two or more amino acids linked together using peptide (amide) bond . PeptideRGD is a peptide that has the ability to bind to integrin receptors  which arWang et al. formulated SOR and quercetin loaded lipid coated PLGA nanoparticles and used RGD conjugated DSPE-PEG as the targeting moiety. Targeted nanoparticles exhibited higher cellular uptake in HepG2 cells when compared to non-targeted combination loaded nanoparticles, non-targeted SOR loaded nanoparticles, and free drugs both the combination and SOR alone .RGD conjugated DSPE-PEG was used by Fei et al. to modify arsenic trioxide loaded liposomal shell-mesoporous silica core hybrid nanoparticles. In vitro study was conducted in αvβ3 positive HepG2 and αvβ3 negative MCF-7 and L02. Targeted nanoparticles exhibited significantly more uptake in HepG2 than MCF-7 and L02. Non-liposome shell mesoporous silica nanoparticles showed lower uptake due to the nanoparticles aggregation on cell surface impeding their uptake. Targeted and non-targeted nanoparticles exhibited enhanced bioavailability and prolonged residence time than non-liposome shell mesoporous silica nanoparticles and free drug due to lower drug leakage in healthy rats. H22 tumor bearing mice showed higher accumulation in tumor tissue by all nanoparticles. Targeted nanoparticles exhibited the highest accumulation in the tumor tissue .iRGD peptide (CRGDK/RGPDC) is a combination between RGD sequence motif and cryptic CendR motif . This seSP94 (SFSIIHTPILPL) is an HCC targeting peptide that was identified using phage display technique . It was Wu et al. enhanced targeting ability of DOX and vinorelbine co-loaded liposomes using SP94 conjugated to DSPE-PEG-N-hydroxysuccinimide(NHS). The ability of SP94 to enhance targeting was explored using DOX alone as the model drug. Targeted liposomes showed improved cellular uptake in SK-HEP‑1 cells when compared to pegylated liposomes. Targeted liposomes exhibited higher tumor accumulation while showing similar uptake to pegylated liposomes in normal organs in Mahlavu tumor bearing mice .The effectiveness of SP94 modification was compared to the effectiveness of galactose modification in enhancing the targeting effect by Jiang et al.. SP94 or galactose was conjugated to DSPE-PEG2000 to modify C14 alkyl chain norcantharimide derivative loaded liposomes. Both galactose and SP94 modified liposomes were able to enhance cellular uptake in HepG2 cells compared to conventional liposomes. However, SP94 modification showed completely different uptake pattern than galactose modified liposomes. SP94 modified liposomes were able to internalize within the cells at a much faster rate than galactose modified liposomes and then decline over time. While galactose modified liposomes showed increased accumulation over time. The authors ascribed SP94 faster uptake to its possible ability to allow the ligands to bind to multiple binding sites through the enhancement of clustering and mobility of ligands. This effect is attributed to the SP94 binding to peg-terminal “brush” on the fluid lipid membrane. SP94 modified liposomes were able to enhance drug concentration in the tumor tissue while decreasing its accumulation in normal organs in H22 tumor bearing mice .To assess the efficacy of different peptides in targeting HCC, Wu et al. conjugated L-peptide, SP94 peptide, and PC5-52 peptide to liposomal iron oxide nanoparticles and DOX loaded liposomes. L-peptide, which is an anti-cancer cell membrane, SP94 peptide, and PC5-52-peptide, which is anti-tumor endothelia. L-peptide and SP94 showed comparable cellular uptake in HepG2 and Huh7. Cellular uptake increased when combining the two peptides. L-peptide was also able to avoid binding to normal hepatocytes, same as SP94. Drug loaded liposomes showed some toxicity signs in the liver, kidney, and spleen. However, L-peptide or SP94 or both peptides modified drug loaded liposomes showed no signs of toxicity in HepG2 tumor bearing mice. The in vivo model also demonstrated the ability to enhance the chemotherapeutic effect of DOX when PC5-52-peptide modified drug loaded liposomes is co-administered with either L-peptide or SP94 modified drug loaded liposomes. The authors concluded from the in vivo results that combining anti-tumor and anti-endothelial peptide is more effective than a combination of two anti-tumor peptides .TAT (GRKKRRQRRRPPQ) is