The carotenoid pathway in plants has been altered through metabolic engineering to enhance their nutritional value and generate keto-carotenoids, which are widely sought after in the food, feed, and human health industries. In this study, the aim was to produce keto-carotenoids by manipulating the native carotenoid pathway in tobacco plants through chloroplast engineering. Transplastomic tobacco plants were generated that express a synthetic multigene operon composed of three heterologous genes, with Intercistronic Expression Elements (IEEs) for effective mRNA splicing. The metabolic changes observed in the transplastomic plants showed a significant shift towards the xanthophyll cycle, with only a minor production of keto-lutein. The use of a ketolase gene in combination with the lycopene cyclase and hydroxylase genes was a novel approach and demonstrated a successful redirection of the carotenoid pathway towards the xanthophyll cycle and the production of keto-lutein. This study presents a scalable molecular genetic platform for the development of novel keto-carotenoids in tobacco using the Design-Build-Test-Learn (DBTL) approach. This study corroborates chloroplast metabolic engineering using a synthetic biology approach for producing novel metabolites belonging to carotenoid class in industrially important tobacco plant. The synthetic multigene construct resulted in producing a novel metabolite, keto-lutein with high accumulation of xanthophyll metabolites. This figure was drawn using BioRender ( https://www.biorender.com ).

Gene editing technology is regarded as a good news to save patients with genetic diseases because of its significant function of specifically changing genetic information. From zinc-finger proteins to transcription activator-like effector protein nucleases gene editing tools are constantly updated. At the same time, scientists are constantly developing a variety of new gene editing therapy strategies, in order to promote gene editing therapy from various aspects and realize the maturity of the technology as soon as possible. In 2016, CRISPR-Cas9-mediated CAR-T therapy was the first to enter the clinical trial stage, indicating that the use of CRISPR-Cas system as the blade of the genetic lancet to save patients is officially on the schedule. The first challenge to achieve this exciting goal is to improve the security of the technology. This review will introduce the gene security issues faced by the CRISPR system as a clinical treatment tool, the current safer delivery methods and the newly developed CRISPR editing tools with higher precision. Many reviews summarize the means of improving the security of gene editing therapy and the comprehensive delivery method, while few articles focus on the threat of gene editing to the genomic security of the treatment target. Therefore, this review focuses on the risks brought by gene editing therapy to the patient genome, which provides a broader perspective for exploring and improving the security of gene editing therapy from two aspects of delivery system and CRISPR editing tools.

Resistance against glucocorticoids which are used to reduce inflammation and treatment of a number of diseases, including leukemia, is known as the first stage of treatment failure in acute lymphoblastic leukemia. Since these drugs are the essential components of chemotherapy regimens for ALL and play an important role in stop of cell growth and induction of apoptosis, it is important to identify genes and the molecular mechanism that may affect glucocorticoid resistance. In this study, we used the GSE66705 dataset and weighted gene co-expression network analysis (WGCNA) to identify modules that correlated more strongly with prednisolone resistance in type B lymphoblastic leukemia patients. The PPI network was built using the DEGs key modules and the STRING database. Finally, we used the overlapping data to identify hub genes. out of a total of 12 identified modules by WGCNA, the blue module was find to have the most statistically significant correlation with prednisolone resistance and Nine genes including SOD1, CD82, FLT3, GART, HPRT1, ITSN1, TIAM1, MRPS6, MYC were recognized as hub genes Whose expression changes can be associated with prednisolone resistance. Enrichment analysis based on the MsigDB repository showed that the altered expressed genes of the blue module were mainly enriched in IL2_STAT5, KRAS, MTORC1, and IL6-JAK-STAT3 pathways, and their expression changes can be related to cell proliferation and survival. The analysis performed by the WGCNA method introduced new genes. The role of some of these genes was previously reported in the resistance to chemotherapy in other diseases. This can be used as clues to detect treatment-resistant (drug-resistant) cases in the early stages of diseases.

The advantage of an increasing amount of bioinformatics data on leukemias intrigued us to explore the hot-spot mutation profiles and investigate the implications of those hot-spot mutations in patient survival. We retrieved somatic mutations and their distribution in protein domains through data analysis of The Cancer Genome Atlas and cBioPortal databases. After determining differentially expressed mutant genes related to leukemia, we further conducted principal component analysis and single-factor Cox regression analyses. Moreover, survival analysis was performed for the obtained candidate genes, followed by a multi-factor Cox proportional hazard model method for the impacts of the candidate genes on the survival and prognosis of patients with leukemia. At last, the signaling pathways involved in leukemia were investigated by gene set enrichment analysis. There were 223 somatic missense mutation hot-spots identified with pertinence to leukemia, which were distributed in 41 genes. Differential expression in leukemia was witnessed in 39 genes. We found a close correlation between seven genes and the prognosis of leukemia patients, among which, three genes could significantly influence the survival rate. In addition, among these three genes, CD74 and P2RY8 were highlighted due to close pertinence with survival conditions of leukemia patients. Finally, data suggested that B cell receptor, Hedgehog, and TGF-beta signaling pathways were enriched in low-hazard patients. In conclusion, these data underline the involvement of hot-spot mutations of CD74 and P2RY8 genes in survival status of leukemia patients, highlighting their as novel therapeutic targets or prognostic indicators for leukemia patients. Summary of Graphical Abstract: We identified 223 leukemia-associated somatic missense mutation hotspots concentrated in 41 different genes from 2297 leukemia patients in the TCGA database. Differential analysis of leukemic and normal samples from the TCGA and GTEx databases revealed that 39 of these 41 genes showed significant differential expression in leukemia. These 39 genes were subjected to PCA analysis, univariate Cox analysis, survival analysis, multivariate Cox regression analysis, GSEA pathway enrichment analysis, and then the association with leukemia survival prognosis and related pathways were investigated.

