Novel neutralizing SARS-CoV-2-specific mAbs offer detection of RBD linear epitopes.

BACKGROUND: To stop the spread of the COVID-19 disease, it is crucial to create molecular tools to investigate and diagnose COVID-19. Current efforts focus on developing specific neutralizing monoclonal antibodies (NmAbs) elicited against the receptor-binding domain (RBD). METHODS: In the present study, recombinant RBD (rRBD) protein was produced in E. coli, followed by immunizing mice with purified rRBD. ELISA was applied to screen the hybridomas for positive reactivity with rRBD protein. The linear and conformational epitopes of the mAbs were subsequently identified using western blot. Finally, the reactivity, affinity, and neutralization activity of the purified mAbs were evaluated using ELISA. RESULTS: All mAbs exhibited similar reactivity trends towards both eukaryotic RBD and prokaryotic rRBD in ELISA. Among them, 2E7-D2 and 2B4-G8 mAbs demonstrated higher reactivity than other mAbs. Additionally, in western blot assays, these two mAbs could detect reducing and non-reducing rRBD, indicating recognition of linear epitopes. Notably, five mAbs effectively blocked rRBD- angiotensin-converting enzyme 2 (ACE2) interaction, while two high-affinity mAbs exhibited potent neutralizing activity against eukaryotic RBD. CONCLUSION: In the current study, we generated and characterized new RBD-specific mAbs using the hybridoma technique that recognized linear and conformational epitopes in RBD with neutralization potency. Our mAbs are novel candidates for diagnosing and treating SARS-CoV-2.

Engineering chimeric autoantibody receptor T cells for targeted B cell depletion in multiple sclerosis model: An in-vitro study.

Background: Recent evidence suggests that B cells and autoantibodies have a substantial role in the pathogenesis of Multiple sclerosis. T cells could be engineered to express chimeric autoantibody receptors (CAARs), which have an epitope of autoantigens in their extracellular domain acting as bait for trapping autoreactive B cells. This study aims to assess the function of designed CAAR T cells against B cell clones reactive to the myelin basic protein (MBP) autoantigen. Methods: T cells were transduced to express a CAAR consisting of MBP as the extracellular domain. experimental autoimmune encephalomyelitis (EAE) was induced by injecting MBP into mice. The cytotoxicity, proliferation, and cytokine production of the MBP-CAAR T cells were investigated in co-culture with B cells. Results: MBP-CAAR T cells showed higher cytotoxic activity against autoreactive B cells in all effector-to-target ratios compared to Mock T cell (empty vector-transduced T cell) and Un-T cells (un-transduced T cell). In co-cultures containing CAAR T cells, there was more proliferation and inflammatory cytokine release as compared to Un-T and Mock T cell groups. Conclusion: Based on these findings, CAAR T cells are promising for curing or modulating autoimmunity and can be served as a new approach for clone-specific B cell depletion therapy in multiple sclerosis.

COVID-19 pandemic impact on screening and diagnosis of prostate cancer: a systematic review.

INTRODUCTION: The healthcare level has been greatly affected by the COVID-19 pandemic compared with before the outbreak. This study aimed to review the impact of COVID-19 on the screening and diagnosis of prostate cancer (PCa). METHOD: The current study was designed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020. The keywords used to perform the search strategy were COVID-19 and prostate neoplasms. The four primary electronic databases comprising PubMed/MEDLINE, Web of Science, Scopus and Embase were searched until 1 September 2022. After screening and selecting studies through the EndNote software, data were extracted from each included study by two independent authors. All studies were evaluated according to Newcastle-Ottawa Scale quality assessment tool. RESULTS: As a result, 40 studies were included, categorised into two subjects. The majority of studies indicated a significant decrease in screening prostate-specific antibody tests during the COVID-19 pandemic compared with the pre-pandemic period, leading to delays in cancer diagnosis. The decrease in the number of diagnosed cases with low/intermediate stages to some extent was more than those with advanced stages. The PCa screening and diagnosis reduction ranged from nearly 0% to 78% and from 4.1% to 71.7%, respectively. CONCLUSION: Our findings showed that during the COVID-19 lockdown, delays in PCa screening tests and diagnoses led to the negative health effects on patients with PCa. Thus, it is highly recommended performing regular cancer screening to reduce the impact of the COVID-19 lockdown. PROSPERO REGISTRATION NUMBER: CRD42021291656.

