Genome-wide characterization and functional analysis of the melon TGA gene family in disease resistance through ectopic overexpression in Arabidopsis thaliana.

TGA-binding (TGA) transcription factors, characterized by the basic region/leucine zipper motif (bZIP), have been recognized as pivotal regulators in plant growth, development, and stress responses through their binding to the as-1 element. In this study, the TGA gene families in melon, watermelon, cucumber, pumpkin, and zucchini were comprehensively characterized, encompassing analyses of gene/protein structures, phylogenetic relationships, gene duplication events, and cis-acting elements in gene promoters. Upon transient expression in Nicotiana benthamiana, the melon CmTGAs, with typical bZIP and DOG1 domains, were observed to localize within the nucleus. Biochemical investigation revealed specific interactions between CmTGA2/3/5/8/9 and CmNPR3 or CmNPR4. The CmTGA genes exhibited differential expression patterns in melon plants in response to different hormones like salicylic acid, methyl jasmonate, and ethylene, as well as a fungal pathogen, Stagonosporopsis cucurbitacearum that causes gummy stem blight in melon. The overexpression of CmTGA3, CmTGA8, and CmTGA9 in Arabidopsis plants resulted in the upregulation of AtPR1 and AtPR5 expression, thereby imparting enhanced resistance to Pseudomonas syringae pv. Tomato DC3000. In contrast, the overexpression of CmTGA7 or CmTGA9 resulted in a compromised resistance to Botrytis cinerea, coinciding with a concomitant reduction in the expression levels of AtPDF1.2 and AtMYC2 following infection with B. cinerea. These findings shed light on the important roles of specific CmTGA genes in plant immunity, suggesting that genetic manipulation of these genes could be a promising avenue for enhancing plant immune responses.

Ectopic expression of OsWOX9A alters leaf anatomy and plant architecture in rice.

MAIN CONCLUSION: Ectopic expression of OsWOX9A induces narrow adaxially rolled rice leaves with larger bulliform cells and fewer large veins, probably through regulating the expression of auxin-related and expansin genes. The WUSCHEL-related homeobox (WOX) family plays a pivotal role in plant development by regulating genes involved in various aspects of growth and differentiation. OsWOX9A (DWT1) has been linked to tiller growth, uniform plant growth, and flower meristem activity. However, its impact on leaf growth and development in rice has not been studied. In this study, we investigated the biological role of OsWOX9A in rice growth and development using transgenic plants. Overexpression of OsWOX9A conferred narrow adaxially rolled rice leaves and altered plant architecture. These plants exhibited larger bulliform cells and fewer larger veins compared to wild-type plants. OsWOX9A overexpression also reduced plant height, tiller number, and seed-setting rate. Comparative transcriptome analysis revealed several differentially expressed auxin-related and expansin genes in OsWOX9A overexpressing plants, consistent with their roles in leaf and plant development. These results indicate that the ectopic expression of OsWOX9A may have multiple effects on the development and growth of rice, providing a more comprehensive picture of how the WOX9 subfamily contributes to leaf development and plant architecture.

TAD border deletion at the Kit locus causes tissue-specific ectopic activation of a neighboring gene.

Topologically associated domains (TADs) restrict promoter-enhancer interactions, thereby maintaining the spatiotemporal pattern of gene activity. However, rearrangements of the TADs boundaries do not always lead to significant changes in the activity pattern. Here, we investigated the consequences of the TAD boundaries deletion on the expression of developmentally important genes encoding tyrosine kinase receptors: Kit, Kdr, Pdgfra. We used genome editing in mice to delete the TADs boundaries at the Kit locus and characterized chromatin folding and gene expression in pure cultures of fibroblasts, mast cells, and melanocytes. We found that although Kit is highly active in both mast cells and melanocytes, deletion of the TAD boundary between the Kit and Kdr genes results in ectopic activation only in melanocytes. Thus, the epigenetic landscape, namely the mutual arrangement of enhancers and actively transcribing genes, is important for predicting the consequences of the TAD boundaries removal. We also found that mice without a TAD border between the Kit and Kdr genes have a phenotypic manifestation of the mutation - a lighter coloration. Thus, the data obtained shed light on the principles of interaction between the 3D chromatin organization and epigenetic marks in the regulation of gene activity.