The effector proteins produced by plant pathogens are one of the essential components of host-pathogen interaction. Despite being important, most of the effector proteins remain unexplored due to the diversity in their primary sequence generated by the high selection pressure of the host immune system. However to maintain the primary function in the infection process, these effectors may tend to maintain their native protein fold to perform the corresponding biological function. In the present study, unannotated candidate secretory effector proteins of sixteen major plant fungal pathogens were analyzed to find the conserved known protein folds using homology, ab initio, and Alpha Fold/Rosetta Fold protein dimensional (3D) structure approaches. Several unannotated candidate effector proteins were found to match various known conserved protein families potentially involved in host defense manipulation in different plant pathogens. Surprisingly a large number of plant Kiwellin proteins fold like secretory proteins (> 100) were found in studied rust fungal pathogens. Many of them were predicted as potential effector proteins. Furthermore, template independent modelling using Alpha Fold/Rosetta Fold analysis and structural comparison of these candidates also predicted them to match with plant Kiwellin proteins. We also found plant Kiwellin matching proteins outside rusts including several non-pathogenic fungi suggesting the broad function of these proteins. One of the highest confidently modeled Kiwellin matching candidates effectors, Pstr_13960 (97.8%), from the Indian P. striiformis race Yr9 was characterized using overexpression, localization, and deletion studies in Nicotiana benthamiana. The Pstr_13960 suppressed the BAX-induced cell death and localized in the chloroplast. Furthermore, the expression of the Kiwellin matching region (Pst_13960_kiwi) alone suppressed the BAX-induced cell death in N. benthamiana despite the change of location to the cytoplasm and nucleus, suggesting the novel function of the Kiwellin core fold in rust fungi. Molecular docking showed that Pstr_13960 can interact with plant Chorismate mutases (CMs) using three loops conserved in plant and rust Kiwellins. Further analysis of Pstr_13960 showed to contain Intrinsically disordered regions (IDRs) in place of the N-terminal β1/β2 region found in plant Kiwellins suggesting the evolution of rust Kiwellins-like effectors (KLEs). Overall, this study reports the presence of a Kiwellin protein-like fold containing a novel effector protein family in rust fungi depicting a classical example of the evolution of effectors at the structure level as Kiwellin effectors show very low significant similarity to plant Kiwellin at the sequence level.

An increasing number of studies reveal the deleterious effects of isoflurane (Iso) exposure during pregnancy on offspring cognition. However, no effective therapeutic strategy for Iso-induced deleterious effects has been well developed. Angelicin exerts an anti-inflammatory effect on neurons and glial cells. This study investigated the roles and mechanism of action of angelicin in Iso-induced neurotoxicity in vitro and in vivo. After exposing C57BL/6 J mice on embryonic day 15 (E15) to Iso for 3 and 6 h, respectively, neonatal mice on embryonic day 18 (E18) displayed obvious anesthetic neurotoxicity, which was revealed by the elevation of cerebral inflammatory factors and blood-brain barrier (BBB) permeability and cognitive dysfunction in mice. Angelicin treatment could not only significantly reduce the Iso-induced embryonic inflammation and BBB disruption but also improve the cognitive dysfunction of offspring mice. Iso exposure resulted in an increase of carbonic anhydrase (CA) 4 and aquaporin-4 (AQP4) expression at both mRNA and protein levels in vascular endothelial cells and mouse brain tissue collected from neonatal mice on E18. Remarkably, the Iso-induced upregulation of CA4 and AQP4 expression could be partially reversed by angelicin treatment. Moreover, GSK1016790A, an AQP4 agonist, was used to confirm the role of AQP4 in the protective effect of angelicin. Results showed that GSK1016790A abolished the therapeutic effect of angelicin on Iso-induced inflammation and BBB disruption in the embryonic brain and on the cognitive function of offspring mice. In conclusion, angelicin may serve as a potential therapeutic for Iso-induced neurotoxicity in neonatal mice by regulating the CA4/AQP4 pathway.