Quantitative measurement of CA 15-3 cancer biomarker using an electrochemical aptasensor based on the electrodeposition of Au thin film on cauliflower-like rGO-MoS2 nanocomposite.

The present research was conducted to design and construct an electrochemical aptasensor for evaluating carbohydrate antigen 15-3 (CA15-3) as a biomarker for breast cancer. The aptasensor has been fabricated by a gold thin film (AuTF) electrodeposited on a cauliflower-like reduced graphene oxide-molybdenum sulfide nanocomposite (rGO-MoS2). The modified electrode's surface was used to immobilize the thiolated aptamer, which was subsequently treated with CA 15-3 antigen. The aptasensor fabrication process was assessed using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). This research also applied EIS to the quantitative measurement of CA 15-3 antigen by the proposed aptasensor. The interfacial charge transfer resistance (Rct) alteration before and after incubation of CA 15-3 by the immobilized aptamer was considered a signal for the quantitative measurement of CA 15-3. A linear concentration ranging from 5.0 to 200.0 U mL-1 with a detection limit of 3.0 × 10-1 U mL-1 was obtained for CA 15-3 using the EIS method. This designed aptasensor indicates satisfactory repeatability and stability, good selectivity, and high sensitivity. Moreover, clinical samples were assayed by the prepared aptasensor and compared with the ELISA method, yielding acceptable results. The recovery and relative standard deviation (RSD) of CA 15-3 in human serum samples were in the range 95.0 to 107.0% and 3.5 to 7.5%, respectively.

Effect of siRNA-mediated silencing of p53R2 gene on sensitivity of T-ALL cellsto Daunorubicin.

INTRODUCTION: p53R2 is a p53-inducible protein that, as one of the subunits of ribonucleotide reductase, plays an important role in providing dNTPs for DNA repair. Although p53R2 is associated with cancer progression, its role in T-cell acute lymphoblastic leukemia (T-ALL) cells is unknown. Therefore, in this study, we evaluated the effect of p53R2 silencing on double-stranded DNA breaks, apoptosis and cell cycle of T-ALL cells treated with Daunorubicin. METHODS: Transfection was performed using Polyethyleneimine (PEI). Gene expression was measured using real-time PCR and protein expression was evaluated using Western blotting. Cell metabolic activity and IC50 were calculated using MTT assay, formation of double-stranded DNA breaks was checked using immunohistochemistry for γH2AX, and cell cycle and apoptosis were evaluated using flow cytometry. RESULTS: We found that p53 silencing synergistically inhibited the growth of T-ALL cells by Daunorubicin. p53R2 siRNA in combination with Daunorubicin but not alone increases the rate of DNA double-strand breaks in T-ALL cells. In addition, p53R2 siRNA significantly increased Daunorubicin-induced apoptosis. p53R2 siRNA also caused a non-significant increase in cells in G2 phase. CONCLUSION: The results of the present study showed that silencing of p53R2 using siRNA can significantly increase the antitumor effects of Daunorubicin on T-ALL cells. Therefore, p53R2 siRNA has the potential to be used as an adjuvant therapy in combination with Daunorubicin in T-ALL.

Exosomal delivery of 7SK long non-coding RNA suppresses viability, proliferation, aggressiveness and tumorigenicity in triple negative breast cancer cells.