Ectopic MYBL2-Mediated Regulation of Androglobin Gene Expression.

Androglobin (ADGB) is a highly conserved and recently identified member of the globin superfamily. Although previous studies revealed a link to ciliogenesis and an involvement in murine spermatogenesis, its physiological function remains mostly unknown. Apart from FOXJ1-dependent regulation, the transcriptional landscape of the ADGB gene remains unexplored. We, therefore, aimed to obtain further insights into regulatory mechanisms governing ADGB expression. To this end, changes in ADGB promoter activity were examined using luciferase reporter gene assays in the presence of a set of more than 475 different exogenous transcription factors. MYBL2 and PITX2 resulted in the most pronounced increase in ADGB promoter-dependent luciferase activity. Subsequent truncation strategies of the ADGB promoter fragment narrowed down the potential MYBL2 and PITX2 binding sites within the proximal ADGB promoter. Furthermore, MYBL2 binding sites on the ADGB promoter were further validated via a guide RNA-mediated interference strategy using reporter assays. Chromatin immunoprecipitation (ChIP)-qPCR experiments illustrated enrichment of the endogenous ADGB promoter region upon MYBL2 and PITX2 overexpression. Consistently, ectopic MYBL2 expression induced endogenous ADGB mRNA levels. Collectively, our data indicate that ADGB is strongly regulated at the transcriptional level and might have functions beyond ciliogenesis.

Ectopic expression in commonly used transgenic Drosophila GAL4 driver lines.

Transgenic tools such as the GAL4/UAS system in Drosophila have been used extensively to induce spatiotemporally controlled changes in gene expression and tissue-specific expression of a range of transgenes. We previously discovered unexpected expression of the commonly used dilp2-GAL4 line in tracheal tissue which significantly impacted growth phenotypes. We realized that few GAL4 lines have been thoroughly characterized, particularly when considering transient activity that may have significant impact on phenotypic readouts. Here, we characterized a further subset of 12 reportedly tissue-specific GAL4 lines commonly used in genetic studies of development, growth, endocrine regulation, and metabolism. Ten out of 12 GAL4 lines exhibited ectopic activity in other larval tissues, with seven being active in the larval trachea. Since this ectopic activity may result in phenotypes that do not depend on the manipulation in the intended target tissue, it is recommended to carefully analyze the outcome while taking this aspect into consideration.

Ectopic expression of HaPEPC1 from the desert shrub Haloxylon ammodendron confers drought stress tolerance in Arabidopsis thaliana.

Phosphoenolpyruvate carboxylase (PEPC) plays a crucial role in the initial carbon fixation process in C4 plants. However, its nonphotosynthetic functions in Haloxylon ammodendron, a C4 perennial xerohalophytic shrub, are still poorly understood. Previous studies have reported the involvement of PEPC in plant responses to abiotic stresses such as drought and salt stress. However, the underlying mechanism of PEPC tolerance to drought stress has not been determined. In this study, we cloned the C4-type PEPC gene HaPEPC1 from H. ammodendron and investigated its biological function by generating transgenic Arabidopsis plants with ectopic expression of HaPEPC1. Our results showed that, compared with WT (wild-type) plants, ectopic expression of HaPEPC1 plants exhibited significantly greater germination rates and chlorophyll contents. Furthermore, under drought stress, the transgenic plants presented increased root length, fresh weight, photosynthetic capacity, and antioxidant enzyme activities, particularly ascorbate peroxidase and peroxidase. Additionally, the transgenic plants exhibited reduced levels of malondialdehyde, H2O2 (hydrogen peroxide), and O2- (superoxide radical). Transcriptome analysis indicated that ectopic expression of HaPEPC1 primarily regulated the expression of genes associated with the stress defence response, glutathione metabolism, and abscisic acid (ABA) synthesis and signalling pathways in response to drought stress. Taken together, these findings suggest that the ectopic expression of HaPEPC1 enhances the reduction of H2O2 and O2- in transgenic plants, thereby improving reactive oxygen species (ROS) scavenging capacity and enhancing drought tolerance. Therefore, the HaPEPC1 gene holds promise as a candidate gene for crop selection aimed at enhancing drought tolerance.

fhl2b mediates extraocular muscle protection in zebrafish models of muscular dystrophies and its ectopic expression ameliorates affected body muscles.

