Expression Of Selected Genes Research Articles

The single-cell landscape of kidney immune cells reveals transcriptional heterogeneity in early diabetic kidney disease

The pathogenesis of diabetic kidney disease (DKD) involves multifactorial processes that converge to initiate and advance the disease. Although DKD is not typically classified as an inflammatory glomerular disease, mounting evidence supports the involvement of kidney inflammation as a key contributor in DKD pathogenesis, particularly through macrophages. However, detailed identification and corresponding phenotypic changes of macrophages in DKD remain poorly understood. To capture the gene expression changes in specific macrophage cell subsets in early DKD, we performed single-cell transcriptomic analysis of CD45-enriched kidney immune cells from type 1 diabetic OVE26 mice at two time points during the disease development. We also undertook a focused analysis of mononuclear phagocytes (macrophages and dendritic cells). Our results show increased resident and infiltrating macrophage subsets in the kidneys of mice with diabetes over time, with heightened expression of pro-inflammatory or anti-inflammatory genes in a subset-specific manner. Further analysis of macrophage polarization states in each subset in the kidneys showed changes consistent with the continuum of activation and differentiation states, with gene expression tending to shift toward undifferentiated phenotypes but with increased M1-like inflammatory phenotypes over time. By deconvolution analysis of RNAseq samples and by immunostaining of biopsies from patients with DKD, we further confirmed a differential expression of select genes in specific macrophage subsets essentially recapitulating the studies in mice. Thus, our study provides a comprehensive analysis of macrophage transcriptomic profiles in early DKD that underscores the dynamic macrophage phenotypes in disease progression.

Pax6 limits the competence of developing cerebral cortical cells to respond to inductive intercellular signals.

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here, we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single-cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors' competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6's main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side effects of morphogens engaged contemporaneously in other essential functions.

Effect of miR-195 inhibition on human skeletal muscle-derived stem/progenitor cells.

The application of a circulating miR-195 inhibitor could be a helping factor in the in vitro model of human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs). Previously, microRNA-195 (miR-195) expression has been reported to be a negative factor for myogenesis. The study aimed to obtain anti-apoptotic and anti-aging effects in in vitro cultured myoblasts and to improve their ability to form myotubes by suppressing miR-195 expression. Human wild-type (WT) SkMDS/PC cells incubated with control (nonspecific) miRNA inhibitor and miR-195-inhibited SkMDS/PCs were studied. Functional assays (myotube formation and cell aging), antioxidant, and myogenic gene expression analyses were performed at two time points, at the seventh and eleventh cell passages. Myotube formation was found to be almost 2-fold higher in the miR-195-inhibited SkMDS/PCs population (P < 0.05) compared to WT cells. miR-195 inhibition did not appear to affect cell aging or rejuvenate human SkMDS/PCs. Antioxidant (SOD3 and FOXO) gene expression was augmented in the miR-195-inhibited SkMDS/PCs population, but no positive effect on the remaining antioxidant genes (SOD1, SOD2, and catalase) was observed. A significant increase in MyoD gene expression with a concomitant decrease in MyoG (P < 0.05) was further documented in miR-195- -inhibited SkMDS/PCs compared to WT cells (the eleventh cell passage). The performed studies may lead to the preconditioning of myogenic stem cells to extend their potential for pro-regenerative activity. The miR-195 inhibitor may serve as a conditioning factor augmenting selective antioxidant gene expression and proliferative potential of SkMDS/PCs, but it does not have an impact on cell aging and/ or apoptosis.

