Loss of Y in regulatory T lymphocytes in the tumor micro-environment of primary colorectal cancers and liver metastases.

Male sex is a risk factor for colorectal cancer (CRC) with higher illness burden and earlier onset. Thus, we hypothesized that loss of chromosome Y (LOY) in the tumor micro-environment (TME) might be involved in oncogenesis. Previous studies show that LOY in circulating leukocytes of aging men was associated with shorter survival and non-hematological cancer, as well as higher LOY in CD4 + T-lymphocytes in men with prostate cancer vs. controls. However, nothing is known about LOY in leukocytes infiltrating TME and we address this aspect here. We studied frequency and functional effects of LOY in blood, TME and non-tumorous tissue. Regulatory T-lymphocytes (Tregs) in TME had the highest frequency of LOY (22%) in comparison to CD4 + T-lymphocytes and cytotoxic CD8 + T-lymphocytes. LOY score using scRNA-seq was also linked to higher expression of PDCD1, TIGIT and IKZF2 in Tregs. PDCD1 and TIGIT encode immune checkpoint receptors involved in the regulation of Tregs function. Our study sets the direction for further functional research regarding a probable role of LOY in intensifying features related to the suppressive phenotype of Tregs in TME and consequently a possible influence on immunotherapy response in CRC patients.

Drug repositioning for immunotherapy in breast cancer using single-cell analysis.

Immunomodulatory peptides, while exhibiting potential antimicrobial, antifungal, and/or antiviral properties, can play a role in stimulating or suppressing the immune system, especially in pathological conditions like breast cancer (BC). Thus, deregulation of these peptides may serve as an immunotherapeutic strategy to enhance the immune response. In this meta-analysis, we utilized single-cell RNA sequencing data and known therapeutic peptides to investigate the deregulation of these peptides in malignant versus normal human breast epithelial cells. We corroborated our findings at the chromatin level using ATAC-seq. Additionally, we assessed the protein levels in various BC cell lines. Moreover, our in-house drug repositioning approach was employed to identify potential drugs that could positively impact the relapse-free survival of BC patients. Considering significantly deregulated therapeutic peptides and their role in BC pathology, our approach aims to downregulate B2M and SLPI, while upregulating PIGR, DEFB1, LTF, CLU, S100A7, and SCGB2A1 in BC epithelial cells through our drug repositioning pipeline. Leveraging the LINCS L1000 database, we propose BRD-A06641369 for B2M downregulation and ST-4070043 and BRD-K97926541 for SLPI downregulation without negatively affecting the MHC complex as a significantly correlated pathway with these two genes. Furthermore, we have compiled a comprehensive list of drugs for the upregulation of other selected immunomodulatory peptides. Employing an immunotherapeutic approach by integrating our drug repositioning pipeline with single-cell analysis, we proposed potential drugs and drug targets to fortify the immune system against BC.

Size matters: the impact of nucleus size on results from spatial transcriptomics.

BACKGROUND: Visium Spatial Gene Expression (ST) is a method combining histological spatial information with transcriptomics profiles directly from tissue sections. The use of spatial information has made it possible to discover new modes of gene expression regulations. However, in the ST experiment, the nucleus size of cells may exceed the thickness of a tissue slice. This may, in turn, negatively affect comprehensive capturing the transcriptomics profile in a single slice, especially for tissues having large differences in the size of nuclei. METHODS: Here, we defined the effect of Consecutive Slices Data Integration (CSDI) on unveiling accurate spot clustering and deconvolution of spatial transcriptomic spots in human postmortem brains. By considering the histological information as reference, we assessed the improvement of unsupervised clustering and single nuclei RNA-seq and ST data integration before and after CSDI. RESULTS: Apart from the escalated number of defined clusters representing neuronal layers, the pattern of clusters in consecutive sections was concordant only after CSDI. Besides, the assigned cell labels to spots matches the histological pattern of tissue sections after CSDI. CONCLUSION: CSDI can be applied to investigate consecutive sections studied with ST in the human cerebral cortex, avoiding misinterpretation of spot clustering and annotation, increasing accuracy of cell recognition as well as improvement in uncovering the layers of grey matter in the human brain.

4-1BBL-containing leukemic extracellular vesicles promote immunosuppressive effector regulatory T cells.

Chronic and acute myeloid leukemia evade immune system surveillance and induce immunosuppression by expanding proleukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse, and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector proleukemic Tregs. Using mouse model of leukemia-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, and IL21R and included 2 distinct effector Treg (eTreg) subsets: CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive Tregs and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms.

Improvement of the performance of anticancer peptides using a drug repositioning pipeline.