In muscular dystrophies, muscle fibers loose integrity and die, causing significant suffering and premature death. Strikingly, the extraocular muscles (EOMs) are spared, functioning well despite the disease progression. Although EOMs have been shown to differ from body musculature, the mechanisms underlying this inherent resistance to muscle dystrophies remain unknown. Here, we demonstrate important differences in gene expression as a response to muscle dystrophies between the EOMs and trunk muscles in zebrafish via transcriptomic profiling. We show that the LIM-protein Fhl2 is increased in response to the knockout of desmin, plectin and obscurin, cytoskeletal proteins whose knockout causes different muscle dystrophies, and contributes to disease protection of the EOMs. Moreover, we show that ectopic expression of fhl2b can partially rescue the muscle phenotype in the zebrafish Duchenne muscular dystrophy model sapje, significantly improving their survival. Therefore, Fhl2 is a protective agent and a candidate target gene for therapy of muscular dystrophies.

Ectopic Expression of TIGIT in Lung Adenocarcinoma and Its Clinical Significance.

This study aimed to investigate the T cell immunoreceptor with ITIM and Ig domains (TIGIT) expression in lung adenocarcinoma (LUAD). TIGIT expression was measured by western blot, reverse transcription quantitative polymerase chain reaction. Seventy-two paired surgical specimens were collected from patients with stage I-IV LUAD. The expression of TIGIT in surgical specimens was determined using immunohistochemistry. TIGIT was overexpressed in LUAD tissues. Moreover, overexpressed TIGIT was significantly associated with advanced clinical staging, lymph node metastasis, distant metastasis, and TP53 mutations in LUAD. Moreover, high expression of TIGIT was negatively correlated with purity of CD4+ T cells. High rations of TIGIT+CD4+ T cells predicted poor overall survival of LUAD patients. Additionally, high ratios of TIGIT+CD4+ T cells is closely related to CD4+ T cell depletion. Taken together, TIGIT was overexpressed in LUAD patients. High levels of TIGIT induced the alteration of CD4+ T cell based immunomodulation and predicted poor prognosis of LUAD patients. Therefore, TIGIT can be potential biomarker for LUAD.

Ectopic Expression of PtrLBD39 Retarded Primary and Secondary Growth in Populus trichocarpa.

Primary and secondary growth of trees are needed for increments in plant height and stem diameter, respectively, affecting the production of woody biomass for applications in timber, pulp/paper, and related biomaterials. These two types of growth are believed to be both regulated by distinct transcription factor (TF)-mediated regulatory pathways. Notably, we identified PtrLBD39, a highly stem phloem-specific TF in Populus trichocarpa and found that the ectopic expression of PtrLBD39 in P. trichocarpa markedly retarded both primary and secondary growth. In these overexpressing plants, the RNA-seq, ChIP-seq, and weighted gene co-expression network analysis (WGCNA) revealed that PtrLBD39 directly or indirectly regulates TFs governing vascular tissue development, wood formation, hormonal signaling pathways, and enzymes responsible for wood components. This regulation led to growth inhibition, decreased fibrocyte secondary cell wall thickness, and reduced wood production. Therefore, our study indicates that, following ectopic expression in P. trichocarpa, PtrLBD39 functions as a repressor influencing both primary and secondary growth.

Ectopic Expression of the Executor-Type R Gene Paralog Xa27B in Rice Leads to Spontaneous Lesions and Enhanced Disease Resistance.