the transcriptional activator protein in HIV-1. It is a cationic cell penetrating peptide which consists of arginine and lysine residues . TAT -liMei et al. formulated multistage liposomes composed of cleavable PEG, RGD, and TAT. Long chain cleavable PEG was employed as the first stage to achieve passive targeting. The second stage composed of RGD conjugated to medium chain PEG to recognize and bind to HCC cells. The third and inner stage is TAT conjugated to short chain PEG to enhance cellular internalization. Cellular uptake was assessed using HeLa cells (low expression of integrin receptors) and HepG2 cells (high expression of integrin receptors). RGD modification improved cellular uptake in HepG2 compared to HeLa cells. TAT co-modification showed synergistic effect in improving cellular uptake in HepG2 cells. Cysteine addition to cells  exhibited higher cellular uptake for cleavable PEG liposomes suggesting that the removal of the outer PEG layer exposed the targeting ligands and enhanced cellular uptake. Multistage liposomes exhibited high dependence on clathrin-dependent uptake pathway. HepG2 tumor bearing mice demonstrated the ability of PEG coating to allow the liposomes to evade the reticuloendothelial system (RES). The in vivo model exhibited higher ability of multistage liposomes to internalize into the tumor tissue with higher stability .Zhao et al. prepared phase-transformation lipid nanoparticles entrapping 10-hydroxycamptothecin and coated with liquid perfluoropentane. The prepared nanoparticles possess the ability to transform into lipid microbubbles upon exposure to external ultrasound radiation with a specific intensity. The prepared nanoparticles were modified using cysteine flanked TAT (CG-TAT-GC) to enhance cellular internalization as well as hyaluronic acid to add target cellular specificity through binding to CD44. Double modified nanoparticles were able to adhere to SMMC-7721 cells which overexpress CD44. However, cysteine flanked TAT modified nanoparticles were not able to adhere to SMMC-7721 cells. SMMC-7721 tumor spheroid showed the ability of the double modified nanoparticles to penetrate the 3D tumor. Yet, hyaluronic acid modified nanoparticles were not able to penetrate the 3D tumor efficiently. SMMC-7721 tumor bearing mice were used to assess the targeting ability in vivo. Double targeted nanoparticles exhibited higher accumulation in the tumor site when compared to non-targeted nanoparticles as well as cysteine flanked TAT modified nanoparticles .Aptamers are defined as nucleic acid molecules that have the ability to bind to specific targets through folding into complex 3D structures that mimic antibodies . Ding etOther less explored active targeting approaches using lipidic nanoparticles are summarized in Table HCC is considered to be one of the most challenging diseases worldwide with less-than-optimal treatment outcomes using chemotherapeutic agents. Lipidic nanoparticles gained significant attention due to their stability, biocompatibility, and their ability to decrease undesirable side effects. Utilizing lipidic nanoparticles significantly enhances the cytotoxic activity of the used anti-neoplastic agents. Further enhancements in the efficiency of lipid based nanoparticles can be achieved through various targeting techniques. Targeting approaches drastically enhance the tumor uptake of the intended anti-neoplastic agents while minimizing the effect on normal tissues. Lipid based nanoparticles hold great promise in improving the treatment outcomes of anti-neoplastic agents for HCC. However, further improvements are required to achieve higher number of clinically approved medications. A special focus should be directed toward decreasing the possible risk of toxicity. Alternatives to the possibly toxic cationic lipids as well as solutions to achieve the encouraging effects of positively charged lipidic nanoparticles without the associated toxicity risk should be attained to further enhance the safety profile of the prepared formulations. Another area of focus to further increase bench to bedside translation is the methods of preparation used. Novel, green, and ecofriendly methods of preparation that do not depend on organic solvents will further improve the scalability of production and decrease production costs while eliminating the risk of the presence of toxic residuals.