Several studies have elucidated the pivotal function that long noncoding RNAs (lncRNAs) exerted on the initiation and development of various human carcinomas, encompassing non-small cell lung cancer (NSCLC). In spite of the fact that lncRNA MAPKAPK5 antisense RNA 1 (MAPKAPK5-AS1) has already been investigated by researchers and confirmed to play oncogenic roles in colorectal cancer, the underlying regulatory function of MAPKAPK5-AS1 in NSCLC cells still remain unclear. In our research, we found that MAPKAPK5-AS1 was expressed at high levels in NSCLC cells. Biological functional assays unclosed that downregulation of MAPKAPK5-AS1 repressed proliferative and migratory capacities whereas promoted apoptotic level in NSCLC cells. Molecular mechanism experiments confirmed that, in NSCLC cells, MAPKAPK5-AS1 combined with miR-515-5p and negatively modulated miR-515-5p expression level. Besides, calcium-binding protein 39 (CAB39) expression level was verified to be negatively modulated by miR-515-5p whereas positively modulated by MAPKAPK5-AS1 in NSCLC cells. Furthermore, rescued-function assays disclosed that inhibited miR-515-5p expression or overexpressed CAB39 could restore the suppressive influence of MAPKAPK5-AS1 silence on NSCLC progression. In summary, MAPKAPK5-AS1 upregulates CAB39 expression level to facilitate NSCLC progression by sequestering miR-515-5p, providing promising biomarkers for NSCLC treatment.

The occurrence of allergy, a type I hypersensitivity reaction, is rising exponentially all over the world. Sometimes, allergy proves to be fatal for atopic patients, due to the occurrence of anaphylaxis. This study is aimed to find an anti-allergic agent that can inhibit the binding of IgE to Human High Affinity IgE Receptor (FCεRI), thereby preventing the degranulation of mast cells. A considerable number of potential anti-allergic compounds were assessed for their inhibitory strength through ADMET studies. AUTODOCK was used for estimating the binding energy between anti-allergic compounds and FCεRI, along with the interacting amino acids. The docked pose showing favorable binding energy was subjected to molecular dynamics simulation study. Marrubiin, a diterpenoid lactone from Lamiaceae, and epicatechin-3-gallate appears to be effective in blocking the Human High Affinity IgE Receptor (FCεRI). This in-silico study proposes the use of marrubiin and epicatechin-3-gallate, in the downregulation of allergic responses. Due to the better inhibition constant, future direction of this study is to analyze the safety and efficacy of marrubiin in anti-allergic activities through in-vivo clinical human trials.

Heterologous functional expression of the recombinant lipases is typically a bottleneck due to the expression in the insoluble fraction as inclusion bodies (IBs) which are in inactive form. Due to the importance of lipases in various industrial applications, many investigations have been conducted to discover suitable approaches to obtain functional lipase or increase the expressed yield in the soluble fraction. The utilization of the appropriate prokaryotic and eukaryotic expression systems, along with the suitable vectors, promoters, and tags, has been recognized as a practical approach. One of the most powerful strategies to produce bioactive lipases is using the molecular chaperones co-expressed along with the target protein's genes into the expression host to produce the lipase in soluble fraction as a bioactive form. The refolding of expressed lipase from IBs (inactive) is another practical strategy which is usually carried out through chemical and physical methods. Based on recent investigations, the current review simultaneously highlights strategies to express the bioactive lipases and recover the bioactive lipases from the IBs in insoluble form.

Neisseria gonorrhoeae (NG) is the second most common bacterial sexually transmitted infection (STI) worldwide. Gonorrhoea is a very serious infection because if untreated, it can lead to significant ramifications to reproductive, maternal, & newborn health and increase transmission of HIV. Infections are very often asymptomatic and symptoms when present manifest differently in men and women. The cornerstone of gonorrhoea control is to assure rapid diagnosis and prompt treatment of patients to prevent the onward spread of infection. The resource-rich settings are utilizing nucleic acid amplification tests (NAATs) for diagnosis, whereas resource-limited settings like ours where laboratory infrastructure is lacking, reliance is placed on syndromic approach. In view of the limitations of each, there is a compelling need for development of a point of care test (POCT). Aptamers offer such potential. These are short oligonucleotides that bind to its target with high affinity and specificity and therefore can be maneuvred for use in diagnostics. In this study, we performed live cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) to select 12 single-stranded DNA (ssDNA) aptamers that bind strongly to a cocktail of Neisseria gonorrhoeae strains, with Kd values ranging from 8.58 to 596 nM. Gold nanoparticle (GNP) assay revealed that one of the aptamers, E8 19 was highly specific for Neisseria gonorrhoeae (Kd = 24.5 nM). More importantly, it did not demonstrate any binding to Neisseria meningitidis and commensal Neisseria sp. The identified aptamer holds much promise for the development of a rapid test for diagnosis of gonorrhoea.