Plant disease resistance (R) gene-mediated effector-triggered immunity (ETI) is usually associated with hypersensitive response (HR) and provides robust and race-specific disease resistance against pathogenic infection. The activation of ETI and HR in plants is strictly regulated, and improper activation will lead to cell death. Xa27 is an executor-type R gene in rice induced by the TAL effector AvrXa27 and confers disease resistance to Xanthomonas oryzae pv. oryzae (Xoo). Here we reported the characterization of a transgenic line with lesion mimic phenotype, designated as Spotted leaf and resistance 1 (Slr1), which was derived from rice transformation with a genomic subclone located 5,125 bp downstream of the Xa27 gene. Slr1 develops spontaneous lesions on its leaves caused by cell death and confers disease resistance to both Xoo and Xanthomonas oryzae pv. oryzicola. Further investigation revealed that the Slr1 phenotype resulted from the ectopic expression of an Xa27 paralog gene, designated as Xa27B, in the inserted DNA fragment at the Slr1 locus driven by a truncated CaMV35Sx2 promoter in reverse orientation. Disease evaluation of IRBB27, IR24, and Xa27B mutants with Xoo strains expressing dTALE-Xa27B confirmed that Xa27B is a functional executor-type R gene. The functional XA27B-GFP protein was localized to the endoplasmic reticulum and apoplast. The identification of Xa27B as a new functional executor-type R gene provides additional genetic resources for studying the mechanism of executor-type R protein-mediated ETI and developing enhanced and broad-spectrum disease resistance to Xoo through promoter engineering. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Ectopic expression of the transcription factor ONECUT3 drives a complex karyotype in myelodysplastic syndromes.

Chromosomal instability is a prominent biological feature of myelodysplastic syndromes (MDS), with over 50% of patients with MDS harboring chromosomal abnormalities or a complex karyotype (CK). Despite this observation, the mechanisms underlying mitotic and chromosomal defects in MDS remain elusive. In this study, we identified ectopic expression of the transcription factor ONECUT3, which is associated with CKs and poorer survival outcomes in MDS. ONECUT3-overexpressing cell models exhibited enrichment of several notable pathways, including signatures of sister chromosome exchange separation and mitotic nuclear division with the upregulation of INCENP and CDCA8 genes. Notably, dysregulation of chromosome passenger complex (CPC) accumulation, besides the cell equator and midbody, during mitotic phases consequently caused cytokinesis failure and defective chromosome segregation. Mechanistically, the homeobox (HOX) domain of ONECUT3, serving as the DNA binding domain, occupied the unique genomic regions of INCENP and CDCA8 and transcriptionally activated these 2 genes. We identified a lead compound, C5484617, that functionally targeted the HOX domain of ONECUT3, inhibiting its transcriptional activity on downstream genes, and synergistically resensitized MDS cells to hypomethylating agents. This study revealed that ONECUT3 promoted chromosomal instability by transcriptional activation of INCENP and CDCA8, suggesting potential prognostic and therapeutic roles for targeting high-risk MDS patients with a CK.

Ectopic expression of an Old Yellow Enzyme (OYE3) gene from Saccharomyces cerevisiae increases the tolerance and phytoremediation of 2-nitroaniline in rice.

2-nitroaniline (2-NA) is an environmental pollutant and has been extensively used as intermediates in organic synthesis. The presence of 2-NA in the environment is not only harmful for aquatic life but also mutagenic for human beings. In this study, we constructed transgenic rice expressing an Old Yellow Enzyme gene, ScOYE3, from Saccharomyces cerevisiae. The ScOYE3 transgenic plants were comprehensively investigated for their biochemical responses to 2-NA treatment and their 2-NA phytoremediation capabilities. Our results showed that the rice seedlings exposed to 2-NA stress, showed growth inhibition and biomass reduction. However, the transgenic plants exhibited strong tolerance to 2-NA stress compared to wild-type plants. Ectopic expression of ScOYE3 could effectively protect transgenic plants against 2-NA damage, which resulted in less reactive oxygen species accumulation in transgenic plants than that in wild-type plants. Our phytoremediation assay revealed that transgenic plants could eliminate more 2-NA from the medium than wild-type plants. Moreover, omics analysis was performed in order to get a deeper insight into the mechanism of ScOYE3-mediated 2-NA transformation in rice. Altogether, the function of ScOYE3 during 2-NA detoxification was characterized for the first time, which serves as strong theoretical support for the phytoremediation potential of 2-NA by Old Yellow Enzyme genes.