Inflammatory Bowel Disease (IBD) is a challenging medical condition that is driven by various genetic and environmental factors. Therapeutic opportunities for this disease remain limited due to the lack of in-depth understanding of the pathogenetic mechanisms and actionable targets driving the disease. Analysis of telomere dysfunctional mice and patients with genetic defects in telomere maintenance unexpectedly revealed phenotypes mirroring those observed in IBD. Molecular characterization of this model identified a pathway driven by telomere DNA damage-mediated activation of the ATM/cABL/YAP1 pathway, which directly regulates genes central to IBD pathogenesis and amenable to therapeutic intervention. This review summarizes the evidence correlating telomere dysfunction with IBD and colitis-associated cancer and proposes therapeutic opportunities for such inflammatory conditions targeting this newly identified pathway. Inflammatory bowel disease, mainly comprised of Crohn’s disease and ulcerative colitis, is a challenging condition associated with both genetic and non-genetic drivers. The mechanisms that drive IBD are not well understood, making it difficult to identify therapeutic targets of pathogenetic relevance . In courTelomeres are nucleoprotein structures at the ends of chromosomes that protect and maintain chromosomal integrity together with the shelterin protein complex. During cell division, chromosomes must be replicated; however, the replication machinery cannot fully copy to the very end of linear human chromosomes. Telomerase Reverse Transcriptase (TERT) and its RNA component (TERC) act in a coordinate manner to elongate telomeres to solve the end replication problem, where TERC provides the RNA template for telomere repeat synthesis . GeneticA systematic phenotypic and histological analysis of the telomere dysfunctional mouse model  revealed a significant inflammatory phenotype specific to the large intestine with remarkably negligible effect on the small intestine [in silico analysis of the promoter for pro-IL18 revealed various transcription factor binding sites. In particular YAP1 caught our attention given a recent report on a SNP associated with TEF binding element in IBD patient epithelium [In vivo treatment of telomere dysfunctional mice with inhibitors for YAP1 (verteporfin) or caspase-1 (Ac-YVAD) or with broad spectrum antibiotics (trimethoprim-sulfa) ameliorated disease burden and suppressed the ATM/YAP1/IL18 axis revealing cooperation between the two axes telomere dysfunction mediated DNA damage and the microbiome activated inflammasome pathway in driving inflammatory response and highlighting YAP1 as a common orchestrator. Interestingly, we found that DNA damage (irradiation) could also activate this pathway. This study also establishes the important role of intestinal epithelial cells in driving such diseases that may cooperate with the immune cells in exacerbating the disease and driving relapse. To summarize, telomere dysfunction can initiate an inflammatory response through DNA damage mediated activation of the YAP1/pro-IL18 axis in the presence of the gut microbiome , a condition known to be associated with telomere dysfunction and aid in identifying viable therapeutic targets for such chronic conditions with limited therapeutic options.


These findings change the field of FSH-FSHR biology, call for paradigm shift, explain FSHR expression on cancer cells in multiple organs and provide straightforward explanations for various existing conundrums including extragonadal expression of FSHR.Follicle stimulating hormone (FSH) is secreted by the anterior pituitary and acts on the germ cells indirectly through Granulosa cells in ovaries and Sertoli cells in the testes. Extragonadal action of FSH has been reported but is still debated. Adult tissues harbor two populations of stem cells including a reserve population of primitive, small-sized, pluripotent very small embryonic-like stem cells (VSELs) and slightly bigger, tissue-specific progenitors which include ovarian stem cells (OSCs) in ovaries, spermatogonial stem cells (SSCs) in testes, endometrial stem cells (EnSCs) in uterus and hematopoietic stem cells (HSCs) in the bone marrow. Data has accumulated in animal models showing FSHR expression on both VSELs and progenitors in ovaries, testes, uterus and bone marrow and eventually gets lost as the cells differentiate further. FSH exerts a direct action on the stem/progenitor cells via alternatively spliced FSHR-3 rather than the canonical FSHR-1. FSH stimulates VSELs to undergo asymmetrical cell divisions to self-renew and give rise to the progenitors that in turn undergo symmetrical cell divisions and clonal expansions followed by differentiation into specific cell types. Excessive self-renewal of VSELs results in cancer and this explains ubiquitous expression of embryonic markers including nuclear OCT-4 along with FSHR in cancerous tissues. Focus of this review is to compile published data to support this concept. FSHR expression in stem/progenitor cells was confirmed by immuno-fluorescence, Western blotting, in situ hybridization and by quantitative RT-PCR. Two different commercially available antibodies  were used to confirm specificity of FSHR expression along with omission of primary antibody and pre-incubation of antibody with immunizing peptide as negative controls. Western blotting allowed detection of alternatively spliced FSHR isoforms. Oligoprobes and primers specific for Fshr-1 and Fshr-3 were used to study these alternately-sliced isoforms by in situ hybridization and their differential expression upon FSH treatment by qRT-PCR. To conclude, stem/progenitor cells in adult tissues express FSHR and directly respond to FSH via FSHR-3.  