Creation of an in vitro model of GM1 gangliosidosis by CRISPR/Cas9 knocking-out the GLB1 gene in SH-SY5Y human neuronal cell line.

GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of ß-galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH-SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock-out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP-CHOP websites, and high-efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH-SY5Y cell line) by electroporation. After 48 h, GFP+ cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X-gal and Periodic acid-Schiff (PAS) staining, and qPCR were used to confirm the knock-out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock-out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH-SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non-transfected cell, X-gal staining resulted in no blue color in GLB1 gene knock-out cells indicating the absence of ß-galactosidase enzyme activity in these cells. Real-time PCR (qPCR) results confirmed the RNA-Seq analysis outcomes on the GEO data set and following the GLB1 gene knock-out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1-NEU1-CTSA complex gene was involved in suppressed proliferation and invasion ability of knock-out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH-SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease.

The emerging role of microRNA in regulating the mTOR signaling pathway in immune and inflammatory responses.

The mechanistic/mammalian target of rapamycin (mTOR) is considered to be an atypical protein kinase that plays a critical role in integrating different cellular and environmental inputs in the form of growth factors, nutrients and energy and, subsequently, in regulating different cellular events, including cell metabolism, survival, homeostasis, growth and cellular differentiation. Immunologically, mTOR is a critical regulator of immune function through integrating numerous signals from the immune microenvironment, which coordinates the functions of immune cells and T cell fate decisions. The crucial role of mTOR in immune responses has been lately even more appreciated. MicroRNAs (miRNAs) are endogenous, small, noncoding single-stranded RNAs that act as molecular regulators involved in multiple processes during immune cells development, homeostasis, activation and effector polarization. Several studies have recently indicated that a range of miRNAs are involved in regulating the phosphoinositide 3-kinase/protein kinase B/mTOR (PI3K/AKT/mTOR) signaling pathway by targeting multiple components of this signaling pathway and modulating the expression and function of these targets. Current evidence has revealed the interplay between miRNAs and the mTOR pathway circuits in various immune cell types. The expression of individual miRNA can affect the function of mTOR signaling to determine the cell fate decisions in immune responses through coordinating immune signaling and cell metabolism. Dysregulation of the mTOR pathway/miRNAs crosstalk has been reported in cancers and various immune-related diseases. Thus, expression profiles of dysregulated miRNAs could influence the mTOR pathway, resulting in the promotion of aberrant immunity. This review summarizes the latest information regarding the reciprocal role of the mTOR signaling pathway and miRNAs in orchestrating immune responses.

Genetic immunization against toxoplasmosis: A review article.

Toxoplasma gondii is a protozoan coccidian parasite belonging to Phylum Apicomplexa and is the causative agent of toxoplasmosis as a zoonotic disease around the world. It is one of the most important protozoa which is transmitted via various routes and infects several warm-blooded animals. The seroprevalence of T. gondii infection is high worldwide and leads to clinical, psychological, and economic problems. At present, available drug therapy for toxoplasmosis has severe side effects, so the development of new anti-toxoplasma drugs or effective vaccines is mandatory. Therefore, different measures have been taken for the development of anti-toxoplasmosis vaccines, and various studies have shown that DNA vaccines could be one of the most successful approaches against the intracellular parasite, T. gondii. Many of these studies have evaluated the efficacy of immunogenicity and different aspects of the DNA vaccines for toxoplasmosis including single genes or multi-gene plasmids with or without adjuvants. Most of the literature confirms that DNA vaccines containing different antigens of the toxoplasma parasite can induce suitable immune response and protection in acute or chronic toxoplasmosis. Therefore, in this review article, we aimed to discuss the current status of DNA vaccines as a new immunization method against toxoplasmosis.

The roles of lncRNAs and miRNAs in pancreatic cancer: a focus on cancer development and progression and their roles as potential biomarkers.

