Changes In Relative Expression Research Articles

Lactobacillus delbrueckii ameliorates Aeromonas hydrophila-induced oxidative stress, inflammation, and immunosuppression of Cyprinus carpio huanghe var NF-κB/Nrf2 signaling pathway

Aeromonas hydrophila (A. hydrophila) is one of the most pathogenic disease-causing bacteria, and causes massive death of animals including fish. Thus, strategies are being sought to ameliorate the impact of A. hydrophila. In this study, we have evaluated the ameliorative potential of dietary Lactobacillus delbrueckii (L. delbrueckii). The fishes were divided into the control group, an A. hydrophila group (A. hydrophila), and an L. delbrueckii group (A. hydrophila + 1*107 CFU/g L. delbrueckii). The results showed that A. hydrophila increased reactive oxygen species (ROS) content. However, dietary supplementation with L. delbrueckii prevented oxidative damage caused by elevated levels of ROS. The toxic effects of A. hydrophila on superoxide dismutase (SOD) activity, glutathione-S-transferase (GST), glutathione peroxidase (GPx), and glutathione reductase (GR), along with the levels of glutathione (GSH), were mitigated by dietary L. delbrueckii (P < 0.05). Also, Dietary L. delbrueckii induced the expression of antioxidant-related genes (sod, cat, gpx, gst, NF-E2-related factor 2 (nrf2), Kelch-like-ECH-and associated protein 1a (keap1a)) in the intestine of fish (P < 0.05). Furthermore, L. delbrueckii increased A. hydrophila-induced lysozyme, ACP, C3, and C4 decline. The mRNA expression levels of interleukin 1β (il-1β), interleukin 8 (il-8), tumour necrosis factor α (tnf-α), and nuclear transcription factor-κB p65 (nf-κb p65) were significantly elevated by A. hydrophila. In contrast, the relative mRNA expression levels of inhibitor factor κBα (iκbα) in the intestine were decreased by A. hydrophila (P < 0.05). However, L. delbrueckii prevented A. hydrophila-induced the relative mRNA expression changes. These present results demonstrate that dietary L. delbrueckii alleviated A. hydrophila-induced oxidative stress, immunosuppression, inflammation, and apoptosis in common Cyprinus carpio.

The C. elegans Myc-family of transcription factors coordinate a dynamic adaptive response to dietary restriction.

Dietary restriction (DR), the process of decreasing overall food consumption over an extended period of time, has been shown to increase longevity across evolutionarily diverse species and delay the onset of age-associated diseases in humans. In Caenorhabditis elegans, the Myc-family transcription factors (TFs) MXL-2 (Mlx) and MML-1 (MondoA/ChREBP), which function as obligate heterodimers, and PHA-4 (orthologous to FOXA) are both necessary for the full physiological benefits of DR. However, the adaptive transcriptional response to DR and the role of MML-1::MXL-2 and PHA-4 remains elusive. We identified the transcriptional signature of C. elegans DR, using the eat-2 genetic model, and demonstrate broad changes in metabolic gene expression in eat-2 DR animals, which requires both mxl-2 and pha-4. While the requirement for these factors in DR gene expression overlaps, we found many of the DR genes exhibit an opposing change in relative gene expression in eat-2;mxl-2 animals compared to wild-type, which was not observed in eat-2 animals with pha-4 loss. Surprisingly, we discovered more than 2000 genes synthetically dysregulated in eat-2;mxl-2, out of which the promoters of down-regulated genes were substantially enriched for PQM-1 and ELT-1/3 GATA TF binding motifs. We further show functional deficiencies of the mxl-2 loss in DR outside of lifespan, as eat-2;mxl-2 animals exhibit substantially smaller brood sizes and lay a proportion of dead eggs, indicating that MML-1::MXL-2 has a role in maintaining the balance between resource allocation to the soma and to reproduction under conditions of chronic food scarcity. While eat-2 animals do not show a significantly different metabolic rate compared to wild-type, we also find that loss of mxl-2 in DR does not affect the rate of oxygen consumption in young animals. The gene expression signature of eat-2 mutant animals is consistent with optimization of energy utilization and resource allocation, rather than induction of canonical gene expression changes associated with acute metabolic stress, such as induction of autophagy after TORC1 inhibition. Consistently, eat-2 animals are not substantially resistant to stress, providing further support to the idea that chronic DR may benefit healthspan and lifespan through efficient use of limited resources rather than broad upregulation of stress responses, and also indicates that MML-1::MXL-2 and PHA-4 may have distinct roles in promotion of benefits in response to different pro-longevity stimuli.

Heterotrophic bacteria trigger transcriptome remodelling in the photosynthetic picoeukaryote Micromonas commoda.