Existing dogma that FSH action is limited to Granulosa cells in ovaries and Sertoli cells in the testes needs an urgent revision.Two populations of stem cells exist in multiple adult tissues including quiescent VSELs and slightly bigger, tissue-specific progenitors including OSCs in ovaries, SSCs in testes, EnSCs in the uterus, HSCs in the bone marrow and so on in other adult organs as well. These stem/progenitor cells express FSHR which eventually gets degraded as cells differentiate further into tissue-specific mature cell types. FSH acts directly on the VSELs that exist in all adult tissues including ovaries, testes, uterus and bone marrow. FSH exerts a direct effect on the stem/progenitor cells and promotes asymmetrical, symmetrical divisions and clonal expansion. FSH effects on extragonadal tissues including uterus and bone marrow stem/progenitor cells is clearly delineated in this review.In addition to the existing dogma that FSH exerts an indirect action on the germ cells via Sertoli cells in testes or granulosa cells in the ovaries, FSH directly affects testicular and ovarian stem cells.FSH action on the stem/progenitor cells is mediated via Fshr-3 which is alternately spliced, growth factor type-1 receptor that acts via calcium signaling and the ERK/MAPK pathway.Stem cells are involved in both endometrial regeneration and hematopoiesis in bone marrow and FSH has a potential role in both these processes.Expression of FSHR on VSELs and OSCs in the ovaries explains why commercially available ovarian cancer cell lines when treated with FSH do not show increase in cAMP. Gonadotropin theory with a potential role of stem cells in the OSE could possibly explain initiation of ovarian cancer.Our findings explain extragonadal expression of FSHR in the uterus. FSH exerts direct action on both endometrial and myometrial stem cells. Various uteropathies have a stem cell basis and this explains FSHR expression in clinical samples of endometriosis, endometrial cancers as well as myomas.Several folds increased Fshr-3 in testicular cancer samples is intriguing and suggests a role of FSH in initiating testicular cancer similar to gonadotropin theory that exists for ovarian cancer.It is textbook information, after almost 90 years of research since follicle-stimulating hormone (FSH) was first reported in 1930s, that FSH is secreted by the anterior pituitary and acts on granulosa cells in the ovarian pre-antral follicles and on Sertoli cells in the testes , 3. PublPresent review is a compilation of the work published since 2013 suggesting that FSHR are expressed on the tissue-resident stem/progenitor cells in multiple organs including bone marrow and reproductive tissues ovaries, testes and uterus. These results call for a paradigm shift in the field since evidently FSH exerts a direct action on the stem cells via Fshr-3 rather than the canonical Fshr-1 isoform. Tissue-resident stem cells that exist in adult tissues are the very small embryonic-like stem cells (VSELs) and were recently reviewed –8. FSHR It is widely believed and textbook information that FSH acts on Granulosa cells in the ovaries and Sertoli cells in the testes . HoweverWe published an article in 2012 . These rThese findings of FSH action on the OSE cells become more intriguing since ovarian stem cells are reported to reside in the OSE. Presence of stem cells in adult ovaries were initially reported by Tilly’s group  in adultIn a review describing various aspects of the ovarian stem cells , we haveStem cells located in sheep OSE cells, collected by gentle scraping, were found to express FSHR (besides stem cell markers) whereas the epithelial cells remained distinctly negative Fig.  24]. Ex. Ex24]. Immuno-localization studies were carried out further for FSHR expression in sheep ovarian sections as well as by confocal microscopy on sheep OSE cell smears Fig. . FSHR exFSH stimulates stem cells proliferation and clonal expansion. It was intriguing to note how stem cells story which was main focus of our lab research gradually got intertwined with FSH-FSHR biology. Further experiments revealed that when sheep OSE cell smears . The chemoablated ovaries were removed and cultured in vitro with and without FSH (5mIU) for 7 days and later studied for the effect of FSH on stem cell markers and proliferation was marked by studying BrdU uptake. An increased expression of stem/progenitor cells  was observed along with proliferation in OSE evidenced by BrdU uptake. This was accompanied by the formation of germ cell nests  and expression of Scp3 which was not detected in chemoablated ovaries without FSH treatment [Fshr-3 transcripts have been detected in mice OSE cells and not canonical Fshr-1 . Studiesreatment .Fig. 8EfThis is entirely a new and novel FSH-FSHR biology within the mammalian ovaries and delineated by our group for the first time. FSHR are expressed on the OSE cells, to be more precise on the stem cells that reside in the OSE whereas the epithelial