Reverse Transcription-polymerase Chain Reaction Analysis Research Articles

FoxO3 Regulates Mouse Bone Mesenchymal Stem Cell Fate and Bone-Fat Balance During Skeletal Aging.

Age-related osteoporosis is characterized by an imbalance between osteogenic and adipogenic differentiation in bone mesenchymal stem cells (BMSCs). Forkhead box O 3 (FoxO3) transcription factor isinvolved in lifespan and cell differentiation. In this study, we explore whether FoxO3 regulates age-related bone loss and marrow fat accumulation. The expression levels of FoxO3 in BMSCs during aging were detected invivo and in vitro. To explore the role of FoxO3 in osteogenic and adipogenic differentiation, primary BMSCs were isolated from young and aged mice. FoxO3 expression was modulated by adenoviral vector transfection. The role of FoxO3 in bone-fat balance was evaluated by alizarin red S staining, oil red O staining, quantitative reverse transcription-polymerase chain reaction, Western blot, and histological analysis. Age-related bone loss and fat deposit are associated with downregulation of FoxO3. Overexpression of FoxO3 alleviated age-related bone loss and marrow fat accumulation in aged mice. Mechanistically, FoxO3 reduced adipogenesis and enhanced osteogenesis of BMSCs via downregulation of PPAR-γ and Notch signaling, respectively. In conclusion, FoxO3 is an essential factor controlling the fate of BMSCs and is a potential target for the prevention of age-related osteoporosis.

Identification and map-based cloning of an EMS-induced mutation in wheat gene TaSP1 related to spike architecture.

A candidate gene TaSP1 related to spike shape was cloned, and the gene-specific marker was developed to efficiently track the superior haplotype in common wheat. Spike shape, an important factor that affects wheat grain yield, is mainly defined by spike length (SPL), spikelet number (SPN), and compactness. Zhoumai32 mutant 1160 (ZM1160), a mutant obtained from ethyl methane sulfonate (EMS) treatment of hexaploid wheat variety Zhoumai32, was used to identify and clone the candidate gene that conditioned the spike shape. Genetic analysis of an F2 population derived from a cross of ZM1160 and Bainong207 suggested that the compact spike shape in ZM1160 was controlled by a single recessive gene, and therefore, the mutated gene was designated as Tasp1. With polymorphic markers identified through bulked segregant analysis (BSA), the gene was mapped to a 2.65-cM interval flanked by markers YZU0852 and MIS46239 on chromosome 7D, corresponding to a 0.42-Mb physical interval of Chinese spring (CS) reference sequences (RefSeq v1.0). To fine map TaSP1, 15 and seven recombinants were, respectively, screened from 1599 and 1903 F3 plants derived from the heterozygous F2 plants. Finally, TaSP1 was delimited to a 21.9Kb (4,870,562 to 4,892,493bp) Xmis48123-Xmis48104 interval. Only one high-confidence gene TraesCS7D02G010200 was annotated in this region, which encodes an unknown protein with a putative vWA domain. Quantitative reverse transcription PCR (qRT-PCR) analysis showed that TraesCS7D02G010200 was mainly expressed in the spike. Haplotype analysis of 655 wheat cultivars using the candidate gene-specific marker Xg010200p2 identified a superior haplotype TaSP1b with longer spike and more spikelet number. TaSP1 is beneficial to the improvement in wheat spike shape.

Influences of Ipomoea batatas Anti-Cancer Peptide on Tomato Defense Genes.