AIMS: RN7SK (7SK), a highly conserved non-coding RNA, serves as a transcription regulator via interaction with a few proteins. Despite increasing evidences which support the cancer-promoting roles of 7SK-interacting proteins, limited reports address the direct link between 7SK and cancer. To test the hypothetic suppression of cancer by overexpression of 7SK, the effects of exosomal 7SK delivery on cancer phenotypes were studied. MATERIALS AND METHODS: Exosomes derived from human mesenchymal stem cells were loaded with 7SK (Exo-7SK). MDA-MB-231, triple negative breast cancer (TNBC), cell line was treated with Exo-7sk. Expression levels of 7SK were evaluated by qPCR. Cell viability was assessed via MTT and Annexin V/PI assays as well as qPCR assessment of apoptosis-regulating genes. Cell proliferation was evaluated by growth curve analysis, colony formation and cell cycle assays. Aggressiveness of TNBCs was evaluated via transwell migration and invasion assays and qPCR assessment of genes regulating epithelial to mesenchymal transition (EMT). Moreover, tumor formation ability was assessed using a nude mice xenograft model. KEY FINDINGS: Treatment of MDA-MB-231 cells with Exo-7SK resulted in efficient overexpression of 7SK; reduced viability; altered transcription levels of apoptosis-regulating genes; reduced proliferation; reduced migration and invasion; altered transcription of EMT-regulating genes; and reduced in vivo tumor formation ability. Finally, Exo-7SK reduced mRNA levels of HMGA1, a 7SK interacting protein with master gene regulatory and cancer promoting roles, and its bioinformatically-selected cancer promoting target genes. SIGNIFICANCE: Altogether, as a proof of the concept, our findings suggest that exosomal delivery of 7SK may suppress cancer phenotypes via downregulation of HMGA1.

Evaluation of Apoptosis, Cell Proliferation and Cell Cycle Progression by Inactivation of the NEAT1 Long Noncoding RNA in a Renal Carcinoma Cell Line Using CRISPR/Cas9.

Background: Long noncoding RNAs (lncRNAs) play an important role in cellular mechanisms including transcription, translation, and apoptosis. NEAT1 is one of the essential types of lncRNAs in humans that can bind to active genes and modify their transcription. NEAT1 upregulation in various forms of cancer such as kidney cancer has been reported. Kidney cancer accounts for approximately 3% of all cancers worldwide and occurs almost twice as often in men as in women. Objectives: This study has been performed to knockout the NEAT1 gene using the CRISPR/Cas9 technique in the Renal Cell Carcinoma ACHN cell line and to evaluate its effects on cancer progression and apoptosis. Material and Methods: Two specific (single guide RNA (sgRNA) sequences for the NEAT1 gene were designed by CHOPCHOP software. These sequences were then cloned into plasmid pSpcas9, and recombinant vectors PX459-sgRNA1 and PX459-sgRNA2 were generated. ACHN cells were transfected using recombinant vectors carrying sgRNA1 and sgRNA2. The expression level of apoptosis-related genes was assessed by real-time PCR. Annexin, MTT and cell scratch tests were performed to evaluate the survival, proliferation, and migration of the knocked out cells, respectively. Results: The results have shown successful knockout of the NEAT1 gene in the cells of the treatment group. Expressions of P53, BAK, BAX and FAS genes in the cells of the treatment group (NEAT1 knockout) showed significant increases in expression compared to the cells of the control group (P <0.01). Additionally, decreased expression of BCL2 and survivin genes was observed in knockout cells compared to the control group (p <0.05). In addition, in the cells of the treatment group compared to control cells, a significant decrease in cell viability, ability to migrate and cell growth and proliferation was observed. Conclusion: Inactivation of the NEAT1 gene in ACHN cell line using CRISPR/Cas9 technology elevated apoptosis and reduced cell survival and proliferation which makes it a novel target for kidney cancer therapeutics.

TLR3 stimulation improves the migratory potency of adipose-derived mesenchymal stem cells through the stress response pathway in the melanoma mouse model.

BACKGROUND: Mesenchymal stem cells (MSCs) are utilized as a carrier of anti-tumor agents in targeted anti-cancer therapy. Despite the improvements in this area, there are still some unsolved issues in determining the appropriate dose, method of administration and biodistribution of MSCs. The current study aimed to determine the influence of toll-like receptor 3 (TLR3) stimulation on the potential of MSCs migration to the neoplasm environment in the mouse melanoma model. METHODS AND RESULTS: Adipose-derived MSCs (ADMSCs) were isolated from the GFP+ transgenic C57BL/6 mouse and treated with different doses (1 µg/ml and 10 µg/ml) of polyinosinic-polycytidylic acid, the related TLR3 agonist, at various time points (1 and 4 h). Following the treatment, the expression of targeted genes such as α4, α5, and ß1 integrins and TGF-ß and IL-10 anti-inflammatory cytokines was determined using real-time PCR. In vivo live imaging evaluated the migration index of the intraperitoneally (IP) injected treated ADMSCs in a lung tumor-bearing mouse (C57BL/6) melanoma model (n = 5). The presented findings demonstrated that TLR3 stimulation enhanced both migration of ADMSCs to the tumor area compared with control group (n = 5) and expression of α4, α5, and ß1 integrins. It was also detected that the engagement of TLR3 resulted in the anti-inflammatory behavior of the cells, which might influence the directed movement of ADMSCs. CONCLUSION: This research identified that TLR3 activation might improve the migration via the stimulation of stress response in the cells and depending on the agonist concentration and time exposure, this activated pathway drives the migratory behavior of MSCs.