Pancreatic ductal adenocarcinoma (PDAC) is among the most penetrative malignancies affecting humans, with mounting incidence prevalence worldwide. This cancer is usually not diagnosed in the early stages. There is also no effective therapy against PDAC, and most patients have chemo-resistance. The combination of these factors causes PDAC to have a poor prognosis, and often patients do not live longer than six months. Because of the failure of conventional therapies, the identification of key biomarkers is crucial in the early diagnosis, treatment, and prognosis of pancreatic cancer. 65% of the human genome encodes ncRNAs. There are different types of ncRNAs that are classified based on their sequence lengths and functions. They play a vital role in replication, transcription, translation, and epigenetic regulation. They also participate in some cellular processes, such as proliferation, differentiation, metabolism, and apoptosis. The roles of ncRNAs as tumor suppressors or oncogenes in the growth of tumors in a variety of tissues, including the pancreas, have been demonstrated in several studies. This study discusses the key roles of some lncRNAs and miRNAs in the growth and advancement of pancreatic carcinoma. Because they are involved not only in the premature identification, chemo-resistance and prognostication, also their roles as potential biomarkers for better management of PDAC patients.

The application of peroxidase mimetic nanozymes in cancer diagnosis and therapy.

In recent decades, scholarly investigations have predominantly centered on nanomaterials possessing enzyme-like characteristics, commonly referred to as nanozymes. These nanozymes have emerged as viable substitutes for natural enzymes, offering simplicity, stability, and superior performance across various applications. Inorganic nanoparticles have been extensively employed in the emulation of enzymatic activity found in natural systems. Nanoparticles have shown a strong ability to mimic a number of enzyme-like functions. These systems have made a lot of progress thanks to the huge growth in nanotechnology research and the unique properties of nanomaterials. Our presentation will center on the kinetics, processes, and applications of peroxidase-like nanozymes. In this discourse, we will explore the various characteristics that exert an influence on the catalytic activity of nanozymes, with a particular emphasis on the prevailing problems and prospective consequences. This paper presents a thorough examination of the latest advancements achieved in the domain of peroxidase mimetic nanozymes in the context of cancer diagnosis and treatment. The primary focus is on their use in catalytic cancer therapy, alongside chemotherapy, phototherapy, sonodynamic therapy, radiation, and immunotherapy. The primary objective of this work is to offer theoretical and technical assistance for the prospective advancement of anticancer medications based on nanozymes. Moreover, it is anticipated that this will foster the investigation of novel therapeutic strategies aimed at achieving efficacious tumor therapy.

The Sero-Prevalence of Hepatitis B and C Viruses in Municipal Waste Collectors in Southwest of Iran.

Aims: The present study aimed to investigate the frequency of hepatitis B virus (HBV) and hepatitis C virus (HCV) serological markers among waste collectors in the municipality of Shiraz city, southwest of Iran, 2018. Settings and Design: In this cross-sectional study, a total of 385 waste collectors from all 10 districts of Shiraz city, southwest of Iran were enrolled. A questionnaire was used to gather occupational and demographic information as well as awareness about viral hepatitis. Methods and Material: Their blood samples were collected, and the sera were investigated for the presence of hepatitis B surface antigen (HBsAg), anti-HBs antibody, and anti-HCV antibody using enzyme-linked immunosorbent assay (ELISA). Results: All the participants were men with a mean age of 41 ± 8 years. Out of 385 participants, 6 (1.5%) subjects were positive for HBsAg, indicative of HBV infection. Moreover, 38 (9.9%) had a protective level of anti-HBs antibodies, while more than 90% had a low level of anti-HBs antibodies. All participants were negative for HCV antibodies. Conclusion: According to these findings, evaluation of HBsAg and anti-HBs levels is recommended in the healthy program of waste collector workers (WCWs). However, this occupation might not be a risk factor for the acquisition of HBV and especially HCV infections.

Screening and identification of potential biomarkers for pancreatic cancer: An integrated bioinformatics analysis.