cells remain distinctly negative. Since cancer involves selective expansion of stem cells, FSHR has been reported on ovarian cancer stem cells. FSH acts via Fshr-3 on the stem cells via ERK/MAPK to bring about proliferation of stem cells.To conclude, stem cells residing in the mouse, rabbit, marmoset, sheep and human ovarian surface epithelium express FSHR. Besides acting on the granulosa cells, FSH acts directly on the ovarian stem cells and stimulates them to undergo proliferation, asymmetrical, symmetrical divisions and clonal expansion to form germ cell nests and this action is mediated via Fshr3 in mice, sheep and human ovaries. FORKO mice discussed above  developeFSHR isoforms have been reported in sheep  and humaWe have recently reported a simple and robust protocol to enrich both the populations of stem cells from mice testes along with their detailed characterization . FSHR exTesticular stem cells smears were prepared from chemoablated testes. Fig. As shown in Fig. Further Western blotting experiment on proteins extracted from Sertoli cells and intact testes after FSH treatment when hybridized with Abcam FSHR antibody showed presence of 75 kDa band for canonical FSHR-1 in Sertoli cells but in intact testis, two bands of 75 and 40KDa were detected. 75 kDa band was not detected in intact testes after FSH treatment because as such the protein amount may be too less but we clearly observed the band in protein extracted from Sertoli cells. However, FSH treatment affected stem/progenitor cells and FSHR-3 and other isoforms showed increased expression and were detected by Western blot Fig. .Fig. 13WExpression of FSHR on the stem cells was also confirmed by in situ hybridization using oligoprobes. Both Fshr-1 and Fshr-3 were found expressed on the stem cells. FSH treatment resulted in active transcription of Fshr-3 on dividing stem cells compared to Fshr-1 mRNA Fig. .Fig. 14FExpression of Fshr1 and Fshr3 were also studied by qRT-PCR in different treatment groups Fig. . It was Fshr1 and Fshr3 was studied on D100 by qRT-PCR  or diethylstilbestrol (2 μg/pup/day on days 1–5) and later studies were undertaken on D100 of adult life. Treatment affected the testicular stem cells and resulted in pathologies including disrupted spermatogenesis, reduced sperm counts, infertility and tumor-like changes were observed in DES treated group . ExpressPCR Fig. . Fshr-3 The results compiled above are intriguing since FSHR expression is expected only on the Sertoli cells as per existing understanding . Resultsfunctional expression of FSHR in endometriotic lesions. FSHR is reported in uterine myomas also [Uterus is considered as an end organ for the steroid hormones estrogen (E) and progesterone (P) to act. These hormones are secreted by the ovaries under the influence of pituitary gonadotropins and thus it is generally believed that FSH exerts an indirect effect on the uterus. However, few reports have challenged this existing dogma in the field. Increased expression of FSHR was reported in the secretory phase human endometrium compared to proliferative phase , 60. Latmas also . This bomas also , 11 who Our group has reported two populations of stem cells including VSELs and slightly bigger EnSCs in adult mouse uterus and recently published a robust protocol to enrich them from mice uterus . VSELs sMyometrium was atrophied after bilateral ovariectomy and both perimetrium and myometrium were clearly visualized in the ovariectomized uterus along with small spherical stem cells Fig. , arrow. A careful scrutiny of the H&E stained sections of atrophied endometrium Fig.  after biFSH treatment exerted a marked effect on the endometrial epithelial cells which showed hypertrophy and hyperplasia Fig. . Small sUterine stem cells can be enriched by spinning at 1000 g and visualized after H&E staining . The steThe stem cells enriched from adult uterus were used to prepare smears and studied for expression of PCNA and FSHR Fig. . Two disTo conclude, two populations of stem cells exist in adult mouse uterus which express FSHR and FSH exerts direct effect on the uterine stem cells similar to in the ovaries and testes. Early differentiating progenitors express embryonic markers as well as FSHR. We have recently shown that neonatal endocrine disruption affects uterine stem cells differentiation and progenitors increase in numbers associated with increased FSHR expression . MoreoveHematopoietic system also harbors two populations of stem cells including VSELs and hematopoietic stem cells (HSCs) , 70, altFunctional studies were reported in vivo by our group on adult mice bone marrow showing a direct effect of FSH on hematopoiesis . TreatmeEffect of FSH treatment was also studied on stem cells in bone marrow cell smears. For this 5-FU treated mice were further treated with FSH (5 IU) on days 4 & 5 and sacrificed on Day 6. Bone marrow was flushed out and used to make cell smears Fig. . One couSimilarly, VSELs in mouse prostate are regulated by FSH and only Fshr3 is expressed in adult prostate and not the canonical Fshr1 (unpublished data by our group). These results are in contradiction with recent scRNAseq study  which neIn view of the research findings compiled in this review, it becomes imperative to appreciate a wider landscape for FSH action which is so far limited to Sertoli cells in testes and Granulosa cells in ovaries.