Engineered anti-EGFRvIII targeted exosomes induce apoptosis in glioblastoma multiforme.

BACKGROUND: The drug delivery for treatment of glioblastoma multiforme (GBM) has been unsatisfactory mainly due to the drug resistance and low targeting efficiency. The selective targeting of GBM cells and using a cocktail of therapeutic agents to synergistically induce apoptosis may help enhance the drug delivery. METHODS: In this study, mesenchymal stem cells (MSCs) were engineered to produce exosomes, i.e. nanosized natural vesicles presenting anti-EGFRvIII (ab139) antibody on their surface while encapsulating two apoptosis-inducing gene therapy agents, i.e. cytosine deaminase (CDA) and miR-34a. Exosomes were characterised for their size, morphology, protein content and markers using dynamic light scattering and nanoparticle tracking analysis, cryo-TEM, Western blotting, respectively. miR-34a overexpression and Lamp2-ab139 protein expression were analysed using real-time PCR and flow cytometry, respectively. The armed exosomes were delivered to EGFRvIII positive GBM cells (U87EGFRvIII) as well as wild type cell line (U87), which was EGFRvIII negative. Apoptosis was quantified using flow cytometry in both EGFRvIII negative and positive U87 cells, receiving one gene therapy agent (either CDA or miR-34a) or a combination of them (CDAmiR). RESULTS: Spherical shape exosomes with an average diameter of 110 nm and a membrane thickness of 6.5 nm were isolated from MSCs. Lamp2-ab139 was successfully expressed on the surface of transfected cells and their secreted exosomes. Induced apoptosis rates was significantly higher in U87EGFRvIII cells than for U87 cells, indicating selectivity. The cell death rate was 6%, 9% and 12% in U87, 13%, 21% and 40% in U87EGFRvIII cells for CDA, miR-34a and CDAmiR treatment respectively, showing a higher apoptosis rate in the cells receiving both drugs compared to when single therapy was applied. CONCLUSION: The experimental findings clearly show the improved apoptosis rate of GBM cells when treated by engineered exosomes armed with two gene therapy agents and targeted towards EGFRvIII antigen.

Combination of androgen receptor inhibitor enzalutamide with the CDK4/6 inhibitor ribociclib in triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) that currently lacks specific therapy options. Thus, chemotherapy continues to be the primary treatment, and developing novel targets is a top clinical focus. The androgen receptor (AR) has emerged as a therapeutic target in a subtype of TNBC, with substantial clinical benefits shown in various clinical studies. Numerous studies have shown that cancer is associated with changes in components of the cell cycle machinery. Although cell cycle cyclin-dependent kinase (CDK) 4/6 inhibitors are successful in the treatment of ER-positive BC, they are not helpful in the treatment of patients with TNBC. We investigated the possibility of combining CDK4/6 inhibitor(ribociclib) with AR inhibitor(enzalutamide) in the AR-positive TNBC cell line. Ribociclib showed an inhibitory effect in TNBC cells. Additionally, we found that enzalutamide reduced cell migration/invasion, clonogenic capacity, cell cycle progression, and cell growth in AR-positive cells. Enzalutamide therapy could increase the cytostatic impact of ribociclib in AR+ TNBC cells. Furthermore, dual inhibition of AR and CDK4/6 demonstrated synergy in an AR+ TNBC model compared to each treatment alone.