The use of anticancer peptides (ACPs) as an alternative/complementary strategy to conventional chemotherapy treatments has been shown to decrease drug resistance and/or severe side effects. However, the efficacy of the positively-charged ACP is inhibited by elevated levels of negatively-charged cell-surface components which trap the peptides and prevent their contact with the cell membrane. Consequently, this decreases ACP-mediated membrane pore formation and cell lysis. Negatively-charged heparan sulphate (HS) and chondroitin sulphate (CS) have been shown to inhibit the cytotoxic effect of ACPs. In this study, we propose a strategy to promote the broad utilization of ACPs. In this context, we developed a drug repositioning pipeline to analyse transcriptomics data generated for four different cancer cell lines (A549, HEPG2, HT29, and MCF7) treated with hundreds of drugs in the LINCS L1000 project. Based on previous studies identifying genes modulating levels of the glycosaminoglycans (GAGs) HS and CS at the cell surface, our analysis aimed at identifying drugs inhibiting genes correlated with high HS and CS levels. As a result, we identified six chemicals as likely repositionable drugs with the potential to enhance the performance of ACPs. The codes in R and Python programming languages are publicly available in https://github.com/ElyasMo/ACPs_HS_HSPGs_CS. As a conclusion, these six drugs are highlighted as excellent targets for synergistic studies with ACPs aimed at lowering the costs associated with ACP-treatment.

Antibacterial effects assessment on some livestock pathogens, thermal stability and proposing a probable reason for different levels of activity of thanatin.

There is a continuing need to prevent the increasing use of common antibiotic and find the replacement to combat the drug/antibiotic resistant bacteria such as antimicrobial peptides (AMPs) such as thanatin peptide. In this study, recombinant thanatin peptide was expressed in the HEK293 cell line. Then the antimicrobial properties of this peptide on some poultry and farm animal's pathogen strains were assessed. The thermal-stability of thanatin was predicted in various temperatures through in silico analysis. Afterwards, according to Minimum Inhibitory Concentration (MIC) results, Escherichia coli and Pseudomonas aeruginosa were chosen to test the hypothesis of LptA/LptD-thanatin interaction, computationally. Relative amino acid sequences and crystallography structures were retrieved and missed tertiary structures were predicted. The interaction of thanatin with LptA and LptD of Escherichia coli and Pseudomonas aeruginosa were analyzed subsequently. The antibacterial activity of thanatin peptide was evaluated between 6.25 and 100 µg/mL using minimum inhibitory concentration. Also, the amounts of minimum bactericidal concentrations (MBC) were between 12.5 and 200 µg/mL. The bioinformatics analysis followed by the in vitro assessment, demonstrated that thanatin would be thermally stable in the body temperature of poultry and farm animals. Thanatin could penetrate to the outer membrane domain of LptD in Escherichia coli and it could block the transition path of this protein while the entrance of LptD in Pseudomonas aeruginosa was blocked for thanatin by extra residues in comparison with Escherichia coli LptD. In addition, the quality of interaction, with regard to the number and distance of interactions which leads to higher binding energy for thanatin and LptD of Escherichia coli was much better than Pseudomonas aeruginosa. But the site and quality of interaction for thanatin and LptA was almost the same for Escherichia coli and Pseudomonas aeruginosa. Accordingly, thanatin can prevent the assembly of LptA periplasmic bridge in both pathogens. The antibacterial and thermal stability of the thanatin peptide suggested that thanatin peptide might serve as a natural alternative instead of common antibiotics in the veterinary medicine. The outcome of this in silico study supports the MIC results. Therefore, a probable reason for different level of activity of thanatin against Escherichia coli and Pseudomonas aeruginosa might be the quality of LptA/LptD-thanatin interaction.

Applications of Genome-Wide Screening and Systems Biology Approaches in Drug Repositioning.

Modern drug discovery through de novo drug discovery entails high financial costs, low success rates, and lengthy trial periods. Drug repositioning presents a suitable approach for overcoming these issues by re-evaluating biological targets and modes of action of approved drugs. Coupling high-throughput technologies with genome-wide essentiality screens, network analysis, genome-scale metabolic modeling, and machine learning techniques enables the proposal of new drug-target signatures and uncovers unanticipated modes of action for available drugs. Here, we discuss the current issues associated with drug repositioning in light of curated high-throughput multi-omic databases, genome-wide screening technologies, and their application in systems biology/medicine approaches.

Current Status of COVID-19 Therapies and Drug Repositioning Applications.

The rapid and global spread of a new human coronavirus (SARS-CoV-2) has produced an immediate urgency to discover promising targets for the treatment of COVID-19. Drug repositioning is an attractive approach that can facilitate the drug discovery process by repurposing existing pharmaceuticals to treat illnesses other than their primary indications. Here, we review current information concerning the global health issue of COVID-19 including promising approved drugs and ongoing clinical trials for prospective treatment options. In addition, we describe computational approaches to be used in drug repurposing and highlight examples of in silico studies of drug development efforts against SARS-CoV-2.

Exploring membrane proteins of Leishmania major to design a new multi-epitope vaccine using immunoinformatics approach.