This study investigates the impact of IbACP (Ipomoea batatas anti-cancer peptide) on defense-related gene expression in tomato leaves, focusing on its role in plant defense mechanisms. Previously, IbACP was isolated from sweet potato leaves, and it was identified as a peptide capable of inducing an alkalinization response in tomato suspension culture media. Additionally, IbACP was found to regulate the proliferation of human pancreatic adenocarcinoma cells. Elucidate IbACP's molecular influence on defense-related gene expression in tomato leaves using next-generation sequencing analysis. To assess the impact of IbACP on defense-related gene expression, transcriptome data were analyzed, encompassing various functional categories such as photosynthesis, metabolic processes, and plant defense. Semi-quantitative reverse-transcription polymerase chain reaction analysis was employed to verify transcription levels of defense-related genes in tomato leaves treated with IbACP for durations ranging from 0 h (control) to 24 h. IbACP induced jasmonic acid-related genes (LoxD and AOS) at 2 h, with a significant up-regulation of salicylic acid-dependent gene NPR1 at 24 h. This suggested a temporal antagonistic effect between jasmonic acid and salicylic acid during the early hours of IbACP treatment. Downstream ethylene-responsive regulator genes (ACO1, ETR4, and ERF1) were consistently down-regulated by IbACP at all times. Additionally, IbACP significantly up-regulated the gene expressions of suberization-associated anionic peroxidases (TMP1 and TAP2) at all time points, indicating enhanced suberization of the plant cell wall to prevent pathogen invasion. IbACP enhances the synthesis of defense hormones and up-regulates downstream defense genes, improving the plant's resistance to biotic stresses.

IKK2 modulates the innate immune response of Zhikong scallop (Chlamys farreri) to bacterial and viral stimuli through the MyD88-dependent Toll-like receptor signaling pathway

Members of the inhibitor of nuclear factor kappa-B kinase (IKK) family are pivotal components of the innate immune signaling pathway responsible for regulating immune and inflammatory responses. Nevertheless, the various roles that IKKs play in the physiology of invertebrates, particularly marine mollusks, remain largely unexplored. In this study, an IKK gene was cloned from the Zhikong scallop, Chlamys farreri, and denoted as CfIKK2. CfIKK2 belongs to the TANK-binding kinase 1 (TBK1)/IKKε family. Real-time quantitative reverse transcription-polymerase chain reaction analysis showed that CfIKK2 was expressed in various scallop tissues and that its transcript levels substantially increased under different pathogenic stress conditions (including lipopolysaccharide, peptidoglycan, poly(I:C), acute viral necrosis virus, and Vibrio challenge). Co-immunoprecipitation experiments revealed that CfIKK2 interacts with MyD88 (a crucial adaptor in the Toll-like receptor signaling pathway) proteins through its C-terminal TBK1 coiled-coil domain 1. Moreover, CfIKK2 has been shown to interact with itself. In HEK293T cells, CfIKK2 overexpression resulted in an augmentation of mitogen-activated protein kinase phosphorylation. Furthermore, overexpression of CfIKK2 resulted in the activation of interferon reporter genes. In summary, our findings offer valuable insights into the mechanisms through which CfIKK2 responds to stress generated by pathogen-associated molecular patterns, participates in Toll-like receptor signaling, and ultimately activates some of the key cytokines involved in innate immunity in C. farreri. As a critical signaling molecule and immunoregulatory factor, CfIKK2 plays a vital role in the innate immune system of the Zhikong scallop.

In vitro proliferation and differentiation of mouse spermatogonial stem cells in decellularized human placenta matrix.

Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three-dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1-positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two-dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35-day mark, the percentage of Prm1-positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio-scaffold for enhancing mouse in vitro spermatogenesis.

Circular RNA KIAA0564 Serves as a Competitive Endogenous RNA for MicroRNA-424-5p, Mediating the Expression of Lysine Demethylase 4a, Thereby Facilitating Intervertebral Disc Degeneration.