Epigenetic remodeling of downstream enhancer regions is linked to selective expression of the IL1F10 gene in differentiated human keratinocytes

Interleukin (IL)-38, encoded by the IL1F10 gene, is a member of the IL-1 family of cytokines. IL-38 is constitutively expressed in epithelia in healthy humans, and in particular in epidermal keratinocytes in the skin. IL-38 expression is closely correlated with keratinocyte differentiation. The aim of this study was to further characterize the regulation of IL1F10 expression and the mechanisms involved in its selective induction in differentiated human keratinocytes. We observed coordinated expression of two IL1F10 transcripts, transcribed from two different promoters, upon differentiation of primary human keratinocytes. Using ENCODE datasets and ChIP-qPCR on ex vivo isolated normal human epidermis, we identified regulatory regions located downstream of the IL1F10 gene, which displayed features of differentiated keratinocyte-specific enhancers. Expression of the IL1F10 gene was linked to changes in the epigenetic landscape at these downstream enhancer regions in human epidermis. Overexpression of the transcription factors KLF4 and TAp63β in an immortalized normal human keratinocyte (iNHK) cell line promoted the expression of mRNA encoding the differentiation markers keratin 10 and involucrin, and of IL1F10. ChIP-qPCR experiments indicated that KLF4 and TAp63β overexpression also modified the chromatin state of the proximal downstream enhancer region, suggesting a role for KLF4 and TAp63β in directly or indirectly regulating IL1F10 transcription.In conclusion, expression of the IL1F10 gene in differentiated keratinocytes in normal human epidermis involves coordinated transcription from two promoters and is linked to epigenetic remodeling of enhancer regions located downstream of the gene.

Exploring the Role of Sodium-Glucose Cotransporter as a New Target for Cancer Therapy

To evaluate the effects of SGLT2 inhibitors on the proliferation, tumorigenesis, migration, colony formation, apoptosis, selected gene expression pattern, and combination with known chemotherapeutic drugs in different human cancer cell lines. The antiproliferative and combined effects of SGLT2 inhibitors were evaluated by MTT assay. Cell migration was assessed using wound-healing and colony formation assays. Apoptosis assay was conducted using annexin V-FITC/ propidium iodide staining. SGLT2 gene expression was determined using real-time PCR. Canagliflozin, dapagliflozin, and ipragliflozin significantly inhibited the growth of different cancer cell lines in a dose and time-dependent manner. IC50 values after 48 hours of treatment with canagliflozin, ipragliflozin, and dapagliflozin ranged from 41.97 µM to 69.49 µM, 63.67 µM to 255.80 µM, and 167.7 µM to 435.70 µM in the examined cancer cell lines, respectively. The combined treatment of SGLT2 with doxorubicin and raloxifene separately resulted in a synergistic effect in Caco-2 and A-549 cell lines. On the other hand, the combination of SGLT2 inhibitors with cisplatin resulted in an antagonistic effect in A-549, Du-145, and Panc-1 cell lines. Canagliflozin and ipragliflozin inhibited cell migration and colony formation ability at IC50 and Sub-IC50 in the examined cancer cell lines. Canagliflozin and ipragliflozin significantly induced apoptosis at IC50 and Double-IC50 in the Du-145 cell line compared to the control. Real-time PCR showed that the treatment with 0.1 IC50 and 0.2 IC50 of both canagliflozin and ipragliflozin resulted in diminished RNA expression of SGLT2, VEGF, and Bcl-2 genes in the Du-145 cell line. SGLT2 inhibitors have antiproliferation, anti-tumorigenesis, and anti-migration effects and may induce apoptosis in cancer cells. In addition, treatment with SGLT2 inhibitors resulted in the downregulation of selected genes in the Du-145 cell line.

DNA methylation dynamics in response to abiotic and pathogen stress in plants.

DNA methylation is a dynamic epigenetic mechanism that plays a significant role in gene expression and also maintains chromatin stability. The process is conserved in both plants and animals, and crucial for development and stress responses. Differential DNA methylation during adverse environmental conditions or pathogen attack facilitates the selective expression of defense-related genes. Both stress-induced DNA hypomethylation and hypermethylation play beneficial roles in activating the defense response. These DNA marks may be carried to the next generation making the progenies 'primed' for abiotic and biotic stress responses. Over the recent years, rapid advancements in the area of high throughput sequencing have enabled the detection of methylation status at genome levels in several plant species. Epigenotyping offers an alternative tool to plant breeders in addition to conventional markers for the selection of the desired offspring. In this review, we briefly discuss the mechanism of DNA methylation, recent understanding of DNA methylation-mediated gene regulation during abiotic and biotic stress responses, and stress memory in plants.