Marine biogeochemical cycles are built on interactions between surface ocean microbes, particularly those connecting phytoplankton primary producers to heterotrophic bacteria. Details of these associations are not well understood, especially in the case of direct influences of bacteria on phytoplankton physiology. Here we catalogue how the presence of three marine bacteria (Ruegeria pomeroyi DSS-3, Stenotrophomonas sp. SKA14 and Polaribacter dokdonensis MED152) individually and uniquely impact gene expression of the picoeukaryotic alga Micromonas commoda RCC 299. We find a dramatic transcriptomic remodelling by M. commoda after 8 h in co-culture, followed by an increase in cell numbers by 56 h compared with the axenic cultures. Some aspects of the algal transcriptomic response are conserved across all three bacterial co-cultures, including an unexpected reduction in relative expression of photosynthesis and carbon fixation pathways. Expression differences restricted to a single bacterium are also observed, with the Flavobacteriia P. dokdonensis uniquely eliciting changes in relative expression of algal genes involved in biotin biosynthesis and the acquisition and assimilation of nitrogen. This study reveals that M. commoda has rapid and extensive responses to heterotrophic bacteria in ways that are generalizable, as well as in a taxon specific manner, with implications for the diversity of phytoplankton-bacteria interactions ongoing in the surface ocean.

Systematic identification and characterization of microRNAs with target genes involved in high night temperature stress at the filling stage of rice.

High night temperature stress is one of the main environmental factors affecting rice yield and quality. More and more evidence shows that microRNA (miRNA) plays an important role in various abiotic stresses. However, the molecular network of miRNA regulation on rice tolerance to high night temperatures remains unclear. Here, small RNA, transcriptome and degradome sequencing were integrated to identify differentially expressed miRNAs, genes, and key miRNA-target gene pairs in rice heat-sensitive and heat-tolerant lines at the filling stage suffering from high night temperature stress. It was discovered that there were notable differences in the relative expression of 102 miRNAs between the two rice lines under stress. Meanwhile, 5263 and 5405 mRNAs were differentially expressed in the heat-sensitive line and heat-tolerant line, and functional enrichment analysis revealed that these genes were involved in heat-related processes and pathways. The miRNAs-mRNAs target relationship was further verified by degradome sequencing. Eventually, 49 miRNAs-222 mRNAs target pairs with reverse expression patterns showed significant relative expression changes between the heat-tolerant and the heat-sensitive line, being suggested to be responsible for the heat tolerance difference of these two rice lines. Functional analysis of these 222 mRNA transcripts showed that high night temperature-responsive miRNAs targeted these mRNAs involved in many heat-related biological processes, such as transcription regulation, chloroplast regulation, mitochondrion regulation, protein folding, hormone regulation and redox process. This study identified possible miRNA-mRNA regulation relationships in response to high night temperature stress in rice and potentially contributed to heat resistance breeding of rice in the future.

Enhanced Fc Receptor Expression In Sea Urchin Spermatozoa By Platelet-Activating Factor

Platelet-activating factor (PAF) is a signaling phospholipid that has a variety of reproductive roles, e.g. sperm capacitation and the acrosome reaction (an exocytotic process). PAF’s mechanism action involves G-protein receptor activation induction of intracellular calcium, which enhances sperm motility. In nonreproductive cells, the Fc receptor (FcR) functions in ligand-triggered transmission signals across the membrane which alters secretion, exocytosis, and increased intracellular calcium. This study looked at the impact of PAF on FcR expression in sea urchin spermatozoa. Sea urchin sperm were exposed to exogenous PAF (10-7M; 15 minutes) and evaluated for FcR activity via flow cytometry. A significant (P=0.01) difference (64.30%) in FcR wave activity between the PAF (mean 3.286) and control (2.0) groups was observed. Within waves, there were more (83.31%) high points (crests) in the PAF (mean 1.571) group than the control (mean 0.857) group. Similarly, there were more (49.96%) low (troughs) points in the PAF (mean 1.714) group than the control (mean 1.143) group. This is the first report to demonstrate a relative positive change in spermatozoa FcR expression when exposed to PAF. The FcR functions in ligand-triggered transmission of PAF via a G-protein coupled receptor mediated pathway leading to physiological alterations. Provided the known mechanism of action of PAF on spermatozoa physiology, the data suggests that the interaction of PAF and FcR play a pivotal role in sperm activity and fertilization potential.

Pre and Postoperative Serum miR-21 Expression Levels in Oral Cancer and it’s Association with Clinical and Histological Parameters: An Observational Study