Production and characterization of a new specific monoclonal antibody against A-isoform of SALL4: A novel emerging testicular cancer marker.

SALL4 transcription factor plays an important role to maintain the pluripotent and self-renewal of embryonic stem cells. It contributes to the growth of many cancers and embryonic development. With the exception of spermatogonia, SALL4 expression is silenced in most adult tissues after birth; nevertheless, it is re-expressed in a subset of different solid malignancies. SALL4 is a new, precise biomarker for testicular germ cell cancers that was just introduced. The whole isoform of SALL4 is called SALL4-A. Regarding the lack of antibody against human SALL4 isoforms, the pattern of expression, the role of each isoform remain unknown. Furthermore, in isoform specific evaluations, we aimed, for the first time, to produce and characterize mAb against human SALL4-A. Immunization of mice were performed with a selected 33-mer synthetic peptide of SALL4-A conjugated with KLH. Hybridoma cells were screened by ELISA for positive reactivity with SALL4-A peptide. From the ascites fluid of mice that had been injected with hybridoma cells, anti-SALL4-A mAbs were isolated using a protein G column. Reactivity of the mAbs was evaluated using the peptide and SALL4-A recombinant protein by ELISA and IHC on testicular cancer tissue as positive control, and normal kidney, stomach and prostate tissues as negative control. The produced mAb could well detect SALL4-A in testicular cancer tissues using IHC, while the reactivity was negative in normal kidney, stomach and prostate tissues. Using ELISA, the mAb affinity for the peptide and SALL4-A recombinant protein was assessed, and it was shown to be reasonably high. The mAb detected SALL4-A in nucleus and cytoplasm of several cancer cells and spermatogonia in testicular cancer tissue. In addition, it could recognize SALL4-A recombinant protein. Our produced monoclonal antibody against isoform-A of human SALL4 can specifically recognize SALL4-A using either IHC or ELISA. We hope that this mAb could help researchers in isoform-specific study of human SALL4.

Impact of COVID-19 pandemic on screening and diagnosis of patients with prostate cancer: a systematic review protocol.

INTRODUCTION: With the exponential progress of patients with COVID-19, unexpected restrictions were directed to limit SARS-CoV-2 dissemination and imposed health-system an entire reformation to diminish transmission risk. These changes likely have caused the full range of cancer screenings and diagnosis gaps. Regardless of the recommendations, prostate cancer (PCa) screening/diagnosis programmes were momentarily postponed. Prostate-specific antigen (PSA) testing has been an inexpensive, low-invasive and relatively precise means of detection for PCa screening that would improve the uncovering of any type of PCa. Unfortunately, a decrease in PSA screening would significantly decrease PCa detection, with non-negligible growth in PCa-specific death. This review is designed to improve our understanding of the impact of the COVID-19 pandemic on the screening and diagnosis of patients with PCa. METHODS AND ANALYSIS: This systematic review will be reported in accordant with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidance. A comprehensive search has been executed through five main electronic databases: PubMed/MEDLINE, Web of Science, Scopus, Embase and ProQuest until 1 March 2022. Besides, grey literature, preprint studies and references of included studies will be searched. The main keywords have been used to perform the search strategy: COVID-19, prostatic neoplasms. All the relevant studies that met the inclusion criteria will be screened, selected and then extracted data by two independent authors. The quality assessment of the included studies will be performed by the Newcastle-Ottawa Scale. In case of any disagreement between the two authors in selecting, extracting data and assessing the quality of included studies, it will be resolved via consensus and checked by the third author. ETHICS AND DISSEMINATION: As this study will be a systematic review without human participants' involvement, there will be no requirement for ethics approval. Findings will be presented at conferences and in a peer-reviewed journal. PROSPERO REGISTRATION NUMBER: CRD42021291656.

Saponin and fluorine-modified polycation as a versatile gene delivery system.