Ectopic expression of the Arabidopsis mutant L3 NB-LRR receptor gene in Nicotiana benthamiana cells leads to cell death.

Nucleotide-binding sites and leucine-rich repeat proteins (NLRs) act as critical intracellular immune receptors. Previous studies reported an Arabidopsis-resistant gene L3 (AT1G15890), which encoded a coiled-coil (CC) NLR that conferred cell death in bacteria; however, its function in planta remains unclear. This study describes a comprehensive structure-function analysis of L3 in Nicotiana benthamiana. The results of the transient assay showed that the L3 CC domain is sufficient for cell-death induction. The first 140 amino acid segment constituted the minimal function region that could cause cell death. The YFP-labeled L3 CC domain was localized to the plasma membrane, which was considered crucial for the function and self-interaction of the L3 CC domain. The results of point mutations analysis showed that L3 CC domain function is affected by mutations in some specific residues, and loss-of-function mutations in the CC domain affected the function of full-length L3. These study results offered considerable evidence to understand the activation mechanism of L3.

Ectopic expression of SaCTP3 from the hyperaccumulator Sedum alfredii in sorghum increases Cd accumulation for phytoextraction.

The Cd tolerance protein SaCTP3, which responds to Cd stress, was identified in Sedum alfredii; however, how to improve the efficiency of phytoremediation of Cd-contaminated soil using the CTP gene remains unknown. In this study, the phytoremediation potential of SaCTP3 of Sedum alfredii was identified. In the yeast Cd-sensitive strain Δycf1 overexpressing SaCTP3, the accumulation of Cd was higher than that in the Δycf1 strain overexpressing an empty vector. Transgenic sorghum plants overexpression SaCTP3 were further constructed to verify the function of SaCTP3. Compared to wild-type plants, the SaCTP3-overexpressing lines exhibited higher Cd accumulation under 500 µM Cd conditions. The average Cd content inSaCTP3-overexpressing plants is more than four times higher than that of WT plants. This was accompanied by an enhanced ability to scavenge ROS, as evidenced by the significantly increased activities of peroxidase, catalase, and superoxide dismutase in response to Cd stress. Pot experiments further demonstrated that SaCTP3 overexpression resulted in improved soil Cd scavenging and photosynthetic abilities. After 20 days of growth, the average Cd content in the soil planted with SaCTP3-overexpressing sorghum decreased by 19.4%, while the residual Cd content in the soil planted with wild-type plants was only reduced by 5.4%. This study elucidated the role of SaCTP3 from S.alfredii, highlighting its potential utility in genetically modifying sorghum for the effective phytoremediation of Cd.

Ectopic Expression of a Truncated Isoform of Hair Keratin 81 in Breast Cancer Alters Biophysical Characteristics to Promote Metastatic Propensity.

Keratins are an integral part of cell structure and function. Here, it is shown that ectopic expression of a truncated isoform of keratin 81 (tKRT81) in breast cancer is upregulated in metastatic lesions compared to primary tumors and patient-derived circulating tumor cells, and is associated with more aggressive subtypes. tKRT81 physically interacts with keratin 18 (KRT18) and leads to changes in the cytosolic keratin intermediate filament network and desmosomal plaque formation. These structural changes are associated with a softer, more elastically deformable cancer cell with enhanced adhesion and clustering ability leading to greater in vivo lung metastatic burden. This work describes a novel biomechanical mechanism by which tKRT81 promotes metastasis, highlighting the importance of the biophysical characteristics of tumor cells.

Ectopic expression of a bacterial thiamin monophosphate kinase enhances vitamin B1 biosynthesis in plants.