Introduction: The patterns of microRNA (miRNA) expression have opened up new avenues in the search for prognostic biomarkers and potential therapeutic targets for various tumours. In oral carcinogenesis research, analysing microRNAs expressed in Oral Squamous Cell Carcinoma (OSCC) unveils a complex network of interest. miR-21 is known to be overexpressed in numerous solid tumours and is linked to the progression of malignancies in hepatocellular carcinomas, breast cancer, and colon carcinomas. Aim: To determine the impact of surgical excision on serum miR-21 expression levels in OSCC cases and to establish correlations with clinicopathological parameters. Materials and Methods: The study was an exploratory prospective observational study conducted with proper institutional ethical approval at the Cancer Research Institute (CRI), Himalayan Institute of Medical Sciences (HIMS), Jolly Grant, Dehradun, Uttarakhand, India. Fifty-six histologically confirmed OSCC cases were enrolled along with 25 healthy subjects as controls from May 2021 to June 2023. The relative fold expression change was calculated using the Livak method (2-∆∆ct). Data analysis was performed using software such as Statistical Package for Social Sciences (SPSS) version 20.0 and MS Excel. Non parametric statistical tests such as Wilcoxon signed-rank test, MannWhitney test, and Kruskal-Wallis test were utilised. Results: Out of 56 sample included, 28 exhibited high expression of miR-21 (p-value&lt;0.001), while 27 cases showed downregulation postsurgery (p-value&lt;0.001), with one sample showing the same level of expression. In cases where the time difference between pre and postsurgery samples was over 25 days, no significant change was observed (p-value=0.06), and similarly, in cases with a time difference below 25 days, no significant difference was noted (p-value=0.14). A significant negative correlation was found between presurgery serum Albumin to Globulin ratio (A:G ratio) and miR-21 expression (p-value &lt;0.05), while no other parameters showed a significant correlation with miR-21. Conclusion: miR-21 expression decreased significantly in some OSCC cases; however, in the majority of cases, it increased postsurgery. A significant correlation was observed between miR-21 expression and the serum A:G ratio.

Structural and Functional Analysis of Crucial Protein Complex in Basal Cell Skin Carcinoma via Network Construction

 Background: The basal-cell carcinoma (BCC) as one of the most common type of skin cancers reported to have an increasing trend during past years. Molecular approaches can be useful to advance diagnosis and treatment goals in this cancer.Materials and Methods: In this sense, one of the recent popular investigations, protein-protein interaction network analysis (PPI), was applied in this study to better facilitate molecular view of BCC.  Cytoscape v3.6.0 and its plug-ins analyzed and explored the topological and annotation features of the constructed network. Result: Among TP53, EGFR, AKT1, ERBB2, HRAS, and CCND1 as central agents of the network, five of them were also present in the first prominent cluster of the network in which considered for further analysis. It is suggested that there are significant related biological processes, actions, and expression changes for this highlighted cluster that may be related to BCC risk.  Conclusion: Therefore, the studied complex of proteins may worth considering for clinical studies and therapeutic interventions after validating by related tests. What is more, among these genes, EBBR2 has more to offer and consequently with additional values.  [GMJ.2018;7:e1271]

MiRNA-369-5p within Small Extracellular Vesicles Correlates with Acute Chest Syndrome in Patients with Sickle Cell Disease

Introduction: Acute chest syndrome (ACS) is one of the leading causes of mortality in patients with sickle cell disease (SCD). Despite this, there are no biomarkers that can distinguish patients who present with a painful vaso-occlusive episode (VOE) and may rapidly develop ACS. Endothelial damage is a critical component of the pathophysiology of SCD. Small extracellular vesicles (EVs) are endocytic-derived vesicles that package protein and nucleic acid cargo for communication between cells. We and others have demonstrated that EVs have unique properties in patients with SCD. We have shown that EVs from SCD patients differentially affect endothelial integrity in vitro. We have previously performed miRNA sequencing and identified 15 miRNA that were differentially expressed based on a history of ACS. In this study we sought to closely assess how three of these miRNA, miR-let7c-5p, miR-122-5p, and miR-369-5p, differ across the timeline of patients with sickle cell, ie. at baseline, during ACS and during VOE. Methods: The SCD biobank at the University of Chicago Comer Children's Hospital and LaRabida Children's Hospital was queried for patients who had multiple blood samples over many years that included at least one each of a baseline (&amp;gt; 4 weeks from an acute event), a hospitalization for ACS and a hospitalization for a VOE. All subjects had been prospectively enrolled, with informed consent provided by parents or patients aged &amp;gt;18 years and assent obtained from subjects aged 9-18 years. Control samples (n=5) were obtained from patients without SCD who received bloodwork for routine check-ups or for follow-up of iron-deficiency anemia. Equal volumes of control samples were pooled for consistency across experiments. EVs were isolated via Total Exosome Isolation Kit (Thermo Fisher Scientific) per the manufacturer's guidelines. RNA was purified from samples using the miRNeasy Micro Kit (Qiagen). For quantitative PCR, complementary DNA was generated with the TaqMan Advanced miRNA cDNA Synthesis kit (Thermo Fisher Scientific). Specified miRNAs (hsa-miR-122-5p, hsa-miR-99a-5p, hsa-miR-369-5p, hsa-miR-let-7c-5p) were analyzed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and TaqMan Advanced miRNA Assay primers (Thermo Fisher Scientific). Samples were analyzed in triplicate on a 7500 Fast Real-Time PCR machine (Applied Biosystems) and threshold cycle values were normalized to cel-miR-39 controls. Relative changes in expression for ACS and VOE samples were calculated using the respective baseline samples or non-SCD controls as reference values and theΔΔ quantification cycle method. Results: To determine whether exosomal miRNA levels correlated with the disease status of patients, normalized samples from all patients were aggregated and then stratified based on sample type (baseline, VOE, ACS, or ACS post transfusion). While there were no statistically significant differences in exosomal expression of miR-let-7c-5p and miR-122-5p between sample types, there was a promising trend for miR-122-5p. There was a 2.6-fold increase in miR-122-5plevels, relative to baseline, when patients experienced a vaso-occlusive episode. In contrast, miR-122-5p levels decreased 2.3-fold, relative to baseline, when patients were hospitalized with acute chest syndrome. Extravesicular miR-369-5p levels were differentially regulated and correlated with the disease status of patients. When patients were hospitalized with a VOE or ACS, there was a 2.9-fold and 10-fold increase in miR-369-5p levels, respectively, compared to their baseline. In addition, there was a statistically significant difference between VOE and ACS samples, which suggests that miR-369-5p levels can distinguish between VOE and ACS events (Fig. 3C). Interestingly, when ACS samples were obtained post-transfusion (ACS ptx), miR-369-5p levels decreased significantly to values near that of VOE samples. Conclusions: In this study, we report that the miRNA-369-5p levels in small EVs are differentially up-regulated in SCD patients during an ACS event. Furthermore, we show that these levels can distinguish patients presenting with a VOE from those who eventually develop ACS. Our findings demonstrate that it is possible to utilize extracellular vesicles and their contents to predict ACS. Further studies should be done so there is a tool to predict ACS and improve the clinical outcomes for patients with SCD.