A growing body of research has confirmed the involvement of circular RNAs (circRNAs) in the regulation of intervertebral disc degeneration (IDD) progression. However, the underlying molecular networks remain largely elusive. This study aimed to explore whether a novel circRNA, named circKIAA0564, affects nucleus pulposus (NP) cell injury and to elucidate its molecular mechanism. Both in vivo and in vitro IDD models were established, and the expression patterns of circKIAA0564/miR-424-5p/lysine demethylase 4a (KDM4A) were evaluated through quantitative reverse transcription PCR and Western blot analysis. Actinomycin D, RNase R, and Northern blotting were utilized to assess the circular structure of circKIAA0564. The Cell Counting Kit-8, flow cytometry, enzyme-linked immunosorbent assay, commercial assay kits, Western blotting, and reactive oxygen species (ROS) probes were employed to assess the inflammatory and oxidative stress status in NP cells and tissues. Hematoxylin and eosin and TUNEL staining were used to evaluate pathological damage in mouse NP tissues. RNA immunoprecipitation and dual-luciferase reporter assays were conducted to assess the direct targeting relationships among circKIAA0564, miR-424-5p, and KDM4A. CircKIAA0564 was found to be abnormally overexpressed in IDD, functioning as a novel circRNA. Knockdown of circKIAA0564 ameliorated interleukin-1 beta (IL-1β)-induced inflammation and oxidative stress in NP cells. The therapeutic effect of circKIAA0564 knockdown on NP cells was reversed by the silencing of miR-424-5p. Overexpression of circKIAA0564 exacerbated IL-1β-induced NP cell injury, a process that was reversed by knockdown of KDM4A. CircKIAA0564 activated the toll-like receptor 4 (TLR4)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)/NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) signaling pathway by regulating the miR-424-5p/KDM4A axis. CircKIAA0564 exacerbates IL-1β-induced inflammation and oxidative stress in NP cells by competitively binding miR-424-5p, thereby mediating KDM4A and activating the TLR4/NF-κB/NLRP3 signaling pathway. These findings provide robust data support for targeted therapy of IDD and the development of future pharmaceuticals.

Interleukin-10 supplementation protects against oxalate-mediated injury in mouse kidneys and bone-marrow derived macrophages

Approximately 10% of the United States population will experience a kidney stone (KS). Dietary oxalate promotes calcium oxalate (CaOx) crystals, which are precursors to CaOx KS. Macrophages rely on Interleukin-10 (IL-10) anti-inflammatory cytokine signaling to phagocytose and remove CaOx crystals. We recently reported that oxalate causes macrophages to become M1-like (pro-inflammatory) as well as have increased Interleukin-6 (IL-6; pro-inflammatory) and reduced IL-10 cytokine secretion. The purpose of this study was to assess the effects of IL-10 supplementation on mouse kidneys and bone-marrow derived macrophages exposed to oxalate. We hypothesized that exogenous IL-10 treatment prevents oxalate-mediated injury in mouse kidneys and bone-marrow derived macrophages (BMDMs). Mice with a C57BL/6 background were fed Hydroxy-L-proline (HLP; an oxalate precursor) for 7 or 14 days ± IL-10 intraperitoneal injections (25 μg/kg for 2 days) before collecting kidneys and BMDMs. Real-Time Quantitative Reverse Transcription PCR, fluorescence assays, and histological analyses were performed. Mice fed HLP had increased IL-6 and M1 macrophage expression in their kidneys over time. These mice also had reduced IL-10 and M2 macrophage gene expression. In contrast, mice fed HLP with IL-10 supplementation had increased M2 macrophage levels as well as reduced inflammation in their kidneys. Consistent with these findings, BMDM isolated from mice fed HLP displayed similar results. These cells also had reduced mitochondrial membrane polarization and lysosomal staining and increased cellular ROS; whereas, exogenous IL-10 treatment protected BMDMs from oxalate mediated injury. These findings suggest that IL-10 signaling may be important for preventing oxalate-induced injury and inflammation in mouse kidneys and BMDMs. NIH R01 DK129885 (TM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Autophagy-related CMTM6 promotes glioblastoma progression by activating Wnt/β-catenin pathway and acts as an onco-immunological biomarker.