Non-coding RNAs, including long-chain non-coding RNA (lncRNA) and micro-RNA (miRNA), have been implicated in osteoporosis (OP) progression by regulating osteoblast-dependent bone metabolism. Herein, we investigated whether LINC01234, miR-513a-5p, and AOX1 regulate osteogenic differentiation and proliferation of human bone marrow mesenchymal stem cells (hMSCs). The expression of LINC01234, miR-513a-5p, and AOX1 was monitored using RT-qPCR or western blotting. Cell proliferation was assessed using a CCK8 assay. Alkaline phosphatase activity (ALP) and alizarin red dye staining were performed to determine osteogenic differentiation. Furthermore, the expression of osteoblast differentiation markers, such as ALP, BMP1 (bone morphogenetic protein 1), osteocalcin (OCN), and osteopontin (OPN), was determined by RT-qPCR. Luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to verify the interplay between miR-513a-5p and LINC01234 or AOX1. Compared with the plasma of healthy controls, LINC01234 and AOX1 were highly expressed in the plasma of patients with OP, whereas miR-513a-5p showed low expression. In contrast, LINC01234 and AOX1 expression displayed a gradual decrease in induced differentiated hMSCs, while miR-513a-5p expression was upregulated with induction time. The predicted binding sites between miR-513a-5p and LINC01234 or AOX1 were verified by luciferase reporter and RIP assays. LINC01234 silencing induced osteogenic differentiation and proliferation in vitro, and miR-513a-5p silencing blunted osteogenic differentiation and proliferation modulated by LINC01234. AOX1 silencing caused by miR-513a-5p enhances osteogenic proliferation and differentiation. LINC01234 sponging of the miR-513a-5p/AOX1 axis impeded the osteogenic differentiation of hMSCs, favoring OP progression.

Circular RNAs (circRNAs) possess important functions in cervical carcinogenesis by operating as competing endogenous RNAs (ceRNAs). Our preliminary bioinformatics predicted the potential circ_0000212/microRNA (miR)-1236-3p/gremlin 1 (GREM1) ceRNA crosstalk. Thus, we further elucidated whether the novel ceRNA crosstalk can participate in cervical cancer development. Circ_0000212, miR-1236-3p and GREM1 were quantified by real-time quantitative polymerase chain reaction (qPCR) and immunoblotting. 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry, and tube formation assay were performed to assess cell proliferation, apoptosis and tube formation, respectively. Transwell assay was used to detect cell migration and invasion. Mouse xenografts were established to evaluate the role of circ_0000212 in vivo. Dual-luciferase reporter assay was performed to verify the direct relationship between miR-1236-3p and circ_0000212 or GREM1. Circ_0000212 expression was elevated in human cervical cancer. Silencing of endogenous circ_0000212 hindered cancer cell proliferation, motility and invasion and induced apoptosis, as well as diminished the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Circ_0000212 silencing also weakened tumor growth in vivo. Mechanistically, circ_0000212 directly bound to miR-1236-3p, and downregulation of miR-1236-3p reversed these effects of circ_0000212 silencing on cell malignant phenotypes and HUVEC tube formation. GREM1 was a direct miR-1236-3p target, and its expression was regulated by circ_0000212 through miR-1236-3p. Moreover, miR-1236-3p upregulation impeded cancer cell malignant phenotypes and HUVEC tube formation by targeting GREM1. Our findings identify a novel ceRNA regulatory network, circ_0000212/miR-1236-3p/GREM1 axis, in cervical carcinogenesis, and provide potential targets that can be explored for therapeutic interventions.

Due to its nutritional value and oil, soybean (Glycine max L.) became an economic crop in India and worldwide. The current study investigated the effect of forest-associated plant growth-promoting rhizobacteria (PGPR) on soybean yield and grain nutrient content. Five potential bacteria were used in this study based on their PGPR traits. The pot assay result with two crops (soybean and chickpea) confirmed the growth promotion activity of the two strains (Bacillus subtilis MpS15 and Paraburkholderia sabiae NvS21). The result showed significant (p < 0.05) enhancement in plant length and biomass with the seed treatment with strains (MpS15 and NvS21) compared to the control. Later both biocompatible potential strains were used in field experiments as individuals and consortia. Seed treatment of consortia significantly improves the nodulation and photosynthetic content more than individual treatments and control. Compared to the control, the co-inoculation of MpS15 and NvS21 increased soybean grain, straw yield, and grain NPK contents. Interestingly, soil parameters (organic carbon, available NPK) showed a strong correlation (p < 0.05) with plant parameters and nutrient uptake. Overall, our study provides strong relationships between soil parameters, microbial inoculum as consortia, and soybean performance, and these strains may be utilized as bioinoculant in future.

Ischemia/reperfusion (I/R) injury is the leading cause of death following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). γδT cells are suggested to aggravate blood-brain barrier (BBB) injury in various pathological processes. We herein investigate the effects of γδT cells inhibitor (UC7-13D5) against I/R injury post-CA/CPR. C57BL/6 mice were subjected to CA through injection of KCL (70 μL of 0.5 mol/L) and cessation of mechanical ventilation followed by CPR. Flow cytometry was performed to measure the proportion of CD3-positive cells after intraperitoneal injection of 200 μg UC7-13D5 at 6 h, 24 h, and 48 h post-resuscitation into mice. Neurological scores and modified neurological severity scores were assessed to examine neurological functions. Brain edema was estimated via brain water content measurements. Immunohistochemistry of caspase-3 and immunofluorescence staining of claudin-1, ZO-1 and CD31 were performed to detect neuronal apoptosis, BBB integrity and angiogenesis. Microvascular morphology in the cortical area was assessed via H&E staining. Oxidative stress was determined by measuring malondialdehyde, myeloperoxidase, xanthine oxidase, superoxide dismutase, and glutathione peroxidase activities. Western blotting was performed to measure the protein levels of Nuclear factor-E2-related factor 2 (Nrf2) and Heme oxygenase-1 (HO-1). UC7-13D5 effectively depleted γδT cells. Inhibition of γδT cells improved neurological deficits and reduced brain edema post-CA/CPR. γδT cells depletion attenuated neuronal apoptosis, BBB disruption and oxidative stress and promoted angiogenesis following CA/CPR. Inhibition of γδT cells facilitated the activation of the Nrf2/HO-1 pathway in CA/CPR-induced mice. Inhibition of γδT cells alleviates neurological deficits and cerebral edema in mice with CA/CPR by inhibiting neuronal apoptosis, BBB disruption and oxidative stress, and promoting angiogenesis via activation of the Nrf2/HO-1 pathway.