Sars-Cov-2 Spike Protein Triggers Inflammasome-Mediated Tissue Factor Activity

Introduction: An exaggerated inflammatory response both locally and systemically mediated by innate immune signaling is a near universal feature of severe COVID-19 illness and has been associated with multi-organ damage and thrombosis. As part of an antiviral response, activation and formation of the intracellular NLRP3 inflammasome leads to expression of proinflammatory IL-1 family cytokines. In macrophages, the NLRP3 inflammasome is linked to the release of tissue factor (TF) bearing microvesicles. Multiple studies provide evidence of NLRP3 inflammasome activation in COVID-19 including increased IL-1β in monocytes, sera, and bronchoalveolar lavage fluid from COVID-19 patients. Patient monocytes bear features of inflammasome activation, which correlate with disease severity (Potere et al. Cardiovascular Research 2023). Given the central role of thromboinflammation in COVID-19, we explored the ability of various coronavirus spike proteins to increase TF activity in a monocytic cell line. Mechanistically, we sought to explore the role of the NLRP3 inflammasome in SARS-CoV-2 spike protein triggered tissue factor generation. Methods: The SARS-CoV-2 α variant S1 subunit (S1α) and Omicron variant BA.1.1.529 S1 (S1O) recombinant proteins were obtained from Raybiotech and OC43, a non-COVID respiratory human coronavirus, recombinant spike protein (OC43 S) from Sino Biological. THP-1 cells at a concentration of 2x10 6/mL were incubated with spike proteins at 37°C in RPMI, supplemented with 10% fetal bovine serum. A specific NLRP3 inflammasome inhibitor (MCC950) was incubated with select samples for 1 hour following addition of the spike protein. The cells were harvested, subjected to RNA extraction and cDNA synthesis by reverse transcription. TF, NLRP3, caspase (CASP)-1, and IL-1β relative mRNA expression was quantified using RT-PCR and normalized to GADPH expression. For quantifying microparticle TF activity, cells were incubated with the spike proteins for 6 hours then microparticles were isolated from the supernatant after centrifugation at 20,000 g for 30 minutes at 4°C, washed and TF activity was measured using a two-stage chromogenic assay kit (Abcam). A monoclonal anti-TF antibody (Santa Cruz) was used to confirm specificity for TF generation. Experiments were performed in triplicate. Data is presented as mean ± standard deviation. The student t test was used to assess the difference between unpaired groups with a value of p &amp;lt;.05 considered statistically significant. Results: Our previous data showed that S1α, but not S1O or OC43 S, leads to increased TF mRNA expression in THP-1 cells in a dose dependent manner. Here we show TF microparticle activity was increased with S1α (20 µg/mL) as compared to incubation of THP-1 cells with S1O or OC43 spike proteins (20 µg/mL), which were comparable to the activity of cells alone ( Figure 1). After treatment with S1α (20 µg/mL) for 1 hour, we found increased expression of TF (10-fold), NLRP3 (2-fold), CASP-1 (3-fold), and IL-1β (3.5-fold). Treatment with S1O and OC43 S did not lead to a significant change in relative mRNA expression of TF, NLRP3, CASP-1, and IL-1β. Following incubation with the S1α spike protein for 1 hour, cells were re-plated in fresh media containing 10 µM MCC950. Treatment with MCC950 significantly reduced expression of TF, NLRP3, CASP-1, and IL-1β ( Figure 2). Conclusion: We confirmed that TF expression induced by the S1α spike protein leads to an increase in TF activity, as assessed by microparticle generation. The NLRP3 inflammasome is a mediator of coagulopathy induced by the spike protein, and TF, CASP-1, and IL-1β expression are abrogated after specific NLRP3 inflammasome inhibition. TF activity and inflammation induced by the coronavirus spike proteins in vitro correlates with the respective clinical phenotype and disease severity of the variants. This suggests that the effects of SARS-CoV-2 on TF and inflammasome activation derive in part from the spike protein, and these assays may be useful in predicting the effects of spike protein variants.