Sarcoidosis is a common disease with the incidence of cardiac involvement varying. Cardiac sarcoidosis should be kept on the differential when young patients present with acute heart failure, conduction abnormalities or new arrhythmia. Cardiac involvement in sarcoidosis must be diagnosed early and treated aggressively. Here we present a patient who presented with shortness of breath and was found to have significant heart failure with reduced ejection fraction caused by sarcoidosis with cardiac involvement. She was treated with optimization of medical therapy for heart failure and eventually required implantable cardioverter defibrillator (ICD) placement. Sarcoidosis is a rare inflammatory disorder of unknown etiology involving multi-organ systems. The cardiovascular system is affected rarely. A key feature of the disease is formation of noncaseating granulomas in the affected organs. In patients with systemic sarcoidosis, it is estimated that about 5% have symptomatic cardiac sarcoidosis (CS) . AdvanceA 40-year-old female with no prior medical history presented to her primary care physician (PCP) for shortness of breath (SOB) on exertion that had progressively worsened over the previous few months. She had a prior CT of her abdomen and it showed multiple pulmonary nodules at the lung bases. This led to further evaluation with CT chest which showed cardiomegaly and numerous scattered pulmonary nodules with the largest being 11 mm  of 30%, mild mitral and tricuspid regurgitation and moderate pulmonary hypertension Figure .One month later she presented to the emergency department for worsening heart failure symptoms including worsening shortness of breath and lower extremity edema. She was symptomatic with New York Heart Association (NYHA) class III heart failure symptoms. Her electrocardiogram (EKG) showed right bundle branch block (RBBB) and first-degree atrioventricular (AV) block Figure .Transthoracic echocardiogram was repeated, and her EF dropped to 14%. Cardiac MRI revealed severe cardiomyopathy with reduced EF estimated at 14% along with a patchy transmural fibrosis. A stress test did show abnormal left ventricular perfusion in a left circumflex distribution. Left heart catheterization was performed and showed no coronary artery disease. She then had a cardiac biopsy that was consistent with cardiac sarcoidosis showing dense interstitial fibrosis with non-necrotizing granulomas with mild inflammation composed of lymphocytes, macrophages and multinucleated giant cells, some containing asteroid bodies  technique. Recently published guidelines acknowledge LGE on Cardiac MRI as a diagnostic criterion .Cardiac involvement can be assessed with endomyocardial biopsy. However, due to patchy myocardial involvement in CS, diagnostic yield of endomyocardial biopsy is only about 20%. Thus, in most cases, diagnosis of CS is made when the patient demonstrates histologically confirmed extracardiac sarcoidosis and has non-histologic clues of CS . HoweverOral corticosteroid is standard treatment. Immunosuppressive combination therapy with methotrexate or azathioprine is considered second-line treatment. A combination of leflunomide and mycophenolate mofetil can be contemplated if the patient does not tolerate first-line treatment due to side effects .Early diagnosis and aggressive treatment of CS are essential in preventing the complications mentioned above, including sudden cardiac death . This case highlights the importance of including CS in the differential diagnosis of a young and otherwise healthy patient presenting with signs and symptoms of new onset heart failure and non-ischemic cardiomyopathy. Optimal medical management of heart failure should also be initiated. These patients are typically started on a prednisone taper and should strongly be considered for automated ICD when left ventricular dysfunction is present. In the case of persistent symptoms despite glucocorticoid therapy, immunosuppressive agents may be of benefit in symptom management.


Providing time and money to adult children may enhance perceived usefulness and consequently the subjective well-being (SWB) of older parents. However, non-reciprocal relationships with children and conflicts with leisure activities could negatively affect parents’ SWB. It was hypothesized that a substantial amount of support to children would be associated with lower SWB when older parents (a) had a low expectation of receiving long-term care from the children, and (b) were engaged in hobbies/learning activities. Life satisfaction and depressive symptoms measured as SWB were predicted based on the Generalized Estimating Equations, using panel data (2012-2017) with a nationwide representative sample of Japanese adults aged 60 years and older . Providing child-rearing support  of 30 hours or more per month was positively associated with SWB regardless of conditions (a) and (b). Hypothesis (b) was partially supported: providing financial support enhanced depressive symptoms among older adults with hobbies/learning.