Breast cancer is one of the most common malignant tumors in women, and causes a large number of cancer-related deaths. The main cause of death of breast cancer patients is tumor recurrence and metastasis. Recent studies show that lncRNA (Long non-coding RNA) plays an important role in breast cancer. However, the overall biological activity and clinical consequences of the lncRNA MIR17HG in breast cancer remain unclear. Thus, we investigate how the MIR17HG/miR-454-3p network impacts breast cancer cell proliferation and migration. Given the TCGA and Oncomine databases, the researchers evaluated variations in MIR17HG expression for the survival rates of breast cancer patients. The influence of MIR17HG on cell proliferation, migration, cell cycle, and the mRNA expression level of miR-454-3p and FAM135A (family with sequence similarity 135 member A) is identified. Luciferase assay was used to detect the regulatory effect of miR-454-3p on the 3'UTR region of FAM135A, and rescue experiments demonstrated that MIR17HG can up-regulate FAM135A expression by competitively binding miR-454-3p. The effect of FAM135A on the cloning and invasion of MCF-7 cells was detected. MIR17HG expression is reduced in breast cancer tissues, and patients with greater levels of MIR17HG expression have a better prognosis. MIR17HG overexpression caused G2/M arrest in breast cancer cells according to a flow cytometry assay. FAM135A knockdown enhances breast cancer cell proliferation and clone creation, as well as two-dimensional and three-dimensional migratory capacities. Patients with high FAM135A expression in their breast cancer had a better prognosis. These novel findings indicate that MIR17HG may be a potential target for breast cancer. Our findings demonstrated that MIR17HG might suppress breast cancer cell proliferation and migration by sponge miR-454-3p through ceRNA(competing endogenous RNAs) mechanism, indicating that targeting MIR17HG may be a feasible therapeutic candidate for breast cancer.

The discovery of new fungal effector proteins is necessary to enable the screening of cultivars for disease resistance. Sequence-based bioinformatics methods have been used for this purpose, but only a limited number of functional effector proteins have been successfully predicted and subsequently validated experimentally. A significant obstacle is that many fungal effector proteins discovered so far lack sequence similarity or conserved sequence motifs. The availability of experimentally determined three-dimensional (3D) structures of a number of effector proteins has recently highlighted structural similarities amongst groups of sequence-dissimilar fungal effectors, enabling the search for similar structural folds amongst effector sequence candidates. We have applied template-based modelling to predict the 3D structures of candidate effector sequences obtained from bioinformatics predictions and the PHI-BASE database. Structural matches were found not only with ToxA- and MAX-like effector candidates but also with non-fungal effector-like proteins-including plant defensins and animal venoms-suggesting the broad conservation of ancestral structural folds amongst cytotoxic peptides from a diverse range of distant species. Accurate modelling of fungal effectors were achieved using RaptorX. The utility of predicted structures of effector proteins lies in the prediction of their interactions with plant receptors through molecular docking, which will improve the understanding of effector-plant interactions.

Brucellosis is one of the neglected endemic zoonoses in the world. Vaccination appears to be a promising health strategy to prevent it. This study used advanced computational techniques to develop a potent multi-epitope vaccine for human brucellosis. Seven epitopes from four main brucella species that infect humans were selected. They had significant potential to induce cellular and humoral responses. They showed high antigenic ability without the allergenic characteristic. In order to improve its immunogenicity, suitable adjuvants were also added to the structure of the vaccine. The physicochemical and immunological properties of the vaccine were evaluated. Then its two and three-dimensional structure was predicted. The vaccine was docked with toll-like receptor4 to assess its ability to stimulate innate immune responses. For successful expression of the vaccine protein in Escherichia coli, in silico cloning, codon optimization, and mRNA stability were evaluated. The immune simulation was performed to reveal the immune response profile of the vaccine after injection. The designed vaccine showed the high ability to induce immune response, especially cellular responses to human brucellosis. It showed the appropriate physicochemical properties, a high-quality structure, and a high potential for expression in a prokaryotic system.