Pancreatic cancer is one of the highly invasive and the seventh most common cause of death among cancers worldwide. To identify essential genes and the involved mechanisms in pancreatic cancer, we used bioinformatics analysis to identify potential biomarkers for pancreatic cancer management. Gene expression profiles of pancreatic cancer patients and normal tissues were screened and downloaded from The Cancer Genome Atlas (TCGA) bioinformatics database. The Differentially expressed genes (DEGs) were identified among gene expression signatures of normal and pancreatic cancer, using R software. Then, enrichment analysis of the DEGs, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, was performed by an interactive and collaborative HTML5 gene list enrichment analysis tool (enrichr) and ToppGene. The protein-protein interaction (PPI) network was also constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) database and ToppGenet web based tool followed by identifying hub genes of the top 100 DEGs in pancreatic cancer using Cytoscape software. Over 2000 DEGs with variable log2 fold (LFC) were identified among 34,706 genes. Principal component analysis showed that the top 20 DEGs, including H1-4, H1-5, H4C3, H4C2, RN7SL2, RN7SL3, RN7SL4P, RN7SKP80, SCARNA12, SCARNA10, SCARNA5, SCARNA7, SCARNA6, SCARNA21, SCARNA9, SCARNA13, SNORA73B, SNORA53, SNORA54 might distinguish pancreatic cancer from normal tissue. GO analysis showed that the top DEGs have more enriched in the negative regulation of gene silencing, negative regulation of chromatin organization, negative regulation of chromatin silencing, nucleosome positioning, regulation of chromatin silencing, and nucleosomal DNA binding. KEGG analysis identified an association between pancreatic cancer and systemic lupus erythematosus, alcoholism, neutrophil extracellular trap formation, and viral carcinogenesis. In PPI network analysis, we found that the different types of histone-encoding genes are involved as hub genes in the carcinogenesis of pancreatic cancer. In conclusion, our bioinformatics analysis identified genes that were significantly related to the prognosis of pancreatic cancer patients. These genes and pathways could serve as new potential prognostic markers and be used to develop treatments for pancreatic cancer patients.

A review: hemocompatibility of magnetic nanoparticles and their regenerative medicine, cancer therapy, drug delivery, and bioimaging applications.

Nanoparticles have demonstrated noteworthy advancements in the management of various complex medical conditions, particularly cancer. In any case, these particles still harbor the potential to improve medicate conveyance to challenging, hard-to-reach loci. The interactions that occur between nanoparticles and red blood cells during their journey throughout the human body, despite exposure to blood, are still not fully understood. Assessment of the ability of nanoparticles to integrate with blood, characterized as nanoparticle compatibility, has been consistently overlooked and undervalued in its import. This review article investigates the effect of nanoparticles on red blood cells, while examining the compatibility of nanoparticles through the angle of hemolysis. This article discusses the main roles of erythrocytes and also provides an informed interpretation of several mechanisms involved in the interaction of nanoparticles and erythrocytes. Throughout the review, significant emphasis is attributed to the investigation of hemocompatibility studies concerning newly designed nanoparticles to promote their successful translation into clinical application. This review article examines the compatibility of magnetic nanoparticles in various fields, including regenerative medicine, cancer therapy, bioimaging, and drug delivery. Our results show that the chemical composition of the nanoparticle surface is a determining factor in hemocompatibility performance and interaction with blood cells. The surface properties of nanoparticles, namely surface charge, geometry, porosity, and surface functionalities of polymers or specific functional groups, represent key determinants of hemocompatibility.

The use of nanoparticles in the treatment of infectious diseases and cancer, dental applications and tissue regeneration: a review.