Glioblastoma multiforme (GBM) is identified as one of the most prevalent and malignant brain tumors, characterized by poor treatment outcomes and a limited prognosis. CMTM6, a membrane protein, has been found to upregulate the expression of programmed cell death 1 ligand 1 protein (PD-L1) and acts as an immune checkpoint inhibitor by inhibiting the programmed death 1 protein/PD-L1 signaling pathway. Recent research has demonstrated a high expression of CMTM6 in GBM, suggesting its potential role in influencing the pathogenesis and progression of GBM, as well as its association with immune cell infiltration in the tumor microenvironment. However, the underlying mechanism of CMTM6 in GBM requires further investigation. Data from cancer patients in The Cancer Genome Atlas, Gene Expression Omnibus and Chinese Glioma Genome Atlas cohorts were consolidated for the current study. Through multi-omics analysis, the study systematically examined the expression profile of CMTM6, epigenetic modifications, prognostic significance, biological functions, potential mechanisms of action and alterations in the immune microenvironment. Additionally, the study investigated CMTM6 expression in GBM cell lines and normal cells using reverse transcription PCR and western blot analysis. The impact of CMTM6 on GBM cell proliferation, migration and invasion was evaluated using a combination of cell counting kit-8 assay, clone formation assay, 5-ethynyl-2'-deoxyuridine incorporation assay, wound healing assay and Transwell assay. In order to explore the mechanism of CMTM6, the Wnt/β-catenin signaling pathway and autophagy-related genes were further verified through western blot analysis. CMTM6 is highly expressed in multiple tumors, particularly GBM. CMTM6 has been shown to be a valuable diagnostic and prognostic biomarker by various bioinformatics approaches. Additionally, CMTM6 plays a pivotal role in the pathogenesis of cancer, specifically GBM, by modulating various biological processes such as DNA methyltransferase expression, RNA modification, copy number variation, genomic heterogeneity, tumor stemness and DNA methylation. The findings of the experiment indicate a significant correlation between elevated CMTM6 expression and the proliferation, invasion, migration and autophagy of GBM cells, with potential key roles mediated through the Wnt/β-catenin signaling pathway. Furthermore, CMTM6 is implicated in modulating tumor immune cell infiltration and is closely linked to the expression of various immune checkpoint inhibitors and immune modulators, particularly within the context of GBM. High levels of CMTM6 expression also enhance the responsiveness of GBM patients to radiotherapy and chemotherapy, thereby offering valuable insights for guiding treatment strategies for GBM. Autophagy-related CMTM6 is highly expressed in various types of cancer, especially GBM, and it can regulate GBM progression through the Wnt/β-catenin signaling pathway and is capable of being used as an underlying target for the diagnosis, treatment selection and prognosis of patients with GBM.

The wheat pathogenesis-related protein (TdPR1.2) enhanced tolerance to abiotic and biotic stresses in transgenic Arabidopsis plants.

In plants, the pathogenesis-related (PR) proteins have been identified as important regulators of biotic and abiotic stresses. PR proteins branch out into 19 different classes (PR1-PR19). Basically, all PR proteins display a well-established method of action, with the notable exception of PR1, which is a member of a large superfamily of proteins with a common CAP domain. We have previously isolated and characterized the first PR1 from durum wheat, called TdPR-1.2. In the current research work, TdPR1.2 gene was used to highlight its functional activities under various abiotic (sodium chloride (100mM NaCl) and oxidative stresses (3mM H2O2), hormonal salicylic acid (SA), abscisic acid (ABA) and jasmonic acid (JA), and abiotic stresses (Botrytis cinerea and Alternaria solani). Enhancement survival index was detected in Arabidopsis transgenic plants expressing TdPR1.2 gene. Moreover, quantitative real-time reverse transcription PCR (qRT-PCR) analysis demonstrated induction of antioxidant enzymes such as catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). It equally revealed a decrease of malondialdehyde (MDA) as well as hydrogen peroxide (H2O2) levels in transgenic Arabidopsis plants compared to control lines, confirming the role of TdPR1.2 in terms of alleviating biotic and abiotic stresses in transgenic Arabidopsis plants. Eventually, RT-qPCR results showed a higher expression of biotic stress-related genes (PR1 and PDF1.2) in addition to a downregulation of the wound-related gene (LOX3 and VSP2) in transgenic lines treated with jasmonic acid (JA). Notably, these findings provide evidence for the outstanding functions of PR1.2 from durum wheat which can be further invested to boost tolerance in crop plants to abiotic and biotic stresses.