Healthcare workers have experienced a significant burden of COVID-19 disease. COVID mRNA vaccines have shown great efficacy in prevention of severe disease and hospitalization due to COVID infection, but limited data is available about acquisition of infection and asymptomatic viral shedding.Fully vaccinated healthcare workers at a tertiary-care academic medical center in Omaha Nebraska who reported a household exposure to COVID-19 infection are eligible for a screening program in which they are serially screened with PCR but allowed to work if negative on initial test and asymptomatic. Serial screening by NP swab was completed every 5-7 days, and workers became excluded from work if testing was positive or became symptomatic. Of the 94 employees who were fully vaccinated at the time of the household exposure to COVID-19 infection, 78 completed serial testing and were negative. Sixteen were positive on initial or subsequent screening. Vaccine failure rate of 17.0% (16/94). Healthcare workers exposed to household COVID positive contactHigh risk household exposures to COVID-19 infection remains a significant potential source of infections in healthcare workers even after workers are fully vaccinated with COVID mRNA vaccines especially those with contact to positive domestic partners. All Authors: No reported disclosures


Chromosomal localization and estimated duplication times illustrated that genes in TIP4 formed a tandem array on the short arm of chromosome 1, resulting from pepper-specific expansion after its divergence with Solanaceae species. Transcriptome analyses under various abiotic stress conditions revealed that transport-, photosystem-, and thylakoid-related genes were generally enriched in expression clusters containing AQP genes in pepper. These results provide valuable genomic resources and insight into the evolutionary mechanism that generate genomic diversity of the AQP gene family in pepper.Aquaporins (AQPs) are known to have a vital role in water transport in all living organisms including agriculturally important crops, but a comprehensive genomic study of AQPs in pepper has not been implemented. Here, we updated previous gene annotations and generated a total of 259 AQP genes from five plants, including pepper. Phylogenetic and motif analyses revealed that a large proportion of pepper AQP genes belong to the specific subgroup of  Th. Th44]. Chromosomal distribution of the re-annotated AQP genes was visualized using MapChart v2.32 . Gene naWe estimated the divergence times of the AQP genes by first identifying duplicated gene pairs with DupGen_Finder pipeline . Next, tTo examine expression profiles of pepper AQP genes, we collected RNA-Seq data for leaf tissues under various abiotic stress  at different times generated from a previous study . We firsp value ≤ 0.05 were clustered using the Mfuzz program [https://www.biobam.com/omicsbox/ (accessed on 5 January 2021)), and enriched GO terms were determined by Fisher’s exact test .We performed expression clustering of the differentially expressed genes (DEGs) in the whole annotated pepper genome, including the newly annotated AQP genes. Expression clustering was calculated from log2 transformation of fold changes between the FPKM values measured in plants under stress conditions and unstressed control plants. The DEGs that had a  program  in an R


Physical activity may preserve cognitive functioning in older adults. This study examined associations between objectively measured physical activity and cognitive functioning. We recruited participants  with (N=26) and without (N=181) cognitive impairment from the University of Kansas Alzheimer’s Disease Center (KU-ADC). We collected cognitive data representing verbal memory, attention, and executive function. Accelerometers (Actigraph GT9X) were used to measure physical activity 24 hours a day for 7 days in a free-living environment. Physical activity was categorized as moderate to vigorous physical activity (MVPA) based on the Freedson (2011) Adult Vector Magnitude cut points. The association between cognitive functioning and total MVPA was evaluated by using multiple regression. We used factor analysis to create three composite scores  from 11 individual cognitive tests. Compared to verbal memory and attention, results indicate that total MVPA was more strongly associated with executive function . These findings are consistent with the literature suggesting that executive function in older adults may benefit from physical activity. Future research should investigate the physiological mechanisms by which MVPA benefits executive function in contrast to types of activity that might benefit verbal memory and attention.


In March 2020 the Centers for Medicare & Medicaid Services (CMS) announced restrictions on visitors and nonessential personnel in nursing homes to protect residents and facilities from SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) outbreaks. At the time, these measures were “temporary” but they continued well into 2021 resulting in a prolonged pause on in-person study activities in a palliative care clinical trial in 12 nursing homes. This session will address the impact of this pause and decisions made to overcome the potential failure of the trial. Of utmost importance was respecting nursing homes rapidly changing context, continued communication with the site leadership, transitioning to phone and video-conference study activities, and designing a retrospective study using existing data to answer a different but similar research question. As clinical researchers move forward implementing trials and complex interventions in nursing homes, we must use the lessons learned to design flexible trial protocols.


Benign anastomotic intestinal strictures are difficult to manage as there may be limited response to dilation. Fully covered self-expanding metal stents have been utilized in small case series; however, stent suturing is required due to the high risk of stent migration. Lumen-apposing metal stents (LAMS) are fully covered and have a novel dumbbell shape that prevents stent migration. Initial reports identify low migration rates and good clinical success rates. This is the first report of perforation following treatment of an ileosigmoid stricture in a 52-year-old female, three weeks after LAMS placement. Lumen-apposing metal stents (LAMS) are fully covered dumb-bell shape stents with flanged ends that were originally designed to drain pancreatic fluid collection . IntestiA 52-year-old female had a past medical history of ischemic pancolitis with transmural necrosis of the splenic flexure, status post subtotal colectomy with end ileostomy and subsequent takedown with ileosigmoid anastomosis. She presented with abdominal pain, nausea and vomiting. CT of the abdomen showed small bowel obstruction at the ileosigmoid anastomosis level Figure . FlexiblThree weeks later, the patient presented to the emergency room with severe abdominal pain. CT of the abdomen showed small bowel obstruction with multiple foci of intraperitoneal air concerning for perforation Figure  and a 6 Anastomotic strictures often present a management challenge. Endoscopic balloon dilation with or without steroid injection is typically the first-line therapy for gastrointestinal (GI) anastomotic strictures. Temporary placement of fully covered SEMS has been utilized for the treatment of upper intestinal tract anastomotic strictures; however, endoscopic suturing of the stent to the intestinal wall to prevent stent migration is required . FrequenYang et al identified a clinical success rate of 64% in a retrospective study of 30 patients treated with LAMS for benign gastrointestinal strictures . Irani eAlthough LAMS placements are well-tolerated procedures, they are not free of complications. Adverse events with LAMS included abdominal pain with subsequent ulceration identified at the time of stent removal from the site of gastrojejunal anastomotic strictures, in two of 25 patients, one to three weeks after stent placement . In anotAdditional adverse events reported in extensive review analysis of the literature available until 2017 were bleeding, vomiting, stent migration, death, and formation of new strictures proximal to the LAMS. In this review, they also included one study in which a perforation happened immediately after the procedure requiring surgery that they considered a technical failure . Here weManaging anastomotic strictures often present a great challenge. Endoscopic balloon dilation with or without steroid injection is first-line therapy. Temporary placing fully covered SEMS is another option; however, has its own limitations and complications. On the other hand, lumen-apposing metal stents (LAMS) provide another approach to manage gastrointestinal strictures. It has great therapeutic potential with its novel dumbbell shape; however, ulceration and perforation are major concerns that require further consideration of the optimal design of the stent design for this indication.