The emergence of nanotechnology as a field of study can be traced back to the 1980s, at which point the means to artificially produce, control, and observe matter on a nanometer level was made viable. Recent advancements in technology have enabled us to extend our reach to the nanoscale, which has presented an unparalleled opportunity to directly target biomolecular interactions. As a result of these developments, there is a drive to arise intelligent nanostructures capable of overcoming the obstacles that have impeded the progress of conventional pharmacological methodologies. After four decades, the gradual amalgamation of bio- and nanotechnologies is initiating a revolution in the realm of disease detection, treatment, and monitoring, as well as unsolved medical predicaments. Although a significant portion of research in the field is still confined to laboratories, the initial application of nanotechnology as treatments, vaccines, pharmaceuticals, and diagnostic equipment has now obtained endorsement for commercialization and clinical practice. The current issue presents an overview of the latest progress in nanomedical strategies towards alleviating antibiotic resistance, diagnosing and treating cancer, addressing neurodegenerative disorders, and an array of applications, encompassing dentistry and tuberculosis treatment. The current investigation also scrutinizes the deployment of sophisticated smart nanostructured materials in fields of application such as regenerative medicine, as well as the management of targeted and sustained release of pharmaceuticals and therapeutic interventions. The aforementioned concept exhibits the potential for revolutionary advancements within the field of immunotherapy, as it introduces the utilization of implanted vaccine technology to consistently regulate and augment immune functions. Concurrently with the endeavor to attain the advantages of nanomedical intervention, it is essential to enhance the unceasing emphasis on nanotoxicological research and the regulation of nanomedications' safety. This initiative is crucial in achieving the advancement in medicine that currently lies within our reach.

A multi-epitope vaccine designed against blood-stage of malaria: an immunoinformatic and structural approach.

Malaria is a complex disease caused by parasites of the genus Plasmodium and is the leading cause of morbidity and mortality worldwide. The most severe form of malaria disease is caused by Plasmodium falciparum. Thus, a combination of different approaches is needed to control malaria. Resistance to first-line drugs and insecticides, on the other hand, makes the need for an effective vaccination more urgent than ever. Because erythrocyte parasites cause the most clinical symptoms, developing a vaccination for this stage of infection might be highly beneficial. In this research, we employed various bioinformatics methods to create an efficient multi-epitope vaccine that induces antibodies against the blood stage of malaria infection. For this purpose, we selected the malaria PfGARP protein as the target here. The B, HTL epitopes, and epitope conservation were predicted. The predicted epitopes (including 5 B and 5 HTL epitopes) were connected using suitable linkers, and the flagellin molecule was used as an adjuvant to improve its immunogenicity. The final construct vaccine with 414 amino acids long was designed. The vaccine's allergenicity, antigenicity, solubility, physicochemical characteristics, 2D and 3D structure modeling, molecular docking, molecular dynamics simulation, in silico cloning, and immunological simulation were tested. In silico immune simulation results showed significantly elevated IgG1 and IgM and T helper cells, INF γ, IL 2, and B-cell populations after the injection of the designed vaccine. These significant computational analyses indicated that our proposed vaccine candidate might activate suitable immune responses against malaria. However, in vitro and in vivo studies are essential for further validation.

B-Cell Epitope Mapping from Eight Antigens of Candida albicans to Design a Novel Diagnostic Kit: An Immunoinformatics Approach.

Invasive candidiasis is an emerging fungal infection and a leading cause of morbidity in health care facilities. Despite advances in antifungal therapy, increased antifungal drug resistance in Candida albicans has enhanced patient fatality. The most common method for Candida albicans diagnosing is blood culture, which has low sensitivity. Therefore, there is an urgent need to establish a valid diagnostic method. Our study aimed to use the bioinformatics approach to design a diagnostic kit for detecting Candida albicans with high sensitivity and specificity. Eight antigenic proteins of Candida albicans (HYR1, HWP1, ECE1, ALS, EAP1, SAP1, BGL2, and MET6) were selected. Next, a construct containing different immunodominant B-cell epitopes was derived from the antigens and connected using a suitable linker. Different properties of the final construct, such as physicochemical properties, were evaluated. Moreover, the designed construct underwent 3D modeling, reverse translation, and codon optimization. The results confirmed that the designed construct could identify Candida albicans with high sensitivity and specificity in serum samples of patients with invasive candidiasis. However, experimental studies are needed for final confirmation. Supplementary Information: The online version contains supplementary material available at 10.1007/s10989-022-10413-1.

Designing of a Novel Fusion Protein Vaccine Candidate Against Human Visceral Leishmaniasis (VL) Using Immunoinformatics and Structural Approaches.