Despite the development of many novel carriers for the delivery of various types of genetic material, the lack of a delivery system with high efficiency and low cytotoxicity is a major bottleneck. Herein, low molecular weight polyethylenimine (PEI1.8k) was functionalized with saponin residues using phenylboronic acid (PBA) as an ATP-responsive cross-linker, and a fluorinated side chain to construct PEI-PBA-SAP-F polycation as a highly efficient delivery vector. This vehicle could transfect small plasmid DNA (∼3 kb) with outstanding efficiency into various cells, including HEK 293T, NIH3T3, A549, PC12, MCF7 and HT-29, as well as robust transfection of a large plasmid (∼9 kb) into HEK 293T cells. The carrier indicated good transfection efficacy even at high concentration of serum and low doses of plasmid. The use of green fluorescent protein (GFP) knock-out analysis demonstrated transfection of different types of CRISPR/Cas9 complexes (Cas9/sgRNA ribonucleoproteins RNP, plasmid encoding Cas9 plus sgRNA targeting GFP, Cas9 expression plasmid plusin vitro-prepared sgRNA). In summary, we report an effective PEI-PBA-SAP-F gene carrier with the appropriate lipophilic/cationic balance for biomedical applications.

Bioinformatic Investigation of Micro RNA-802 Target Genes, Protein Networks, and Its Potential Prognostic Value in Breast Cancer.

Background: An increasing number of studies have suggested that unveiling the molecular network of miRNAs may provide novel therapeutic targets or biomarkers. In this study, we investigated the probable molecular functions that are related to microRNA-802 (miR-802) and evaluated its prognostic value in breast cancer utilizing bioinformatics tools. Methods: PPI network, pathway enrichment and transcription factor analysis were applied to obtain hub genes among overlapping genes of four miRNA target prediction databases. Prognosis value assessments and expression analysis of hub genes using bioinformatics tools, as well as their literature validation were performed. Results: Our results showed a significant correlation of the miR-802 overexpression with poor patient survival rate (BC, p=2.7e-5). We determined 247 target genes significant for GO and KEGG terms. Analysis of TFs by TRUST showed that RUNX3, FOXO3, and E2F1 are possible TFs that regulate the miR-802 expression and target genes network. According to our analysis; 21 genes might have an important function in miR-802 molecular processes and regulatory networks. The result shows that among these 21 genes, 8 genes (CASC3, ITGA4, AGO3, TARDBP, MED13L, SF1, SNRPE and CRNKL1) are positively correlated with patient survival. Therefore these genes could be considered and experimentally evaluated as a prognostic biomarker for breast cancer. Conclusion: The comprehensive bioinformatics study on miR-802 target genes provided insight into miR-802 mediated pathways and processes. Furthermore, representing candidate target genes by prognostic values indicates the potential clinical application of miR-802 in breast cancer.

Poly-L-lysine/hyaluronan nanocarriers as a novel nanosystem for gene delivery.

The present research comes up with a novel DNA-loaded poly-L-lysine (PLL)/hyaluronan (HA) nanocarrier (DNA-loaded PLL/HA NCs) for gene delivery applications, as a promising candidate for gene delivery into diverse cells. A straightforward approach was employed to prepare such a nanosystem through masking DNA-loaded PLL molecules by HA. Fourier-transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), field emission-scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) were used to analyse the interaction of the molecules as well as the physicochemical properties of the NCs. The NCs showed a negative charge of -24 ± 3 mV, with an average size of 138 ± 6 nm, in an ellipsoid-shape with smooth surfaces. The DNA loading efficiency (LE) measured by DNA absorbance was around 95 %. The MTT assay showed that the developed NCs are non-toxic to the cells. Furthermore, the uptake of the DNA-loaded PLL/HA NCs by the human embryonic kidney (HEK)-293T cells was evaluated by a flow cytometry method, and demonstrated high potential cellular uptake over 90% for transferring the gene to HEK-293T cells at the optimised conditions. Therefore, the DNA-loaded PLL/HA NCs are the potent strategy for developing nanosystems for gene delivery applications.

Gold nanorods decorated polycaprolactone/cellulose acetate hybrid scaffold for PC12 cells proliferation.