Transcriptomic analysis reveals that RasGEF1b deletion alters basal and LPS-induced expression of genes involved in chemotaxis and cytokine responses in macrophages

Ras guanine nucleotide exchange factor member 1b (RasGEF1b) of the RasGEF/CDC25 domain-containing family is preferentially expressed by macrophages. However, information is lacking about its role in macrophage function. In this study, we generated mice with ubiquitous deletion of Rasgef1b and used RNA-seq-based transcriptomics to compare the global gene expression in wild-type and knock-out primary bone-marrow-derived macrophages under basal conditions and after lipopolysaccharide (LPS) treatment. Transcriptional filtering identified several genes with significantly different transcript levels between wild-type and knock-out macrophages. In total, 49 and 37 differentially expressed genes were identified at baseline and in LPS-activated macrophages, respectively. Distinct biological processes were significantly linked to down-regulated genes at the basal condition only, and largely included chemotaxis, response to cytokines, and positive regulation of GTPase activity. Importantly, validation by RT-qPCR revealed that the expression of genes identified as down-regulated after LPS stimulation was also decreased in the knock-out cells under basal conditions. We used a luciferase-based reporter assay to showcase the capability of RasGEF1b in activating the Serpinb2 promoter. Notably, knockdown of RasGEF1b in RAW264.7 macrophages resulted in impaired transcriptional activation of the Serpinb2 promoter, both in constitutive and LPS-stimulated conditions. This study provides a small collection of genes that shows relative expression changes effected by the absence of RasGEF1b in macrophages. Thus, we present the first evidence that RasGEF1b mediates the regulation of both steady-state and signal-dependent expression of genes and propose that this GEF plays a role in the maintenance of the basal transcriptional level in macrophages.

Increased pituitary adenylate cyclase-activating peptide genes expression in the prefrontal cortex in schizophrenia in relation to suicide.

Pituitary adenylate cyclase-activating peptide (PACAP) is a stress-related neuropeptide that is produced in several brain areas. It acts by 3 receptors: PACAP type-1 (PAC1), vasoactive intestinal peptide (VIP) -1 and -2 (VPAC1 and 2). Data on polymorphisms in PACAP and PAC1 indicate a relationship of the PACAP system with schizophrenia (SCZ). The prefrontal cortex was chosen to measure PACAP-gene related expression changes, since this is a central structure in the symptoms of schizophrenia (SCZ). We investigated alterations in the expression of the PACAP-related genes by qPCR in the human dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC) of 35 SCZ patients and 34 matched controls in relation to SCZ, suicide, gender and medication. The ACC revealed an upregulation in PACAP, PAC1, VPAC1 and VPAC2 in SCZ suicide (S) completers compared to controls. An increase in PACAP, VPAC1 and VPAC2 expression was also present in the ACC in SCZ-S compared to SCZ patients who died naturally (SCZ-N). In the DLPFC, an increase in PAC1 was found in SCZ-N patients compared to SCZ-S and controls. Moreover, an increase in all PACAP-related genes was present in SCZ-N male patients compared to SCZ-N females. Concluding, expression changes were found in PACAP-related genes in relation to SCZ, suicide and gender. In particular, there was a higher PACAP-related gene expression in SCZ patients in the ACC in relation to suicide and in DLPFC in relation to SCZ. These findings suggest a potential link between PACAP and the pathophysiology of SCZ and suicide. Further research is needed to understand the functional significance and potential clinical applications of these changes.