Leishmaniasis is caused by an obligate intracellular protozoan parasite. The clinical forms of leishmaniasis differ from cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis (VL) which depend on the parasite species and the host's immune responses. There are significant challenges to the available anti-leishmanial drug therapy, particularly in severe forms of disease, and the rise of drug resistance has made it more difficult. Currently, no licensed vaccines have been introduced to the market for the control and elimination of VL. A potential target for use in candidate vaccines against leishmaniasis has been shown to be leishmania Kinetoplastid membrane protein-11 (KMP-11) antigen. In this study, we chose KMP-11 antigen as target antigen in our vaccine construct. In addition, B-type flagellin (fliC) was used as an adjuvant for enhancing vaccine immunogenicity. The GSGSGSGSGSG linker was applied to link the KMP-11 antigen and fliC (KMP-11-fliC) to construct our fusion protein. Bioinformatics approaches such as; 3D homology modeling, CTL, B-cell, MHC class I and II epitopes prediction, allergenicity, antigenicity evaluations, molecular docking, fast simulations of flexibility of docked complex and in silico cloning were employed to analysis and evaluation of various properties of the designed fusion construct. Computational results showed that our engineered structure has the potential for proper stimulation of cellular and humoral immune responses against VL. Consequently, it could be proposed as a candidate vaccine against VL according to these data and after verifying the efficacy of the candidate vaccine through in vivo and in vitro immunological tests.

Recombinant NS3 Protein Induced Expression of Immune Modulatory Elements in Hepatic Stellate Cells During Its Fibrotic Activity.

There is a growing body of studies that show the important role of NS3 protein from hepatitis C virus in fibrosis. However, mechanisms of the effects of this protein on immune modulation of stellate cells remain to be investigated. Herein, the effect of NS3 protein on the expression level of suppressor of cytokine signaling (SOCS)1/3 and interleukin-24 (IL-24)-related genes was investigated in hepatic stellate cell (HSC), LX-2. Recombinant NS3 protein was added to LX-2 HSC culture. Leptin and standard medium treatments were also included in experiments as positive and negative controls, respectively. Total RNA was extracted from each well at 6, 12, and 24 h after NS3 addition. The expression levels of the fibrotic (transforming growth factor beta 1 [TGF-ß], alpha-smooth muscle actin [α-SMA], and COL1A1), inflammatory (IL-6 and IL-24), IL-20R, IL-22R, and immunosuppressive genes (SOCS1 and SOCS3) were evaluated by real-time polymerase chain reaction (PCR). Recombinant NS3 protein induced activated phenotypes of LX-2 with a significant increase in the expression level of α-SMA COL1A1 (p < 0.0001) and TGF-ß. Moreover, this exposure led to a meaningful elevation in the expression of IL-6. Furthermore, compared with leptin (control), after the stellate cell treatment with NS3, SOCS1 and SOCS3 gene expression induced at a comparable level. Compared with the control sample, the NS3 protein significantly increased the expression level of IL-24 and its related receptors, IL-20R and IL-22R. This study not only confirmed the previously proved inflammatory and fibrotic effect of this protein but also indicated that high expression levels of SOCS1, SOCS3, and IL-24 have a significant effect on HSC activation. Therefore, these two molecules can be used as a potential therapeutic target candidate.

Green synthesis of silver nanoparticles toward bio and medical applications: review study.

Development of biologically inspired green synthesis of silver nanoparticles has attracted considerable worldwide attention in matter of medical science and disease treatment. Herein, the green synthesis of silver nanomaterials using organic green sources has been evaluated and discussed. These kinds of materials are widely used for treatment of antibiotic-resistant bacteria, cancer and etc due to their elegant properties compared with other chemical ways and drugs. Moreover, the outcome of green-based approaches were compared with chemical procedures and obtained data were examined via various analyses including UV-visible spectroscopy, scanning electron microscope (SEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscope (TEM), atomic force microscopy (AFM) and Fourier transforms infrared spectroscopy (FT-IR). In this study, variety of green methods were investigated to present a summary of recent achievements toward highlighting biocompatible nanoparticles, all of which can reduce the toxicity of nanoparticles, make them eco-friendly, reduce their side effects and decrease the production cost. The nature of these biological organisms also affect the structure, shape, size and morphology of synthesized nanoparticles.