Keratinocyte-derived S100A9 modulates neutrophil infiltration and affects psoriasis-like skin and joint disease

ObjectivesS100A9, an alarmin that can form calprotectin (CP) heterodimers with S100A8, is mainly produced by keratinocytes and innate immune cells. The contribution of keratinocyte-derived S100A9 to psoriasis (Ps) and psoriatic...

The centromeric histone CenH3 is recruited into the tombusvirus replication organelles.

Tombusviruses, similar to other (+)RNA viruses, exploit the host cells by co-opting numerous host components and rewiring cellular pathways to build extensive virus-induced replication organelles (VROs) in the cytosol of the infected cells. Most molecular resources are suboptimal in susceptible cells and therefore, tomato bushy stunt virus (TBSV) drives intensive remodeling and subversion of many cellular processes. The authors discovered that the nuclear centromeric CenH3 histone variant (Cse4p in yeast, CENP-A in humans) plays a major role in tombusvirus replication in plants and in the yeast model host. We find that over-expression of CenH3 greatly interferes with tombusvirus replication, whereas mutation or knockdown of CenH3 enhances TBSV replication in yeast and plants. CenH3 binds to the viral RNA and acts as an RNA chaperone. Although these data support a restriction role of CenH3 in tombusvirus replication, we demonstrate that by partially sequestering CenH3 into VROs, TBSV indirectly alters selective gene expression of the host, leading to more abundant protein pool. This in turn helps TBSV to subvert pro-viral host factors into replication. We show this through the example of hypoxia factors, glycolytic and fermentation enzymes, which are exploited more efficiently by tombusviruses to produce abundant ATP locally within the VROs in infected cells. Altogether, we propose that subversion of CenH3/Cse4p from the nucleus into cytosolic VROs facilitates transcriptional changes in the cells, which ultimately leads to more efficient ATP generation in situ within VROs by the co-opted glycolytic enzymes to support the energy requirement of virus replication. In summary, CenH3 plays both pro-viral and restriction functions during tombusvirus replication. This is a surprising novel role for a nuclear histone variant in cytosolic RNA virus replication.

Abstract 235: Differential polyamine expression in human pancreatic cancer supports the use of polyamine blockade therapy to modulate the in vivo pancreatic tumor microenvironment

Abstract Pancreatic cancer has a poor five-year survival rate of less than 8% and is projected to be the second leading cause of cancer related deaths in the US by the year 2030. Polyamine metabolism is an underexplored therapeutic target in pancreatic ductal adenocarcinoma (PDAC). The current investigation profiled RNA expression of genes associated with polyamine metabolism, transport and homeostasis in clinical pancreatic cancer samples to reveal evidence for dysregulation of polyamine-based pathways in PDAC. Increased expression of select pro-polyamine genes was associated with poorer patient prognosis in data obtained from the TCGA dataset. Preclinical experimental data supported the use of polyamine blockade therapy (PBT) in pancreatic tumor bearing mice, and revealed that PBT increased survival and decreased tumor weights in comparison to control treatment. PBT therapeutic effects were associated with increased expression of CD86 T cell co-stimulatory marker present on antigen presenting cells in the tumor microenvironment. Collectively, polyamine dysregulation in human pancreatic cancer is associated with poorer patient prognosis, whereas PBT treatment resulted in improved in vivo therapeutic outcomes in part through immune modulation in the tumor microenvironment. Citation Format: Sai Preethi Nakkina, Sarah B. Gitto, Veethika Pandey, Jignesh Parikh, Dirk Geerts, Carlo Maurer, Kenneth P. Olive, Otto Phanstiel, Deborah A. Altomare. Differential polyamine expression in human pancreatic cancer supports the use of polyamine blockade therapy to modulate the in vivo pancreatic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 235.