Molecular characterization and expression profile of the ALDH1A1 gene and its functions in yak luteal cells

The ALDH1A1 gene encodes a cytoplasmic member of the aldehyde dehydrogenase 1 family, which plays an important role in regulating animal reproductive performance, including estrus cycle and embryonic development. The aim of this study was to characterize ALDH1A1 activity in ovaries of 3–5 year-old yaks and to determine its effects on cell proliferation, apoptosis, and progesterone secretion in luteal cells (LCs). The coding sequence (CDS) of the ALDH1A1 gene was cloned by reverse transcription-PCR and immunohistochemical analysis was used to confirm localization of the ALDH1A1 protein in the ovary. To assess the activity of ALDH1A1 in regulating progesterone secretion, si-ALDH1A1 was transfected into LCs in vitro and progesterone levels in LC supernatants were measured by ELISA. The interference efficiency was assessed by real-time quantitative PCR (RT-qPCR) and immunofluorescence staining, and cell proliferation and apoptosis were evaluated by EdU and TUNEL staining, respectively. The cloned ALDH1A1 sequence contained 1462 bp, encoding 487 amino acids. Immunohistochemical analysis showed that ALDH1A1 protein expression, which was significantly higher in LCs, was mainly found in antral follicles and the corpus luteum (CL). The expression of ALDH1A1 mRNA in LCs was effectively inhibited by si-ALDH1A1transfection, and progesterone secretion was markedly decreased along with the significant down-regulation of progesterone pathway-related genes, STAR, CYP11A1, CYP19A1, CYP17A1, 3β-HSD, and HSD17B1. Knockdown of ALDH1A1 mRNA expression decreased cell proliferation and increased apoptosis in LCs. The mRNA expression of the proliferation-related genes, PCNA, CCND1, CCNB1 and CDC25A, was significantly down-regulated, while expression of the apoptosis-promoting CASP3 gene was significantly increased. In summary, we characterized the yak ALDH1A1 gene and revealed that ALDH1A1 knockdown promoted apoptosis, repressed cell proliferation, and decreased progesterone secretion by yak LCs, potentially by regulating the mRNA expression of genes related to proliferation, apoptosis, and progesterone synthesis and secretion.

Genome-wide characterization, transcriptome profiling, and functional analysis of the ALMT gene family in Medicago for aluminum resistance

Aluminum (Al) is the major limiting factor affecting plant productivity in acidic soils. Al3+ ions exhibit increased solubility at a pH below 5, leading to plant root tip toxicity. Alternatively, plants can perceive very low concentrations of Al3+, and Al triggers downstream signaling even at pH 5.7 without causing Al toxicity. The ALUMINUM-ACTIVATED-MALATE-TRANSPORTER (ALMT) family members act as anion channels, with some regulating the secretion of malate from root apices to chelate Al, which is a crucial mechanism for plant Al resistance. To date, the role of the ALMT gene family within the legume Medicago species has not been fully characterized. In this study, we investigated the ALMT gene family in M. sativa and M. truncatula and identified 68 MsALMTs and 18 MtALMTs, respectively. Phylogenetic analysis classified these genes into five clades, and synteny analysis uncovered genuine paralogs and orthologs. The real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that MtALMT8, MtALMT9, and MtALMT15 in clade 2-2b are expressed in both roots and root nodules, and MtALMT8 and MtALMT9 are significantly upregulated by Al in root tips. We also observed that MtALMT8 and MtALMT9 can partially restore the Al sensitivity of Atalmt1 in Arabidopsis. Moreover, transcriptome analysis examined the expression patterns of these genes in M. sativa in response to Al at both pH 5.7 and pH 4.6, as well as to protons, and found that Al and protons can independently induce some Al-resistance genes. Overall, our findings indicate that MtALMT8 and MtALMT9 may play a role in Al resistance, and highlight the resemblance between the ALMT genes in Medicago species and those in Arabidopsis.