Plants and bacteria have distinct pathways to synthesize the bioactive vitamin B1 thiamin diphosphate (TDP). In plants, thiamin monophosphate (TMP) synthesized in the TDP biosynthetic pathway is first converted to thiamin by a phosphatase, which is then pyrophosphorylated to TDP. In contrast, bacteria use a TMP kinase encoded by ThiL to phosphorylate TMP to TDP directly. The Arabidopsis THIAMIN REQUIRING2 (TH2)-encoded phosphatase is involved in TDP biosynthesis. The chlorotic th2 mutants have high TMP and low thiamin and TDP. Ectopic expression of Escherichia coli ThiL and ThiL-GFP rescued the th2-3 mutant, suggesting that the bacterial TMP kinase could directly convert TMP into TDP in Arabidopsis. These results provide direct evidence that the chlorotic phenotype of th2-3 is caused by TDP rather than thiamin deficiency. Transgenic Arabidopsis harboring engineered ThiL-GFP targeting to the cytosol, chloroplast, mitochondrion, or nucleus accumulated higher TDP than the wild type (WT). Ectopic expression of E. coli ThiL driven by the UBIQUITIN (UBI) promoter or an endosperm-specific GLUTELIN1 (GT1) promoter also enhanced TDP biosynthesis in rice. The pUBI:ThiL transgenic rice accumulated more TDP and total vitamin B1 in the leaves, and the pGT1:ThiL transgenic lines had higher TDP and total vitamin B1 in the seeds than the WT. Total vitamin B1 only increased by approximately 25-30% in the polished and unpolished seeds of the pGT1:ThiL transgenic rice compared to the WT. Nevertheless, these results suggest that genetic engineering of a bacterial vitamin B1 biosynthetic gene downstream of TMP can enhance vitamin B1 production in rice.

Ectopic expression of tumor suppressive miR-181c-5p downregulates oncogenic Notch signaling in MDA-MB-231 cells.

Triple negative breast cancer (TNBC) is a very invasive subtype of breast cancer (BCa), this is accounted for 15-20% of all BCa cases. TNBC patients have very limited therapy option due to lack of effective targets and patients shows the worse survival. Therefore, present study has tried to introduce the target based therapy by studying the tumor suppressive role of miR-181c-5p on oncogenic Notch1 signaling. Transient transfection, bioinformatics, qRT-PCR, Notch1 luciferase assay and western blotting techniques were utilized to study the effect of induced expression of miR-181c-5p on oncogenic Notch1 signaling in MDA-MB-231 cells. Results shows that miR-181c-5p mimic increase the expression of miR-181c-5p by 45.26% and 75.96% in 24 and 48 h incubation, respectively (p < 0.0003) in transfected cells. The miR-181c-5p binds at NOTCH1 3' UTR target binding site with a minimum free energy of - 26.0 kcal/mol. The AGO protein showed significant interaction with the miR-181c-5p and miR-181c-5p-NOTCH1 complex. Decreased expression of NOTCH1 by 32.88% and 45.87% (p < 0.0001); and HES1 expression by 14.06% and 53.24% (p < 0.0001) was observed in 24 and 48 h transfected cells respectively. Notch1 promoter luciferase activity was reduced by 25.72% and 46.98% in 24 and 48 h miRNA-mimic transfected cells. Western blot analysis also showed significant reduction in NOTCH1 and HES1 proteins expression. In conclusion, present study suggests that the forced expression of tumor suppressive miR-181c-5p negatively regulates oncogenic Notch1 signaling in TNBC. Negative regulation of Notch1 signaling via miR-181c-5p mimic could be a hopeful therapeutic strategy in TNBC patient treatment.

Ectopic Expression of MADS-Box Transcription Factor VvAGL12 from Grape Promotes Early Flowering, Plant Growth, and Production by Regulating Cell-Wall Architecture in Arabidopsis.