α-Tocopherol Restriction Exacerbated Lipopolysaccharide-Induced Inflammatory Response in α-Tocopherol Transfer Protein-Null Mice

ObjectivesThe α-tocopherol transfer protein-null (Ttpa−/−) mouse model is a valuable tool for studying the molecular and functional consequences of vitamin E (α-tocopherol, αT) deficiency. Our objective was to assess how dietary αT restriction, followed by lipopolysaccharide (LPS) exposure affected the inflammatory response in Ttpa−/− and wild-type (Ttpa+/+) mice.MethodsAfter weaning (3 weeks of age), male Ttpa+/+ and Ttpa−/− littermates (n = 36/genotype) were fed an αT deficient diet ad libitum for 4 weeks. At 7 weeks of age, mice were injected with LPS (1 or 10 µg/mouse) or saline (control) intraperitoneally and sacrificed 4 hours post-injection. Brain and heart IL-6 levels, a marker of inflammatory response, and serum and tissue αT concentrations were measured via ELISA and HPLC-PDA, respectively. Hippocampal Il6, Tnf, and Gpx1 expression, markers of inflammatory and oxidative stress response, were measured via RT-qPCR, and blood immune cell profiles were measured via a hematology analyzer.ResultsαT concentrations in serum and most analyzed tissues were below the limit of detection in Ttpa−/− mice but not Ttpa+/+ mice. Circulating white blood cell levels, particularly lymphocytes, were lower in all LPS groups compared to controls (P < 0.01). The 10 µg LPS groups had elevated IL-6 in the cerebellum and heart compared to controls, confirming an acute inflammatory response (P < 0.01). Hippocampal Il6 and Tnf expression were significantly increased in Ttpa−/− mice that received 10 µg LPS compared to those that received saline (∼20- and ∼3-fold higher, respectively) (P < 0.01). Comparing expression patterns by genotype, the 10 µg LPS-Ttpa−/− mice had ∼2-fold lower Gpx1 and ∼2-fold higher Il6 expression than the 10 µg LPS-Ttpa+/+ mice. Hippocampal Il6 expression was increased by LPS in a dose-dependent manner (P < 0.05).ConclusionsLPS, especially at the higher dose, altered inflammatory markers in the brain, heart, and serum. αT restriction further exacerbated the expression of select hippocampal genes.Funding SourcesAbbott Nutrition via the Center for Nutrition, Learning and Memory, University of Illinois, Urbana-Champaign; Division of Nutritional Sciences Vision 20/20 and Margin of Excellence Research grants.

Preclinical activity of irreversible Menin inhibitor, BMF-219, in chronic lymphocytic leukemia.