BACKGROUND: Circular RNAs (circRNAs) are key regulators of oral squamous cell carcinoma (OSCC) progression. In this study, we aimed to clarify the regulatory roles of circ_0058063 and its effect on tumorigenesis in OSCC. METHODS: Quantitative real-time polymerase chain reaction was conducted to determine the expression levels of microRNA (miR)-145-5p and circ_0058063 in OSCC. Cell viability, adhesion, migration, and epithelial-mesenchymal transition (EMT) of OSCC cells were assessed using cell counting kit-8, cell adhesion, and transwell assays. Western blotting was performed to determine the phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) phosphorylation levels. Xenograft tumor models were constructed to evaluate the tumorigenicity of OSCC cells in vivo. In addition, the interaction between circ_0058063 and miR-145-5p was validated via luciferase reporter and RNA immunoprecipitation assays. RESULTS: Expression levels of circ_0058063 were elevated, whereas those of miR-145-5p were decreased in OSCC. Upregulation of circ_0058063 levels enhanced the viability, adhesion, migration, and EMT of OSCC cells in vitro and promoted tumorigenicity in vivo. Moreover, circ_0058063 promoted OSCC growth by upregulating the PI3K and AKT phosphorylation levels. miR-145-5p overexpression considerably inhibited the PI3K/AKT pathway and decreased OSCC cell viability, adhesion, migration, and EMT. Mechanistically, circ_0058063 sponged miR-145-5p and activated the PI3K/AKT pathway in OSCC cells. CONCLUSION: Our results revealed that circ_0058063 functions as an oncogene via regulation of the PI3K/AKT pathway by targeting miR-145-5p in OSCC, suggesting its potential for OSCC diagnosis and treatment.

Traditional Chinese medicine has been used to treat diabetic foot ulcer (DFU) for a long time. However, the underlying mechanism of Radix arnebiae seu lithospermi ointment (RAS-ointment) has not been revealed. Effects of RAS-ointment treatment were observed in DFU patients. The endogenous competitive RNA mechanism was constructed based on micro-array sequencing and bioinformatics analysis. RT-PCR was used to detected the expression of genes in DFU ulcerated skins and non-ulcerated skins. Dual luciferase and RT-PCR experiments were used to investigate the endogenous competitive RNA mechanism. Based on micro-array sequencing and bioinformatics analysis, we found that SNHG12/NFYC-AS1, hsa-miR-199a-5p and S100A8/S100A7/XDH might form an endogenous competitive RNA mechanism. RT-PCR assay shown that SNHG12, NFYC-AS1, S100A8, S100A7 and XDH were significantly up-regulated, while hsa-miR-199a-5p was significantly down-regulated in DFU ulcerated skins (N = 10) compared with non-ulcerated skins (N = 10). Dual luciferase and RT-PCR experiments showed that SNHG12 or NFYC-AS1 up-regulated the expression of S100A8, S100A7 and XDH by inhibiting hsa-miR-199a-5p in a direct binding way. After 35 days of RAS-ointment treatment, the wound healing of DFU patients was substantially improved and the expression of S100A7 and XDH were reduced expression in DFU patients. In addition, the monomer composition of RAS-ointment, 49070_FLUKA or auraptenol inhibited the expression of S100A7 and XDH in Te317.sk cells. In conclusion, RAS-ointment may be used as an adjunctive therapy for DFU patients.

In order to examine new potential treatment options for the treatment of hepatoblastoma (HB), we identified the differential expression of five-candidate tumor suppressor miRNAs in HB and explored possible regulatory mechanisms of target miRNA molecule. By using real-time quantitative polymerase chain reaction (qPCR), we examined the expression of miRNAs in HB tissues and cells. The effect of has-miR-139-3p mimics on the invasion and migration ability was assessed by transwell assay and scratch-wound assay in HepG2 cells. Subsequently, we analyzed the target genes of miR-139-3p and their enrichment signaling pathways through bioinformatics. qPCR, Western-blot and dual-luciferase assays were further used to assess whether has-miR-139-3p targets Wnt5A. The results showed that hsa-miR-139-3p was significantly decreased in HB cells. Upregulation of hsa-miR-139-3p inhibited the invasive and migratory ability of HepG2. Bioinformatics analysis showed that hsa-miR-139-3p may target Wnt5A to regulate the WNT pathway, which was further confirmed by Western-blot and dual-luciferase assays. Overexpression of Wnt5A can reverse the miR-139-3p mimic-induced declines in the expression of WNT pathway-related proteins and restore the invasion and migration of HepG2. These data indicated that the hsa-miR-139-3p/Wnt5A axis inhibited HB metastasis, suggesting that miR-139-3p and Wnt5A may be potential targets for the treatment of HB.