There is limited data on how endurance training can impact cardiac function and arrhythmogenesis. Intense endurance training has been associated with pathological remodeling of the right ventricle (RV) that can act as a substrate for fatal ventricular arrhythmias in older athletes. A previously healthy 63-year-old female marathon runner presented with symptomatic monomorphic ventricular tachycardia (VT) while exercising. Transthoracic echocardiogram (TTE) demonstrated no structural or functional abnormalities. Electrophysiology study (EPS) with three-dimensional mapping and programmed electrical stimulation was performed demonstrating significant scarring of the RV, including RV outflow tract and RV free wall. VT ablation was successfully performed. Unfortunately, exclusion of arrhythmogenic right ventricular cardiomyopathy (ARVC) was limited due to the lack of cardiac magnetic resonance imaging (MRI). Therefore, a single chamber implantable cardioverter defibrillator (ICD) was placed for secondary prevention. Currently, the clinical significance of exercise-induced ventricular arrhythmias in trained athletes without cardiovascular disease is still unknown. This case highlights the need for investigation with larger studies and longer follow up to help us understand the mechanism of exercise-induced scar formation and standardize our management regarding screening, exercise recommendations, and ICD placement in older athletes. There is limited data on how endurance training can impact cardiac function and arrhythmogenesis. Intense endurance exercise predominantly affects the right ventricle (RV) and may induce chronic structural changes and pathological remodeling that can act as a substrate for fatal ventricular arrhythmias . ManagemA previously healthy 63-year-old woman, who is an avid runner and still trains for and participates in marathons, presented with symptomatic wide complex tachycardia while exercising Figure . Her symOur case showed an unusual substrate for VT localized into the RVOT, inferior and free wall in an older athlete without a prior history of underlying structural heart disease or cardiovascular risk factors. Currently, the clinical significance of exercise-induced ventricular arrhythmias in trained athletes without apparent cardiovascular disease remains unknown. In contrast to our case, in a large consecutive cohort of young and highly trained athletes, ventricular tachyarrhythmias induced by exercise testing were found in 7% . The undFurthermore, this case suggests that EPS and 3D mapping can be important to guide us to the possible underlying mechanism and formulate further treatment plans for similar patients. This was illustrated by a recent study that described a novel clinical entity of an isolated subepicardial RVOT scar as a substrate for fast VT in high-level endurance athletes that can be successfully treated by ablation . Of inteOur observations posit a few important questions. Is endurance training the only exposure to form VT substrate or should we consider genetic testing in all these patients? What is the optimal method to screen asymptomatic older athletes who wish to continue high-intensity exercise? Is there any benefit to defibrillator therapy in this population? What are the best recommendations for symptomatic older athletes post ablation therapy? There is not enough data in the literature to answer these questions. This case highlights the need for investigation with larger studies and longer follow up to help us understand the mechanism of exercise-induced scar formation and standardize our management regarding screening, exercise recommendations, and ICD placement in older athletes.


Only about three million people in France have so far received at least one dose of a Covid-19 vaccine. Those aged over 75 are offered either Pfizer or Moderna vaccines in a vaccination center. Older people with pre-existing conditions can now get AstraZeneca's Covid-19 vaccine. We will provide detailed information later. The symposium has experts from 8 countries. We will use an interview and dialog format, instead of presentations (please refer to the program overview).