7541 Background: Menin is a scaffold protein that drives oncogenic function through transcriptional modulation directed by its various cofactors. A previous report demonstrated that menin regulates a distinct set of gene targets independent of its function with the MLL proteins in hematopoiesis and is essential for B-cell maturation (Li et al. Blood.2013;122(12):2039-46.). Chronic Lymphocytic Leukemia (CLL) is a disease of malignant B lymphocytes, for which standard-of-care agents are generally well tolerated; however, CLL patients with certain genetic backgrounds demonstrate inferior outcomes to these regimens. A major driving feature of CLL is overexpression of the anti-apoptotic marker, BCL2. We previously reported the ability of BMF-219, a selective, irreversible menin inhibitor, to downregulate the expression of BCL2 in acute leukemia cells. Additionally, we have reported the synergy of BCL2-targeted agent, venetoclax, with BMF-219 in potent cell killing of diffuse large B-cell lymphoma (DLBCL) preclinical models, prompting our exploration of BMF-219 activity in CLL. Here, we provide the first preclinical evidence for menin as a therapeutic target in CLL, by demonstrating high potency of BMF-219 against a diverse collection of CLL patient specimens. Methods: A comprehensive panel of CLL samples isolated from patients with Rai Stage 1 to 3 disease, including relapsed or refractory disease, were cultured ex vivo in the presence of BMF-219 or clinical reversible menin inhibitors to assess the antileukemic activity of the compounds. Samples were analyzed for differential gene expression of select targets in response to treatment. Results: BMF-219 demonstrated high potency, achieving &gt; 98% cell lethality at 1 µM exposure in all patient samples tested, with IC50 values in the range of 0.1 to 0.38 µM. Specimens isolated from patients with clinical profiles of high-risk genetic backgrounds associated with inferior outcomes to standard therapy, such as mutations in TP53 and NOTCH1, and chromosomal aberrations such as del(13q), trisomy 12 and complex karyotype, exhibited high sensitivity to BMF-219 treatment. BMF-219 was also highly effective against patient samples with clinical profiles of resistance to bendamustine, ibrutinib and venetoclax therapy. In comparison, clinical reversible menin inhibitors demonstrated no significant activity across all patient samples tested, with incalculable IC50 values and &lt; 15% reduction in cell viability at 1 µM exposure. Expression of select target genes in treated CLL cell lines was explored and will be reported. Conclusions: Collectively, our data demonstrate the potent preclinical activity of BMF-219 against CLL patient specimens harboring various mutational and cytogenetic backgrounds, including categories of high-risk. These data highlight the unique potential of irreversible menin inhibition as a novel therapeutic option for patients with CLL.

Regulation of human T-cell leukemia virus type 1 antisense promoter by myocyte enhancer factor-2C in the context of adult T-cell leukemia and lymphoma.

Adult T-cell leukemia and lymphoma (ATLL) is an intractable T-cell neoplasia caused by a retrovirus, namely human T-cell leukemia virus type 1 (HTLV-1). Patients suffering from ATLL present a poor prognosis and have a dearth of treatment options. In contrast to the sporadic expression of viral transactivator protein Tax present at the 5' promoter region long terminal repeats (LTR), HTLV-1 bZIP gene (HBZ) is encoded by 3'LTR (the antisense promoter) and maintains its constant expression in ATLL cells and patients. The antisense promoter is associated with selective retroviral gene expression and has been an understudied phenomenon. Herein, we delineate the activity of transcription factor MEF (myocyte enhancer factor)-2 family members, which were found to be enriched at the 3'LTR and play an important role in the pathogenesis of ATLL. Of the four MEF isoforms (A to D), MEF-2A and 2C were highly overexpressed in a wide array of ATLL cell lines and in acute ATLL patients. The activity of MEF-2 isoforms were determined by knockdown experiments that led to decreased cell proliferation and regulated cell cycle progression. High enrichment of MEF-2C was observed at the 3'LTR along with cofactors Menin and JunD resulting in binding of MEF-2C to HBZ at this region. Chemical inhibition of MEF-2 proteins resulted in the cytotoxicity of ATLL cells in vitro and reduction of proviral load in a humanized mouse model. Taken together, this study provides a novel mechanism of 3'LTR regulation and establishes MEF-2 signaling a potential target for therapeutic intervention for ATLL.

Molecular Subtyping of Cancer Based on Robust Graph Neural Network and Multi-Omics Data Integration.

Accurate molecular subtypes prediction of cancer patients is significant for personalized cancer diagnosis and treatments. Large amount of multi-omics data and the advancement of data-driven methods are expected to facilitate molecular subtyping of cancer. Most existing machine learning–based methods usually classify samples according to single omics data, fail to integrate multi-omics data to learn comprehensive representations of the samples, and ignore that information transfer and aggregation among samples can better represent them and ultimately help in classification. We propose a novel framework named multi-omics graph convolutional network (M-GCN) for molecular subtyping based on robust graph convolutional networks integrating multi-omics data. We first apply the Hilbert–Schmidt independence criterion least absolute shrinkage and selection operator (HSIC Lasso) to select the molecular subtype-related transcriptomic features and then construct a sample–sample similarity graph with low noise by using these features. Next, we take the selected gene expression, single nucleotide variants (SNV), and copy number variation (CNV) data as input and learn the multi-view representations of samples. On this basis, a robust variant of graph convolutional network (GCN) model is finally developed to obtain samples’ new representations by aggregating their subgraphs. Experimental results of breast and stomach cancer demonstrate that the classification performance of M-GCN is superior to other existing methods. Moreover, the identified subtype-specific biomarkers are highly consistent with current clinical understanding and promising to assist accurate diagnosis and targeted drug development.