Systematic characterization of Gossypium GLN family genes reveals a potential function of GhGLN1.1a regulates nitrogen use efficiency in cotton

The enzyme glutamine synthetase (GLN) is mainly responsible for the assimilation and reassimilation of nitrogen (N) in higher plants. Although the GLN gene has been identified in various plants, there is little information about the GLN family in cotton (Gossypium spp.). To elucidate the roles of GLN genes in cotton, we systematically investigated and characterized the GLN gene family across four cotton species (G. raimondii, G. arboreum, G. hirsutum, and G. barbadense). Our analysis encompassed analysis of members, gene structure, cis-element, intragenomic duplication, and exploration of collinear relationships. Gene duplication analysis indicated that segmental duplication was the primary driving force for the expansion of the GhGLN gene family. Transcriptomic and quantitative real-time reverse-transcription PCR (qRT-PCR) analyses indicated that the GhGLN1.1a gene is responsive to N induction treatment and several abiotic stresses. The results of virus-induced gene silencing revealed that the accumulation and N use efficiency (NUE) of cotton were affected by the inactivation of GhGLN1.1a. This study comprehensively analyzed the GhGLN genes in Gossypium spp., and provides a new perspective on the functional roles of GhGLN1.1a in regulating NUE in cotton.

The Female-Biased General Odorant Binding Protein 2 of Semiothisa cinerearia Displays Binding Affinity for Biologically Active Host Plant Volatiles.

Herbivorous insects rely on volatile chemical cues from host plants to locate food sources and oviposition sites. General odorant binding proteins (GOBPs) are believed to be involved in the detection of host plant volatiles. In the present study, one GOBP gene, ScinGOBP2, was cloned from the antennae of adult Semiothisa cinerearia. Reverse-transcription PCR and real-time quantitative PCR analysis revealed that the expression of ScinGOBP2 was strongly biased towards the female antennae. Fluorescence-based competitive binding assays revealed that 8 of the 27 host plant volatiles, including geranyl acetone, decanal, cis-3-hexenyl n-valerate, cis-3-hexenyl butyrate, 1-nonene, dipentene, α-pinene and β-pinene, bound to ScinGOBP2 (KD = 2.21-14.94 μM). The electrical activities of all eight ScinGOBP2 ligands were confirmed using electroantennography. Furthermore, oviposition preference experiments showed that eight host volatiles, such as decanal, cis-3-hexenyl n-valerate, cis-3-hexenyl butyrate, and α-pinene, had an attractive effect on female S. cinerearia, whereas geranyl acetone, 1-nonene, β-pinene, and dipentene inhibited oviposition in females. Consequently, it can be postulated that ScinGOBP2 may be implicated in the perception of host plant volatiles and that ScinGOBP2 ligands represent significant semiochemicals mediating the interactions between plants and S. cinerearia. This insight could facilitate the development of a chemical ecology-based approach for the management of S. cinerearia.

The Long Noncoding RNA Gall Bladder Cancer-Associated Suppressor of Pyruvate Carboxylase Inhibits the Proliferation, Migration, and Invasion of Colorectal Cancer Cells and Induces Their Apoptosis.

This study aimed to determine the role of the long noncoding RNA (lncRNA) gall bladder cancer-associated suppressor of pyruvate carboxylase (SOD2-1) in the progression of colorectal cancer (CRC). A total of 23 pairs of specimens, including CRC tissues and adjacent normal tissues, were collected, and the expression of lncRNA SOD2-1 (lnc-SOD2-1) was measured. lnc-SOD2-1 function was examined using HCT15 and HCT116 cells. A lnc-SOD2-1 overexpression vector was designed and transfected into both cell lines. MTS and colony formation assays were used to determine cell viability. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays were performed to measure apoptosis. Cell migration and invasion were evaluated using the Transwell assay. Migration and invasion markers were validated using quantitative reverse transcription-polymerase chain reaction and western blot analysis. The results indicated that the expression of lnc-SOD2-1 was downregulated in CRC tissues. lnc-SOD2-1 overexpression evidently decreased cell viability and led to the formation of fewer cell colonies. lnc-SOD2-1 overexpression induced ~ twofold higher apoptosis than the control group. lnc-SOD2-1 overexpression reduced the proportion of migratory and invasive cells to 50% and 75% of the control group, respectively. lnc-SOD2-1 overexpression significantly decreased the expression of matrix metalloproteinase-2 and -9. In conclusion, lnc-SOD2-1 may act as a tumor suppressor that inhibits the proliferation, migration, and invasion of CRC cells and induces their apoptosis.