The MADS-box family, a substantial group of plant transcription factors, crucially regulates plant growth and development. Although the functions of AGL12-like subgroups have been elucidated in Arabidopsis, rice, and walnut, their roles in grapes remain unexplored. In this study, we isolated VvAGL12, a member of the grape MADS-box group, and investigated its impact on plant growth and biomass production. VvAGL12 was found to localize in the nucleus and exhibit expression in both vegetative and reproductive organs. We introduced VvAGL12 into Arabidopsis thaliana ecotype Columbia-0 and an agl12 mutant. The resulting phenotypes in the agl12 mutant, complementary line, and overexpressed line underscored VvAGL12's ability to promote early flowering, augment plant growth, and enhance production. This was evident from the improved fresh weight, root length, plant height, and seed production, as well as the reduced flowering time. Subsequent transcriptome analysis revealed significant alterations in the expression of genes associated with cell-wall modification and flowering in the transgenic plants. In summary, the findings highlight VvAGL12's pivotal role in the regulation of flowering timing, overall plant growth, and development. This study offers valuable insights, serving as a reference for understanding the influence of the VvAGL12 gene in other plant species and addressing yield-related challenges.

Ectopic expression of insulin in a type 1 diabetic rat model by injection of manipulated mesenchymal stem cells with an insulin construct driven by a glucose-sensitive promoter in the port vein.

The treatment of type 1 diabetes through islet cell transplantation is a complex process, facing challenges such as allograft rejections and a limited supply of donors. One potential solution is to utilize the liver as an alternative for natural insulin production, as hepatocytes can secrete proteins and respond to glucose levels. Recent research has shown promising results in using mesenchymal stem cells as a potential cure for diabetes. The study utilized a diabetic rat model, confirmed through blood sugar measurement. A plasmid vector was designed with specific genetic components, synthesized by biotech company, and then Inserted vector into a plasmid with resistance genes and bacterial origin. Bone marrow-derived mesenchymal stem cells (BM-MSCs) were cultured and transfected with the plasmid using Lipofectamine 3000. Polymerase chain reaction was employed to confirm successful transfection using specific primers. For the animal study, 30 male Wistar rats were divided into six groups, each comprising five rats. The control group did not receive any treatment, while the second group received MSCs via Portal Vein Injection. The third group received MSCs transfected with a specific construct via Portal Vein Injection. The fourth group was induced to develop diabetes through streptozotocin (STZ) injection, the fifth group developed diabetes and received untransfected MSCs via Portal Vein Injection, and the sixth group received MSCs transfected with the specific construct via Portal Vein Injection. To manage Pain, appropriate pain control was administered to the rats for 3 days after the surgery. Fixed liver tissues obtained from the euthanized rats were utilized for immunohistochemistry. In this study, immunohistochemical techniques were used to examine insulin expression in different groups of rats. The control groups showed high levels of insulin expression, while the diabetic groups exhibited lower expression. However, there was a significant difference between the diabetic groups treated with MSC and transgenic MSC cells. All groups had similar baseline glucose levels, but the diabetic groups showed a significant increase after STZ injection, whereas the control and MSC groups did not. Postintervention, both the control and MSC groups had similar glucose levels to the post-STZ levels. However, diabetes-induced groups experienced a significant decrease in glucose levels, with the transfected MSCs showing a greater decrease than the untransfected MSCs. The study suggested that treatment with MSCs, especially transfected ones, can effectively reduce glucose levels in rats with diabetes. In this research, rat BM-MSCs were utilized to create insulin-producing mesenchymal cells with glucose-sensitive insulin expression. The cells were transferred to the liver of diabetic rats via portal vein injection, leading to an increase in insulin expression. This study proposes a novel approach for cell therapy and delivery in the treatment of type 1 diabetes.

Ectopic expression of murine CD163 enables cell-culture isolation of lactate dehydrogenase-elevating virus 63 years after its discovery.

IMPORTANCE: Mouse models of viral infection play an especially large role in virology. In 1960, a mouse virus, lactate dehydrogenase-elevating virus (LDV), was discovered and found to have the peculiar ability to evade clearance by the immune system, enabling it to persistently infect an individual mouse for its entire lifespan without causing overt disease. However, researchers were unable to grow LDV in culture, ultimately resulting in the demise of this system as a model of failed immunity. We solve this problem by identifying the cell-surface molecule CD163 as the critical missing component in cell-culture systems, enabling the growth of LDV in immortalized cell lines for the first time. This advance creates abundant opportunities for further characterizing LDV in order to study both failed immunity and the family of viruses to which LDV belongs, Arteriviridae (aka, arteriviruses).