Comparative transcriptome analysis of high- and low-embryogenic Hevea brasiliensis genotypes reveals involvement of phytohormones in somatic embryogenesis

BackgroundRubber plant (Hevea brasiliensis) is one of the major sources of latex. Somatic embryogenesis (SE) is a promising alterative to its propagation by grafting and seed. Phytohormones have been shown to influence SE in different plant species. However, limited knowledge is available on the role of phytohormones in SE in Hevea. The anther cultures of two Hevea genotypes (Yunyan 73477-YT and Reken 628-RT) with contrasting SE rate were established and four stages i.e., anthers (h), anther induced callus (y), callus differentiation state (f), and somatic embryos (p) were studied. UPLC-ESI-MS/MS and transcriptome analyses were used to study phytohormone accumulation and related expression changes in biosynthesis and signaling genes.ResultsYT showed higher callus induction rate than RT. Of the two genotypes, only YT exhibited successful SE. Auxins, cytokinins (CKs), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), gibberellins (GAs), and ethylene (ETH) were detected in the two genotypes. Indole-3-acetic acid (IAA), CKs, ABA, and ETH had notable differences in the studied stages of the two genotypes. The differentially expressed genes identified in treatment comparisons were majorly enriched in MAPK and phytohormone signaling, biosynthesis of secondary metabolites, and metabolic pathways. The expression changes in IAA, CK, ABA, and ETH biosynthesis and signaling genes confirmed the differential accumulation of respective phytohormones in the two genotypes.ConclusionThese results suggest potential roles of phytohormones in SE in Hevea.

Effect of BTS, GEPS and MODENA Extender on DNA Integrity and Relative Expression of HSP70 and Cas3 Gene in HD-K75 Boar Semen

Background: The present study compared the quality characteristics of boar semen diluted with three extenders at different hours of preservation. Methods: A total of 24 ejaculates comprising 6 ejaculates from each of four boars of 10-12 months age maintained under uniform management conditions are being selected for the present study. Semen was collected once weekly from four clinically healthy HD-K75 boars by simple fist method. The ejaculated semen was extended in BTS, GEPS and MODENA extender and maintained at 15°C upto 120 hours of preservation. The DNA integrity and the mean fold change in relative expression of HSP70 and Cas3 gene in BTS, GEPS and MODENA extender is also being studied. Result: The percentage of DNA integrity revealed that no DNA damaged spermatozoa were observed in MODENA extender from 0 to 120 hours of preservation. The relative expression of HSP70 gene elucidated significant difference (P less than 0.01) at 24 hours in three extenders. The relative expression of Cas3 gene in BTS, GEPS and MODENA extender differed significantly (P less than 0.01) at 0, 72 and 96 hours of preservation. The overall expression (fold change) of apoptotic gene was found to be higher in BTS extender followed by MODENA and GEPS.

Combining RNAscope and immunohistochemistry to visualize inflammatory gene products in neurons and microglia.

A challenge for central nervous system (CNS) tissue analysis in neuroscience research has been the difficulty to codetect and colocalize gene and protein expression in the same tissue. Given the importance of identifying gene expression relative to proteins of interest, for example, cell-type specific markers, we aimed to develop a protocol to optimize their codetection. RNAscope fluorescent in situ hybridization (FISH) combined with immunohistochemistry (IHC) in fixed (CNS) tissue sections allows for reliable quantification of gene transcripts of interest within IHC-labeled cells. This paper describes a new method for simultaneous visualization of FISH and IHC in thicker (14-μm), fixed tissue samples, using spinal cord sections. This method's effectiveness is shown by the cell-type-specific quantification of two genes, namely the proinflammatory cytokine interleukin-1beta (IL-1b) and the inflammasome NLR family pyrin domain containing 3 (NLRP3). These genes are challenging to measure accurately using immunohistochemistry (IHC) due to the nonspecificity of available antibodies and the hard-to-distinguish, dot-like visualizations of the labeled proteins within the tissue. These measurements were carried out in spinal cord sections after unilateral chronic constriction injury of the sciatic nerve to induce neuroinflammation in the spinal cord. RNAscope is used to label transcripts of genes of interest and IHC is used to label cell-type specific antigens (IBA1 for microglia, NeuN for neurons). This combination allowed for labeled RNA transcripts to be quantified within cell-type specific boundaries using confocal microscopy and standard image analysis methods. This method makes it easy to answer empirical questions that are intractable with standard IHC or in situ hybridization alone. The method, which has been optimized for spinal cord tissue and to minimize tissue preparation time and costs, is described in detail from tissue collection to image analysis. Further, the relative expression changes in inflammatory genes NLRP3 and IL-1b in spinal cord microglia vs. neurons of somatotopically relevant laminae are described for the first time.

ErbBs in Lens Cell Fibrosis and Secondary Cataract.