Synthetic and natural polymers have recently received considerable attention due to the exclusive potential for supporting the regenerative cellular processes in peripheral nerve injuries (PNIs). Gold nanorods (GNRs) decorated polycaprolactone (PCL)/cellulose acetate (CA) nanocomposite (PCL/CA/GNR) were fabricated via electrospinning to improve PC12 cells attachment and growth or scaffold cues. Transmission electron microscopy (TEM) corroborated the GNR distribution (23 ± 2 nm length and 3/1 Aspect ratio) and suitable average dimension of 800 nm for the fibers; also, scanning electron microscopy (SEM) represented block-free and smooth fibers without perturbation. Because of gold nanorods incorporation, electrical conductivity of PCL/CA/GNR increased ~21%. Water contact angle data emphasized PCL/CA/GNR surface is more wettable that PCL/CA (<90° at 62 s). Real-time PCR technique (RT-PCR) demonstrated overexpression of ß-tubulin and microtubule-associated protein 2 (MAP2) on PCL/CA/GNR compared to PCL/CA composite. Additionally, evaluated of the maturation and neurogenic differentiation of PC12 cells emphasized overexpression of nestin and ß-tubulin by Immunocytochemistry staining onto PCL/CA/GNR in comparison to PCL/CA composite. Notably, these recently developed hybrid scaffolds could be considered for peripheral nerve injury (PNI) regeneration.

Stem cell therapy for COVID-19 pneumonia.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus is a highly contagious microorganism, and despite substantial investigation, no progress has been achieved in treating post-COVID complications. However, the virus has made various mutations and has spread around the world. Researchers have tried different treatments to reduce the side effects of the COVID-19 symptoms. One of the most common and effective treatments now used is steroid therapy to reduce the complications of this disease. Long-term steroid therapy for chronic inflammation following COVID-19 is harmful and increases the risk of secondary infection, and effective treatment remains challenging owing to fibrosis and severe inflammation and infection. Sometimes our immune system can severely damage ourselves in disease. In the past, many researchers have conducted various studies on the immunomodulatory properties of stem cells. This property of stem cells led them to modulate the immune system of autoimmune diseases like diabetes, multiple sclerosis, and Parkinson's. Because of their immunomodulatory properties, stem cell-based therapy employing mesenchymal or hematopoietic stem cells may be a viable alternative treatment option in some patients. By priming the immune system and providing cytokines, chemokines, and growth factors, stem cells can be employed to build a long-term regenerative and protective response. This review addresses the latest trends and rapid progress in stem cell treatment for Acute Respiratory Distress Syndrome (ARDS) following COVID-19.

The most common disease-causing mutation of factor XIII deficiency is corrected by CRISPR/CAS9 gene editing system.

Factor XIII (FXIII) deficiency is one of the most severe congenital bleeding disorders, with an estimated incidence of one person per one million. Patients with severe FXIII deficiency present a wide range of clinical manifestations, including umbilical cord bleeding, intracranial haemorrhage and recurrent miscarriages. Due to the high rate of life-threatening bleeding, primary prophylaxis is mandatory from the time of diagnosis. Although replacement therapy is the most common therapeutic choice, gene therapy remains the only curative option. In the present study, we assessed the efficacy of the clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR/Cas9) system in the correction of the most common FXIII disease-causing mutation (c.562 T > C). A dermal fibroblast was harvested from the human skin biopsy of a young patient with FXIII deficiency. Sanger sequencing was used to confirm the presence of c.562 T>C mutation in the patient and in the harvested fibroblasts. PX459 vector was digested with BbsI restriction enzyme, and after annealing and ligation of two 20-bp guide-RNAs (g-RNAs) close to the PAM (NGG) sequence, the constructed vectors were amplified in Escherichia coli Top 10. Transfection was performed by a nucleofector device, and DNA extraction was performed after puromycin selection and serial dilution from potentially transfected colonies. A 50-bp template oligonucleotide was used to aid homologous repair for correction of the underlying mutation and synonymous mutation as an internal control. The synonymous mutation (AAT to ACT) near the mutation site was used as internal control. Sanger sequencing was done in order to check the gene correction. The c.562 T > C mutation was detected in homozygote state in the primary fibroblasts of the patient and wild-type alleles were confirmed in the normal individual. Colony PCR and sequencing revealed successful cloning of the designed gRNAs. The detected mutation was corrected from a homozygote mutant state (c.562 T > C) to a homozygote wild type in transfected dermal fibroblasts of the patient. The control mutation, as an internal control, was also corrected in the same fibroblasts in the heterozygote manner. The result of the study shows that the CRISPR/CAS9 gene editing system is an effective tool for correction of point mutations in transfected fibroblasts of patients with congenital FXIII deficiency and represents a new, potentially curative, option.