Overexpression of the energy metabolism transcriptome within clonal plasma cells is associated with the pathogenesis and outcomes of patients with multiple myeloma.

Altered energy metabolism and changes in glycolytic and oxidative phosphorylation pathways are hallmarks of all cancer cells. The expression of select genes associated with the production of various enzymes and proteins involved in glycolysis and oxidative phosphorylation were assessed in the clonal plasma cells derived from patients with newly diagnosed multiple myeloma (NDMM) enrolled in the Multiple Myeloma Research Foundation (MMRF) CoMMpass data set. A scoring system consisting of assigning a point for every gene where their fragments per kilobase of transcript per million (FPKM) was above the median yielded a minimum of 0 and a maximum of 12 for the set of genes in the glycolytic and oxidative phosphorylation pathways to create a total energy metabolism molecular signature (EMMS) score. This EMMS score was independently associated with worse progression free survival (PFS) and overall survival (OS) outcomes of patients with NDMM. A higher EMMS score was more likely to be present in clonal plasma cells derived from Multiple myeloma (MM) patients than those from patients with monoclonal gammopathy of undetermined significance (MGUS). This was functionally confirmed by the clonal plasma cells from MM patients having a higher rate of mitochondrial and glycolysis-derived ATP formation than clonal plasma cells from MGUS patients. Thus, this study provides evidence for the effect of energy metabolism within clonal plasma cells on pathogenesis and outcomes of patients with MM. Exploiting the energy-producing metabolic pathways within clonal plasma cells for diagnostic and therapeutic purposes in MM should be explored in the future.

Gene expression changes in Daphnia magna following waterborne exposure to cyanobacterial strains from the genus Nostoc

Cyanobacteria can produce highly potent cyanotoxins, however, limited information is provided about their toxicity mechanisms in exposed aquatic invertebrates at the molecular level. In the present study, the effects of cyanobacterial strains from the genus Nostoc (Nostoc Z1 and Nostoc 2S3B) in Daphnia magna after waterborne exposure were investigated. Examined endpoints included immobilization (survival) in acute toxicity tests and selected gene expression changes (cyp314, cyp360A8, gst, p-gp, vtg) analyzed by the quantitative real-time polymerase chain reaction (RT-PCR). In addition, enzyme-linked immunosorbent assay (ELISA) was performed to determine whether the observed changes could be due to the presence of microcystins, the most widespread group of cyanotoxins. The results of acute toxicity tests have shown only minor changes in survival rates, which have not exceeded 20% after 48 h of exposure to either strain. On the other hand, significant changes were recorded in molecular responses of Daphnia to tested strains. Treatment with the aquatic strain Nostoc Z1 altered the expression levels of all analyzed genes. Both strains caused a significant p-glycoprotein (p-gp) induction at 75 µg ml−1 which suggests the involvement of p-gp mediated multixenobiotic resistance mechanism (MXR) in facilitating excretion of toxic cyanobacterial compounds in daphnids. Additionally, these strains caused an increase in the expression levels of cyp360A8, indicating that genes related to detoxification processes could be sensitive indicators of cyanobacterial toxicity. Statistically significant induction of cyp314, as well as increases in expression of gst and vtg, were observed only after exposure to Nostoc Z1. This study indicates the potential of certain cyanobacterial metabolites to modify the expression of toxicant responsive genes involved in phase I and phase III of the xenobiotic metabolism, as well as possible interference with growth and reproduction in D. magna. Low microcystin concentrations found in both samples suggest that these cyanotoxins were not responsible for the detected toxic effects.