TGFβ-induced epithelial-to-myofibroblast transition (EMyT) of lens cells has been linked to the most common vision-disrupting complication of cataract surgery-namely, posterior capsule opacification (PCO; secondary cataract). Although inhibitors of the ErbB family of receptor tyrosine kinases have been shown to block some PCO-associated processes in model systems, our knowledge of ErbB signaling in the lens is very limited. Here, we investigate the expression of ErbBs and their ligands in primary cultures of chick lens epithelial cells (dissociated cell-derived monolayer cultures [DCDMLs]) and how TGFβ affects ErbB function. DCDMLs were analyzed by immunofluorescence microscopy and Western blotting under basal and profibrotic conditions. Small-molecule ErbB kinase blockers, including the human therapeutic lapatinib, selectively inhibit TGFβ-induced EMyT of DCDMLs. Lens cells constitutively express ErbB1 (EGFR), ErbB2, and ErbB4 protein on the plasma membrane and release into the medium ErbB-activating ligand. Culturing DCDMLs with TGFβ increases soluble bioactive ErbB ligand and markedly alters ErbBs, reducing total and cell surface ErbB2 and ErbB4 while increasing ErbB1 expression and homodimer formation. Similar, TGFβ-dependent changes in relative ErbB expression are induced when lens cells are exposed to the profibrotic substrate fibronectin. A single, 1-hour treatment with lapatinib inhibits EMyT in DCDMLs assessed 6 days later. Short-term exposure to lower doses of lapatinib is also capable of eliciting a durable response when combined with suboptimal levels of a mechanistically distinct multikinase inhibitor. Our findings support ErbB1 as a therapeutic target for fibrotic PCO, which could be leveraged to pharmaceutically preserve the vision of millions of patients with cataracts.

Comparative study of the digestion and metabolism related genes' expression changes during the postnatal food change in different dietary mammals.

The changes in the expression of genes related to digestion and metabolism may be various in different dietary mammals from juvenile to adult, especially, the giant panda (Ailuropoda melanoleuca) and red panda (Ailurus fulgens), which were once carnivores but have shifted to being specialized bamboo eaters, are unique features of their changes are more unclear. To elucidate the changing patterns of gene expression related to digestion and metabolism from juvenile to adult in different dietary mammals, we performed transcriptome analysis of the liver or pancreas in giant and red pandas, herbivorous rabbits (Oryctolagus cuniculus) and macaques (Macaca mulatta), carnivorous ferrets (Mustela putorius furo), and omnivorous mice (Mus musculus) from juvenile to adult. During the transition from juvenile to adulthood, giant and red pandas, as well as rabbits and macaques, show significant upregulation of key genes for carbohydrate metabolism, such as starch hydrolysis and sucrose metabolism, and unsaturated fatty acid metabolism, such as linoleic acid, while there is no significant difference in the expression of key genes for fatty acid β-oxidation. A large number of amino acid metabolism related genes were upregulated in adult rabbits and macaques compared to juveniles. While adult giant and red pandas mainly showed upregulation of key genes for arginine synthesis and downregulation of key genes for arginine and lysine degradation. In adult stages, mouse had significantly higher expression patterns in key genes for starch hydrolysis and sucrose metabolism, as well as lipid and protein metabolism. In contrast to general expectations, genes related to lipid, amino acid and protein metabolism were significantly higher expressed in adult group of ferrets, which may be related to their high metabolic levels. Our study elucidates the pattern of changes in the expression of genes related to digestion and metabolism from juvenile to adult in different dietary mammals, with giant and red pandas showing adaptations associated with specific nutritional limitations of bamboo.

Curcumin derivative NL01 induces ferroptosis in ovarian cancer cells via HCAR1/MCT1 signaling

ObjectiveCurcumin has been shown to have anti-tumor proliferative properties, but its clinical application is limited by its low bioavailability, etc. Derivatives of curcumin have been developed and tested to improve its therapeutic efficacy. Derivative NL01 could induce ferroptosis through the HCAR1/MCT1 pathway. MethodCCK-8 was used to detect curcumin and derivative IC50, crystalline violet staining was used to detect the proliferation inhibition effect of NL01 in ovarian cancer, western blot and qPCR were used to detect downstream related molecular expression changes, Transwell and survival curve assays were used to detect malignant phenotypic. ResultsNL01 inhibited cell growth of Anglne and HO8910PM ovarian cancer cells by 13 times more potent than curcumin and induced ferroptosis of these two cells. we found that NL01 was able to reduce the expression of HCAR1/MCT1 and activate the AMPK signaling pathway, which in turn induced cellular ferroptosis via SREBP1 pathway. Knock-down HCAR1 expression revealed similar phenotype and pathway alterations to NL01 treatment. HCAR1 overexpression promoted a malignant phenotype and resistance to cisplatin in both cancer cells, whereas knockdown of HCAR1 showed the opposite phenotype. Subcutaneous transplantation tumor experiments in nude mice also showed that NL01 induced iron death and inhibited ovarian cancer proliferation. Further study showed that NL01 promoted the downregulation of GPX4 expression, which is related to ferroptosis, and that addition of ferrostatin-1 partially reversed NL01-mediated inhibition of the growth of two cell lines. ConclusionNL01 exhibits better anti-tumor growth properties than curcumin, and NL01 induces ferroptosis in ovarian cancer cells.