We focus on hsa_circ_0084912's role in Cervical cancer (CC) and its molecular pathways. In order to determine the expression of Hsa_circ_0084912, miR-429, and SOX2 in CC tissues and cells, Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were utilized. Cell counting kit 8 (CCK-8), colony formation and Transwell assays were respectively to analyze CC cell proliferation viability, clone formation ability and migration. RNA immunoprecipitation (RIP) assay and dual-luciferase assay were used to assure the targeting correlation among hsa_circ_0084912/SOX2 and miR-429. By using a xenograft tumor model, the hsa_circ_0084912 impact on CC cell proliferation in vivo was confirmed. Hsa_circ_0084912 and SOX2 expressions were aggrandized, however, miR-429 expression was descended in CC tissues and cells. Silencing hsa_circ_0084912 inhibited cell proliferation, colony formation and migration in vitro of CC, meanwhile reducing growth of tumor in vivo. MiR-429 might be sponged by Hsa_circ_0084912 to control SOX2 expression. Hsa_circ_0084912 knockdown impact on the malignant phenotypes of CC cells was restored by miR-429 inhibitor. Moreover, SOX2 silencing eliminated the promotive effects of miR-429 inhibitors on CC cell malignancies. By raising SOX2 expression by targeting miR-429, hsa_circ_0084912 accelerated the development of CC, offering fresh proof that it is a viable target for CC treatment.

Implementation of computational tools in the identification of novel drug targets for Tuberculosis (TB) has been a promising area of research. TB has been a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) localized primarily on the lungs and it has been one of the most successful pathogen in the history of mankind. Extensively arising drug resistivity in TB has made it a global challenge and need for new drugs has become utmost important.The involvement of Nucleoid-Associated Proteins (NAPs) in maintaining the structure of the genomic material and regulating various cellular processes like transcription, DNA replication, repair and recombination makes significant, has opened a new arena to find the drugs targeting Mtb. The current study aims to identify potential inhibitors of NAPs through a computational approach. In the present work we worked on the eight NAPs of Mtb, namely, Lsr2, EspR, HupB, HNS, NapA, mIHF and NapM. The structural modelling and analysis of these NAPs were carried out. Moreover, molecular interaction were checked and binding energy was identified for 2500 FDA-approved drugs that were selected for antagonist analysis to choose novel inhibitors targeting NAPs of Mtb. Drugs including Amikacin, streptomycin, kanamycin, and isoniazid along with eight FDA-approved molecules that were found to be potential novel targets for these mycobacterial NAPs and have an impact on their functions. The potentiality of several anti-tubercular drugs as therapeutic agents identified through computational modelling and simulation unlocks a new gateway for accomplishing the goal to treat TB. Complete framework of the methodology employed in this study to predict inhibitors against mycobacterial NAPs.

Annual global temperature is increasing rapidly. Therefore, in the near future, plants will be exposed to severe heat stress. However, the potential of microRNAs-mediated molecular mechanism for modulating the expression of their target genes is unclear. To investigate the changes of miRNAs in thermo-tolerant plants, in this study, we first investigated the impact of four high temperature regimes including 35/30 °C, 40/35 °C, 45/40 °C, and 50/45 °C in a day/night cycle for 21 days on the physiological traits (total chlorophyll, relative water content and electrolyte leakage and total soluble protein), antioxidant enzymes activities (superoxide dismutase, ascorbic peroxidase, catalase and peroxidase), and osmolytes (total soluble carbohydrates and starch) in two bermudagrass accessions named Malayer and Gorgan. The results showed that more chlorophyll and the relative water content, lower ion leakage, more efficient protein and carbon metabolism and activation of defense proteins (such as antioxidant enzymes) in Gorgan accession, led to better maintained plant growth and activity during heat stress. In the next stage, to investigate the role of miRNAs and their target genes in response to heat stress in a thermo-tolerant plant, the impact of severe heat stress (45/40 °C) was evaluated on the expression of three miRNAs (miRNA159a, miRNA160a and miRNA164f) and their target genes (GAMYB, ARF17 and NAC1, respectively). All measurements were performed in leaves and roots simultaneously. Heat stress significantly induced the expression of three miRNAs in leaves of two accession, while having different effects on the expression of these miRNAs in roots. The results showed that a decrease in the expression of the transcription factor ARF17, no change in the expression of the transcription factor NAC1, and an increase in the expression of the transcription factor GAMYB in leaf and root tissues of Gorgan accession led to improved heat tolerance in it. These results also showed that the effect of miRNAs on the modulating expression of target mRNAs in leaves and roots is different under heat stress, and miRNAs and mRNAs show spatiotemporal expression. Therefore, the simultaneous analysis of miRNAs and mRNAs expressions in shoot and roots is needed to comprehensively understand miRNAs regulatory function under heat stress.

The metastasis is a major reason for the poor prognosis of the patients with prostate cancer (PC). Currently, androgen deprivation therapy (ADT) is the basic method for the treatment of PC regardless of surgery or drug treatments. However, ADT therapy is generally not recommended for patients with advanced/metastatic PC. Herein, we report for the first time a long non-coding RNA (lncRNA)-PCMF1 which promotes the progression of Epithelial-Mesenchymal Transition (EMT) in PC cells. Our data demonstrated that PCMF1 in metastatic PC tissues increased significantly compared to non-metastatic specimens. Mechanism research showed that PCMF1 could competitively bind to hsa-miR-137 instead of the 3' -Untranslated Region (UTR) of Twist Family BHLH Transcription Factor 1 (Twist1) by acting as an endogenous miRNA sponge. Furthermore, we found that silence of PCMF1 effectively blocked the EMT in PC cells by indirectly suppressing Twist1 protein mediated by hsa-miR-137 at post-transcriptional level. In summary, our research shows that PCMF1 promotes the EMT of PC cells by causing the functional inactivation of hsa-miR-137 on Twist1 protein, which is an independent risk factor of PC. PCMF1 knockdown combined with hsa-miR-137 expression is a promising PC-targeted therapy. Furthermore, PCMF1 is also expected to act as a useful marker for predicting malignant changes and assessing the prognosis of PC patients.