Differential expression of the Mycobacterium tuberculosis heat shock protein genes in response to drug-induced stress

Heat shock proteins are essential in maintaining cellular protein function, especially during stress. Their influence in managing drug-induced stress in Tuberculosis is not clearly understood. AimsStudy the expression of select genes of the DnaK/ClpB chaperone network to evaluate their role in stress response in Mycobacterium tuberculosis clinical isolates during exposure to Isoniazid (INH) and Rifampicin (RIF). MethodsSanger sequencing to detect drug-resistant mutations followed by Drug Susceptibility Testing and Minimum Inhibitory Concentration determination. Culturing the bacilli in vitro, exposed to 1/4, 1/2 and 1 × MIC, and RNA quantification of dnaK, dnaJ1, grpE and clpB genes by using Real-time PCR. ResultsSusceptible isolates showed marginal down-regulation of two genes for INH, whereas all genes under-expressed against RIF. INH-resistant isolates had distinct expression profiles for inhA-15 and katG315 mutants. RIF-resistant bacilli did not have significant differential expression. MDR isolate showed up-regulation of all the four genes, with two genes over-expressing (≥4-fold). ConclusionsWe observed characteristic gene expression profiles for each isolate in response to lethal and sub-lethal doses of INH and RIF. This provides insight into the role of DnaK/ClpB chaperone network in managing drug-induced stress and facilitating resistance. Further, the knowledge could provide targets for new drugs and augmenters.

Tgfbr2 in Dental Pulp Cells Guides Neurite Outgrowth in Developing Teeth.

Transforming growth factor β (TGFβ) plays an important role in tooth morphogenesis and mineralization. During postnatal development, the dental pulp (DP) mesenchyme secretes neurotrophic factors that guide trigeminal nerve fibers into and throughout the DP. This process is tightly linked with dentin formation and mineralization. Our laboratory established a mouse model in which Tgfbr2 was conditionally deleted in DP mesenchyme using an Osterix promoter-driven Cre recombinase (Tgfbr2 cko ). These mice survived postnatally with significant defects in bones and teeth, including reduced mineralization and short roots. Hematoxylin and eosin staining revealed reduced axon-like structures in the mutant mice. Reporter imaging demonstrated that Osterix-Cre activity within the tooth was active in the DP and derivatives, but not in neuronal afferents. Immunofluorescence staining for β3 tubulin (neuronal marker) was performed on serial cryosections from control and mutant molars on postnatal days 7 and 24 (P7, P24). Confocal imaging and pixel quantification demonstrated reduced innervation in Tgfbr2 cko first molars at both stages compared to controls, indicating that signals necessary to promote neurite outgrowth were disrupted by Tgfbr2 deletion. We performed mRNA-Sequence (RNA-Seq) and gene onotology analyses using RNA from the DP of P7 control and mutant mice to investigate the pathways involved in Tgfbr2-mediated tooth development. These analyses identified downregulation of several mineralization-related and neuronal genes in the Tgfbr2 cko DP compared to controls. Select gene expression patterns were confirmed by quantitative real-time PCR and immunofluorescence imaging. Lastly, trigeminal neurons were co-cultured atop Transwell filters overlying primary Tgfbr2 f/f DP cells. Tgfbr2 in the DP was deleted via Adenovirus-expressed Cre recombinase. Confocal imaging of axons through the filter pores showed increased axonal sprouting from neurons cultured with Tgfbr2-positive DP cells compared to neurons cultured alone. Axon sprouting was reduced when Tgfbr2 was knocked down in the DP cells. Immunofluorescence of dentin sialophosphoprotein in co-cultured DP cells confirmed reduced mineralization potential in cells with Tgfbr2 deletion. Both our proteomics and RNA-Seq analyses indicate that axonal guidance cues, particularly semaphorin signaling, were disrupted by Tgfbr2 deletion. Thus, Tgfbr2 in the DP mesenchyme appears to regulate differentiation and the cells’ ability to guide neurite outgrowth during tooth mineralization and innervation.