Deciphering antifungal and antibiofilm mechanisms of isobavachalcone against Cryptococcus neoformans through RNA-seq and functional analyses

Cryptococcus neoformans has been designated as critical fungal pathogens by the World Health Organization, mainly due to limited treatment options and the prevalence of antifungal resistance. Consequently, the utilization of novel antifungal agents is crucial for the effective treatment of C. neoformans infections. This study exposed that the minimum inhibitory concentration (MIC) of isobavachalcone (IBC) against C. neoformans H99 was 8 µg/mL, and IBC dispersed 48-h mature biofilms by affecting cell viability at 16 µg/mL. The antifungal efficacy of IBC was further validated through microscopic observations using specific dyes and in vitro assays, which confirmed the disruption of cell wall/membrane integrity. RNA-Seq analysis was employed to decipher the effect of IBC on the C. neoformans H99 transcriptomic profiles. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis was performed to validate the transcriptomic data and identify the differentially expressed genes. The results showed that IBC exhibited various mechanisms to impede the growth, biofilm formation, and virulence of C. neoformans H99 by modulating multiple dysregulated pathways related to cell wall/membrane, drug resistance, apoptosis, and mitochondrial homeostasis. The transcriptomic findings were corroborated by the antioxidant analyses, antifungal drug sensitivity, molecular docking, capsule, and melanin assays. In vivo antifungal activity analysis demonstrated that IBC extended the lifespan of C. neoformans-infected Caenorhabditis elegans. Overall, the current study unveiled that IBC targeted multiple pathways simultaneously to inhibit growth significantly, biofilm formation, and virulence, as well as to disperse mature biofilms of C. neoformans H99 and induce cell death.

The novel candidate gene Grb10 regulates the growth performance of Guizhou white goats

Growth factor receptor binding protein 10 (Grb10) regulates muscle growth but its function in goats is not well studied. This study examined the relationship between Grb10 gene polymorphism and growth traits in Guizhou white goats. Further, the effect of promoter polymorphism on Grb10 transcription was analyzed via a dual-luciferase assay. Quantitative reverse transcription-polymerase chain reaction analysis showed that the expression level of the Grb10 gene was the highest in the longissimus dorsi muscle (P < 0.01) and the lowest in the spleen. Moreover, sequencing results showed that the Grb10 gene had five single nucleotide polymorphisms. The association analysis showed that g.–246 C > T, g.72323 T > C, and g.178634 A > G loci were significantly associated with growth traits (P < 0.05). Linkage disequilibrium analysis of each locus showed that g.72323 T > C and g.72814 T > A loci had the strongest linkage (r2 = 0.482, D' = 0.73). Furthermore, according to an analysis conducted using TFDB4 software, it was predicted that a mutation in the promoter region of the g.–246 C > T locus resulted in the absence of four transcriptional regulatory factors. The results of dual-luciferase reporter assay further demonstrated that mutation of the g.–246 C > T locus reduced the transcriptional activity of the promoter region of the Grb10 gene. These findings suggest that Grb10 gene polymorphism could affect growth traits in goats.

Effects of environmental enrichment on GLUT expression in the visual cortex of amblyopic rats