Leishmaniasis is one of the major global endemic diseases. Among all the different forms of the disease, cutaneous Leishmaniasis has the highest prevalence worldwide. Treatment with current drugs has not had a significant effect on the improvement of the disease. An attempt to replace an appropriate vaccine that can stimulate host cellular immunity and induce the response of Major histocompatibility complex I (MHCI) and Major histocompatibility complex II (MHCII) against Leishmania is essential. Vaccine production remains a challenge despite the use of different antigens for vaccination against Leishmania major. Hence, we were used the immunoinformatics approach to design a new multi-epitope vaccine against L. major using immunogenic outer membrane proteins. Helper T-lymphocyte (HTL) and Cytotoxic T lymphocyte (CTL) epitopes were predicted and for final confirmation of the selected epitopes, docking analysis, and molecular dynamics simulation was performed. Then, GDGDG linker and profilin adjuvant were added to enhance the immunity of vaccines. The designed vaccine was evaluated in terms of molecular weight, PI, immunogenicity, and allergenicity. Moreover, the secondary and three-dimensional structure of the final construct was identified. In silico cloning approach was carried out to improve expression of the vaccine construct. Finally, molecular docking, followed by molecular dynamic was performed to determine the interaction between multi-epitope vaccine and TLR11. We hope that the designed vaccine can be a good candidate for the development of cutaneous leishmaniasis vaccine. but its effectiveness should be assessed in vivo.

In silico and In vitro Investigation of a Likely Pathway for Anti-Cancerous Effect of Thrombocidin-1 as a Novel Anticancer Peptide.

BACKGROUND: Antimicrobial and antifungal activities of Thrombocidin-1 (TC-1) is shown previously, however,.the anti-cancerous feature of this peptide is still uncovered. OBJECTIVE: The objective is to evaluate anti-cancerous feature of recombinant TC-1. METHODS: In this study, based on the significant similarity of rTC-1 and IL-8 in case of coding sequence, tertiary structure, and also docking and molecular dynamic simulation (MD) results with CXCR1, a receptor which has positive correlation with different cancers, a likely pathway for anticancerous effect of rTC-1 was proposed. In addition, the coding sequence of TC-1+6xhistidine (rTC-1) was inserted into the pET22b(+) vector and cloned and expressed by E. coli BL21 and finally purified through nickel affinity column. Afterward, the retrieved rTC-1 was used in MTT assay against mouse colon adenocarcinoma, hepatocellular carcinoma, chondrosarcoma, mouse melanoma, and breast adenocarcinoma cell lines to investigate its probable anticancer application. RESULTS: Docking and MD simulation results showed that rTC-1 and IL-8 share almost the same residues in the interaction with CXCR1 receptor. Besides, the stability of the rTC-1_CXCR11-38 complex was shown during 100ns MD simulation. In addition, the successful expression and purification of rTC-1 depict an 8kD peptide. The IC50 results of MTT assay revealed that rTC-1 has cytotoxic effect on C26-A and SW1353 cancerous cell lines. CONCLUSION: Therefore, apart from probable anti-cancerous effect of rTC-1 on C26-A and SW1353 cell lines, this peptide may be able to mimic the anti-cancerous pathway of IL-8.

Assessment of a Novel Antimicrobial Peptide Against Clinically Isolated Animal Pathogens and Prediction of Its Thermal-Stability.

Multidrug resistance of pathogenic microorganisms is a common problem in the treatment of infections. Therefore, searching for new agents with antimicrobial activities appears to be essential. Thrombocidin-1 (TC-1) is an antimicrobial peptide (AMP) derived from platelets. The present study aimed to produce recombinant TC-1 (rTC-1) with His tag to evaluate its antimicrobial activity and also predict its thermal-stability through molecular dynamic (MD) simulations. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration of rTC-1 against bacterial isolates were determined. Considering the importance of thermal-stability of proteins in their therapeutic applications, thermal-stability of rTC-1 predicted through MD simulation during 25ns at two important temperatures including avian normal body temperature and water boiling temperature at sea level. MIC results revealed that rTC1 had the most and least potency against Salmonella enteritidis and Escherichia coli, respectively. The root-mean-square deviation of rTC-1 during 25ns MD simulations revealed that this protein is stable at avian normal body temperature (40°C) but might lose stability at water boiling temperature at sea level. rTC-1 as an AMP has a good potency against some bacterial pathogens especially Salmonella spp. and E. coli 0157:H7. rTC-1 can be used as an alternative for common antimicrobial agents used in clinical settings.

Recombinant production of a chimeric antimicrobial peptide in E. coli and assessment of its activity against some avian clinically isolated pathogens.

Over the last decades, poultry industry faced to the rapid emergence of multidrug-resistant bacteria as a global concern. Antimicrobial peptide (AMPs) known as potential antibiotic alternative and were considered as a new antimicrobial agent. Current methods of production and purification of AMPs have several limitations such as: costly, time-consuming and killing the producing host cells in recombinant form. In the present study, a chimeric peptide derived from camel lactoferrin was produced in Escherichia coli periplasmic space using a pET-based expression system and its antibacterial activity was determined on some avian pathogens in vitro. A carboxy-terminal polyhistidine tag was used for purification by Ni2+ affinity chromatography with an average yield of 0.42 g/L. The His-tagged chimeric peptide showed different range of antimicrobial activity against clinically isolated avian pathogens with low chicken blood hemolysis activity and high serum stability. Overall, the results of this investigation showed the recombinant chimeric peptide was successfully expressed in pET-based expression system and could be considered as a proper alternative for some currently used antibiotics in poultry industry and drugs veterinary medicine.