The Mlc1 Promoter Directs Müller Cell-specific Gene Expression in the Retina

PurposeBecause the importance of glia in regulating brain functions has been demonstrated, genetic technologies that manipulate glial cell-specific gene expression in the brain have become essential and have made great progress. However, it is unknown whether the same strategy that is used in the brain can be applied to the retina because retinal glia differs from glia in the brain. Here, we aimed to find a method for selective gene expression in Müller cells (characteristic glial cells in the retina) and identified Mlc1 as a specific promoter of Müller cells.MethodsMlc1-tTA::Yellow-Cameleon-NanotetO/tetO (YC-Nano) mice were used as a reporter line. YC-Nano, a fluorescent protein, was ectopically expressed in the cell type controlled by the Mlc1 promotor. Immunofluorescence staining was used to identify the cell type expressing YC-Nano protein.ResultsYC-Nano-positive (+) signals were observed as vertical stalks in the sliced retina and spanned from the nerve fiber layer through the outer nuclear layer. The density of YC-Nano+ cells was higher around the optic nerve head and lower in the peripheral retina. The YC-Nano+ signals colocalized with vimentin, a marker of Müller cells, but not with the cell markers for blood vessels, microglia, neurons, or astrocytes.ConclusionsThe Mlc1 promoter allows us to manipulate gene expression in Müller cells without affecting astrocytes in the retina.Translational RelevanceGene manipulation under control of Mlc1 promoter offers novel technique to investigate the role of Müller cells.

Fat-associated lymphoid clusters as expandable niches for ectopic liver development.

Background and AimsHepatocyte transplantation holds great promise as an alternative approach to whole‐organ transplantation. Intraportal and intrasplenic cell infusions are primary hepatocyte transplantation delivery routes for this procedure. However, patients with severe liver diseases often have disrupted liver and spleen architectures, which introduce risks in the engraftment process. We previously demonstrated i.p. injection of hepatocytes as an alternative route of delivery that could benefit this subpopulation of patients, particularly if less invasive and low‐risk procedures are required; and we have established that lymph nodes may serve as extrahepatic sites for hepatocyte engraftment. However, whether other niches in the abdominal cavity support the survival and proliferation of the transplanted hepatocytes remains unclear.Approach and ResultsHere, we showed that hepatocytes transplanted by i.p. injection engraft and generate ectopic liver tissues in fat‐associated lymphoid clusters (FALCs), which are adipose tissue–embedded, tertiary lymphoid structures localized throughout the peritoneal cavity. The FALC‐engrafted hepatocytes formed functional ectopic livers that rescued tyrosinemic mice from liver failure. Consistently, analyses of ectopic and native liver transcriptomes revealed a selective ectopic compensatory gene expression of hepatic function–controlling genes in ectopic livers, implying a regulated functional integration between the two livers. The lack of FALCs in the abdominal cavity of immunodeficient tyrosinemic mice hindered ectopic liver development, whereas the restoration of FALC formation through bone marrow transplantation restored ectopic liver development in these mice. Accordingly, induced abdominal inflammation increased FALC numbers, which improved hepatocyte engraftment and accelerated the recovery of tyrosinemic mice from liver failure.ConclusionsAbdominal FALCs are essential extrahepatic sites for hepatocyte engraftment after i.p. transplantation and, as such, represent an easy‐to‐access and expandable niche for ectopic liver regeneration when adequate growth stimulus is present.

Prognostic Utility of CpG Island Hypermethylated Phenotype in Early-Stage Invasive Primary Melanomas

In their article in the Journal of Investigative Dermatology, Conway etal. (2021) show the significance of the CpG island methylator phenotype (CIMP) subclass in early-stage invasive primary melanomas, particularly in the lentigo maligna melanoma histological subtype. CIMP melanomas had reduced melanoma-specific survival compared with intermediate and low methylation subtypes. This study emphasizes the importance of epigenomic changes in early-stage primary tumors that are associated with poor prognosis.