As a member of the tumor necrosis factor (TNF) superfamily, the B-cell activating factor (BAFF) plays a crucial role in B-cell survival and differentiation. Overexpression of this protein has been closely linked to autoimmune disorders and some B-cell malignancies. Using monoclonal antibodies (mAbs) against the BAFF soluble domain appears to be a complementary treatment for some of these diseases. This study aimed to produce and develop a specific Nanobody (Nb), a variable camelid antibody domain, against the soluble domain of BAFF protein. After camel immunization with recombinant protein and preparing cDNA from total RNAs separated from camel lymphocytes, an Nb library was developed. Individual colonies capable of binding selectively to rBAFF were obtained by periplasmic-ELISA, sequenced, and expressed in a bacterial expression system. The specificity and affinity of selected Nb were determined and its target identification and functionality were evaluated using flow cytometry.

Cold shock proteins (CSPs) are small, acidic proteins which contain a conserved nucleic acid-binding domain. These perform mRNA translation acting as "RNA chaperones" when triggered by low temperatures initiating their cold shock response. CSP- RNA interactions have been predominantly studied. Our focus will be CSP-DNA interaction examination, to analyse the diverse interaction patterns such as electrostatic, hydrogen and hydrophobic bonding in both thermophilic and mesophilic bacteria. The differences in the molecular mechanism of these contrasting bacterial proteins are studied. Computational techniques such as modelling, energy refinement, simulation and docking were operated to obtain data for comparative analysis. The thermostability factors which stabilise a thermophilic bacterium and their effect on their molecular regulation is investigated. Conformational deviation, atomic residual fluctuations, binding affinity, Electrostatic energy and Solvent Accessibility energy were determined during stimulation along with their conformational study. The study revealed that mesophilic bacteria E. coli CSP have higher binding affinity to DNA than thermophilic G. stearothermophilus. This was further evident by low conformation deviation and atomic fluctuations during simulation.

This study aimed to evaluate the comparative effect of silibinin (SB) on the expression of MiR‑20b and BCL2L11 in T47D and MCF-7 cell lines. Molecular simulation studies were carried out to analyze Erbb2, as a potential target of SB, to direct the breast cancer cells toward apoptosis. At first, cell viability, apoptosis, and cell cycle arrest-inducing capacity of SB were examined using MTT and flow cytometry analysis, respectively. Real-time PCR (RT-PCR) was employed to assess the effect of SB on BCL2L11, Phosphatase and tensin homolog (PTEN), and Caspase 9 mRNarrest-indu. Moreover, alterations in Caspase 9 protein expression were determined using Western blot analysis. Finally, AutoDockVina software was used to dock the SB/ MiR‑20b and SB/ erb-b2 receptor tyrosine kinase 2 (Erbb2) interaction. The obtained data revealed the potent cytotoxicity of SB in both T47D and MCF-7 cells through apoptosis induction and cell cycle arrest. SB-treated cells also showed downregulation of MiR‑20b and high expression of BCL2L11, PTEN, and Caspase 9 mRNA compared to non-treated cancer cells. Computational docking showed a strong interaction between SB/ MiR‑20b and SB/Erbb2. It can be concluded that SB had a strong anti-tumorigenic activity through BCL2L11upregulation and MiR‑20b down expression, maybe through targeting the PTEN and interacting with Erbb2, which resulted in apoptotic induction and cell cycle arrest.

To exploit the rice seed-based oral vaccine against Sjögren's syndrome, altered peptide ligand of N-terminal 1 (N1-APL7) from its M3 muscarinic acetylcholine receptor (M3R) autoantigen was expressed as fusion protein with the representative four types of rice prolamins (16 kDa, 14 kDa, 13 kDa, and 10 kDa prolamins) under the control of the individual native prolamin promoter. The 10kD:N1-APL7 and 14kD:N1-APL7 accumulated at high levels (287 and 58 µg/grain), respectively, whereas production levels of the remaining ones were remarkably low. Co-expression of these fusion proteins did not enhance the accumulation level of N1-APL7 in an additive manner. Downregulation of endogenous seed storage proteins by RNAi-mediated suppression also did not lead to substantial elevation of the co-expressed prolamin:N1-APL7 products. When transgenic rice seeds were subjected to in vitro proteolysis with pepsin, the 10kD:N1-APL7 was digested more quickly than the endogenous 10 kDa prolamin and the 14kD:N1-APL7 deposited in PB-Is. This difference could be explained by the finding that the 10kD:N1-APL7 was unexpectedly localized in the PB-IIs containing glutelins. These results indicated that not only accumulation level but also subcellular localization of inherent prolamins were highly influenced by the liked N1-APL7 peptide.