Trichoderma gamsiiT6085, a biocontrol agent of Fusarium head blight, modulates biocontrol‐relevant defence genes expression in wheat

AbstractTo enhance the framework of the mechanisms of action used by Trichoderma gamsii T6085 for the control of Fusarium head blight (FHB), this work investigated its ability to modulate the expression of defence‐related genes of wheat (Triticum aestivum ‘Apogee’) in response to endophytic colonization of plant tissues. Changes in relative expression of pal1, pr1, pgip2 and lox1 genes were assessed over time in wheat roots, in spikes colonized by T6085 alone and both T6085 and Fusarium graminearum, and in leaves from wheat seedlings root‐inoculated with T6085. Results indicate the ability of T6085 to induce local and systemic defence responses in wheat plants in the presence of one of the causal agents of FHB. There was a general significant up‐regulation of the plant defence‐related genes analysed, especially in the first days after the application of T6085. According to these results, modulation of plant defence genes could be included within the arsenal of mechanisms used by T6085 when applied to wheat, an additional feature of interest in the management of FHB. To evaluate the effect of the plant genotype on the ability of T6085 to endophytically colonize roots, root colonization was assessed on four cultivars of T. aestivum and two cultivars of T. durum. Data showed that roots of only two T. aestivum cultivars were endophytically colonized by T6085, similar to cv. Apogee used here as control, thus demonstrating an effect of the host genotype on the endophytic ability of T6085.

Gene expression of near-isogenic lines (NILs) carrying blast resistance genes Pi9 and Pi54 in the background of rice cultivar Mushk Budji.

Kashmir valley, India is a homeland to rice landraces like Zag, Nunbeoul, Qadirbeigh, Kawkadur, Kamad, Mushk Budji, etc., generally characterized by short grains, aroma, earliness and cold tolerance. Mushk Budji is a commercially important speciality rice known for its taste and aroma, nonetheless, is extremely vulnerable to blast disease. Through the use of the marker-assisted backcrossing (MABC) approach, a set of 24 Near-isogenic lines (NILs) was created, and the lines with the highest background genome recovery were chosen. The expression analysis was carried out for the component genes and other eight pathway genes related to blast resistance. The major blast resistance genes Pi9 (from IRBL-9W) and Pi54 (from DHMAS 70Q 164-1b) were incorporated following simultaneous-but-step-wise MABC. The NILs harbouring genes Pi9 + Pi54, Pi9 and Pi54 expressed resistance to isolate (Mo-nwi-kash-32) under controlled and natural field conditions. The loci controlling ETI (effector triggered immunity) included the gene Pi9 and showed 61.18 and 60.27 fold change in relative gene expression in Pi54 + Pi9 and Pi9 carrying NILs against RP Mushk Budji. Pi54 was up regulated and showed 41 and 21 fold change in relative gene expression for NIL-Pi54 + Pi9 and NIL-Pi54, respectively. Among the pathway genes, LOC_Os01g60600 (WRKY 108) recorded 8 and 7.5 fold up regulation in Pi9 and Pi54 NILs. The NILs showed recurrent parent genome recovery (RPG) per cent of 81.67 to 92.54 and were on par in performance to recurrent parent Mushk Budji. The lines were utilized to study the expression of the loci controlling WRKYs, peroxidases and chitinases that confer overall ETI response.

Effectiveness of analog of Humanin in ameliorating streptozotocin-induced diabetic nephropathy in Sprague Dawley rats

Diabetes mellitus (DM) is associated with numerous complications, including nephropathy, which principally occur due to hyperglycemia-induced oxidative stress and inflammation. Humanin (HN), a novel peptide generated from mitochondria, has anti-oxidant and anti-inflammatory potential as observed in different disease models. However, role of HN in diabetic nephropathy (DN) has not yet been explored. This study aimed to evaluate biochemical and molecular aspects of the effects of HN analog, Humanin-glycine ([S14G]-humanin) on streptozotocin (STZ)-induced rat model of DN. Ninety Sprague Dawley (SD) rats were randomly segregated into three groups – A (control), B (disease control) and C (treatment). DM type-I was induced in group B and C via single intra-peritoneal dose of STZ (45 mg/Kg). Seven days following STZ injection, rats were deemed diabetic if their blood glucose level was > 250 mg/dL. Subsequently, diabetic rats in group C were injected with [S14G]-humanin intra-peritoneally (0.4 mg/Kg/day) for sixteen weeks. Biochemical analysis revealed that diabetic rats had markedly elevated levels of serum glucose, creatinine, BUN, TNF-α, and kidney tissue SOD. Whereas, significant decline was detected in serum insulin and albumin levels. All these parameters were significantly reversed in group C after administering [S14G]-humanin. Moreover, qRT-PCR analysis displayed up-regulation of pro-inflammatory (IL-18, IL-6, IL-1α, IL-1β, TNF-α) and down-regulation of anti-inflammatory cytokines (IL-10, IL-1RN, IL-4) in diabetic rats (group B). [S14G]-humanin treatment significantly reversed the expression of IL-18 and IL-1α, however, change in relative expression of IL-6, IL-1β, TNF-α and anti-inflammatory cytokines was insignificant (group C). Conclusively, the findings of this study depicted potential therapeutic role of [S14G]-humanin in pre-clinical rodent model of DN.