Bioengineering of fibroblast-conditioned polycaprolactone/gelatin electrospun scaffold for skin tissue engineering.

BACKGROUND: Synthetic tissue engineering scaffolds has poor biocompatiblity with very low angiogenic properties. Conditioning the scaffolds with functional groups, coating with biological components, especially extracellular matrix (ECM), is an excellent strategy for improving their biomechanical and biological properties. METHODS: In the current study, a composite of polycaprolactone and gelatin (PCL/Gel) was electrospun in the ratio of 70/30 and surface modified with 1% gelatin-coating (G-PCL/Gel) or plasma treatment (P-PCL/Gel). The surface modification was determined by SEM and ATR-FTIR spectroscopy, respectively. The scaffolds were cultured with fibroblast 3T3, then decellularized during freeze-thawing process to fabricate a fibroblast ECM-conditioned PCL/Gel scaffold (FC-PCL/Gel). The swelling and degaradtion as well as in vitro and in vivo biocompatibility and angiogenic properties of the scaffolds were evaluated. RESULTS: The structure of the surface-modified G-PCL/Gel and P-PCL/Gel were unique and not changed compared with the PCL/Gel scaffolds. ATR-FTIR analysis admitted the formation of oxygen-containing groups, hydroxyl and carboxyl, on the surface of the P-PCL/Gel scaffold. The SEM micrographs and DAPI staining confirmed the cell attachment and the ECM deposition on the platform and successful removal of the cells after decellularization. P-PCL/Gel showed better cell attachment, ECM secretion and deposition after decellularization compared with G-PCL/Gel. The FC-PCL/Gel was considered as an optimized scaffold for further assays in this study. The FC-PCL/Gel showed increased hydrophilic behavior and cytobiocompatibility compared with P-PCL/Gel. The ECM on the FC-PCL/Gel scaffold showed a gradual degradation during 30 days of degradation time, as a small amount of ECM remained over the FC-PCL/Gel scaffold at day 30. The FC-PCL/Gel showed significant biocompatibility and improved angiogenic property compared with P-PCL/Gel when subcutaneously implanted in a mouse animal model for 7 and 28 days. CONCLUSIONS: Our findings suggest FC-PCL/Gel as an excellent biomimetic construct with high angiogenic properties. This bioengineered construct can serve as a possible application in our future pre-clinical and clinical studies for skin regeneration.

Evaluation of CRISPR/Cas9 System Effects on Knocking Out NEAT1 Gene in AGS Gastric Cancer Cell Line with Therapeutic Perspective.

AIM: Gastric cancer (GC) is one of the most common malignant tumors globally, with an increasing incidence rate. Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long non-coding RNA (lncRNAs) responsible for regulating cell cycle progression, apoptosis, cell growth, proliferation, and migration in various cells. The present survey was performed to assess the effects of NEAT1 gene knocking out by CRISPR/Cas9 system in human gastric cancer cells. METHODS: The CRISPR/Cas9 genome editing technique was used to knockout NEAT1 in AGS cells as a gastric cancer model. After the design and construction of the vector, transfection was performed. The expression levels of mRNA, the survival of cells, apoptosis, and cell migration were evaluated by real-time quantitative polymerase chain reaction, flow cytometry, and scratch wound. RESULTS: Degradation of NEAT1 by CRISPR/cas9 significantly suppressed the gene's expression rate, arrested cell cycle in the G0/G1 phase, and a significant reduction in cell number in the S phase (P < 0.05). Degradation of NEAT1 by CRISPR/cas9 also restrained the ability to migrate in transfected cells compared to the control group (P < 0.01). Knockout of NEAT1 via impact on miR-34a gene expression induced apoptosis of AGS cells (P < 0.05) with increasing in the FAS level and total apoptosis (P < 0.001). CONCLUSIONS: Findings suggest that NEAT1 plays a vital role in cellular mechanisms of GC's occurrence and can serve as a new treatment target in GC.