ObjectiveTo investigate the potential changes of glucose metabolism and glucose transporter protein (GLUT) in the visual cortex of formally deprived amblyopic rats, as well as the effects of enriched environments on the levels of nerve conduction and glucose metabolism in the visual cortex of amblyopic rats. Methods36 rats were randomly divided into three groups: CON + SE (n = 12), MD + SE (n = 12) and MD + EE (n = 12). The right eyelids of both MD + SE and MD + EE groups were sutured. After successful modelling, the MD + EE group was maintained in an enriched environment, and the other two groups were kept in the same environment. Pattern visual evoked potentials (PVEP) was used to confirm models' effect, glucose metabolism was analyzed by Micro-PET/CT (18F-FDG), and the protein as well as mRNA expression levels of GLUT were detected by Western Blot and quantitative RT-PCR (quantitative Reverse Transcription-Polymerase Chain Reaction) analyses, site of GLUT expression by immunofluorescence (IF). ResultsAfter suture modelling, both the MD + EE and MD + SE groups objective visual nerve conduction function decreased, the glucose metabolism in the visual cortex was markedly lower. After the enriched environment intervention, it recovered in the MD + EE group. The expression levels of GLUT1 and GLUT3 were increased in the MD + EE group in comparison with the MD + SE group. GLUT1 was primarily expressed on astrocytes and endothelial cells, but GLUT3 was mainly expressed on neurons. ConclusionEnrichment of the environment exhibited a therapeutic effect on amblyopia, which could be related to the enhancement of glucose metabolism and GLUT expression in the visual cortex.

Pepper vein yellow virus P0 protein triggers NbHERC3, NbBax, and NbCRR mediated hypersensitive response.

P0 proteins encoded by the pepper vein yellow virus (PeVYV) are pathogenic factors that cause hypersensitive response (HR). However, the host gene expression related to PeVYV P0-induced HR has not been thoroughly studied. Transcriptomic technology was used to investigate the host pathways mediated by the PeVYV P0 protein to explore the molecular mechanisms underlying its function. We found 12,638 differentially expressed genes (DEGs); 6784 and 5854 genes were significantly upregulated and downregulated, respectively. Transcriptomic and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analyses revealed that salicylic acid (SA) and jasmonic acid (JA) synthesis-related gene expression was upregulated, and ethylene synthesis-related gene expression was downregulated. Ultrahigh performance liquid chromatography-tandem mass spectrometry was used to quantify SA and JA concentrations in Nicotiana benthamiana, and the P0 protein induced SA and JA biosynthesis. We then hypothesized that the pathogenic activity of the P0 protein might be owing to proteins related to host hormones in the SA and JA pathways, modulating host resistance at different times. Viral gene silencing suppression technology was used in N. benthamiana to characterize candidate proteins, and downregulating NbHERC3 (Homologous to E6-AP carboxy-terminus domain and regulator of choromosome condensation-1 dmain protein 3) accelerated cell necrosis in the host. The downregulation of NbCRR reduced cell death, while that of NbBax induced necrosis and curled heart leaves. Our findings indicate that NbHERC3, NbBax, and NbCRR are involved in P0 protein-driven cell necrosis.

Downregulation of NAT1 Expression is Associated with Poor Prognosis and Immune Infiltration in COAD.

An increasing corpus of evidence has identified the involvement of N-acetyltransferase 1 (NAT1), a member of the NAT family, in the progression of various cancers. However, the specific function of NAT1 in colon cancer (COAD) remains elusive. This study aims to decip her the role of NAT1 in COAD and its associated mechanisms. The Tumor Immunity Evaluation Resource (TIMER), The Cancer Genome Atlas (TCGA), and the Gene Expression Omnibus (GEO) databases were employed to assess the NAT1 expression level in COAD. The differential expression between COAD and normal colon tissue was further validated using quantitative real-time reverse-transcription PCR (RT-qPCR) and Western blot (WB) analyses. Additionally, survival analysis of NAT1 in COAD was carried out using the PrognoScan database and TCGA dataset. The functions of NAT1 were explored through gene set enrichment analysis (GSEA) and immuno-infiltration analysis. There was a significant reduction in NAT1 expression in COAD samples compared to normal tissue. Notably, low NAT1 expression in COAD correlated significantly with various clinical parameters such as tumor stage (T stage, N stage, M stage, pathologic stage), primary therapy outcome, carcinoembryonic antigen (CEA) level, and lymphatic invasion. The downregulation of NAT1 was also strongly linked with poor outcomes in overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS). Cox regression analysis highlighted NAT1 as an independent prognostic indicator for overall survival in COAD patients. GSEA results revealed NAT1's involvement in multiple pathways, including the neuroactive ligand-receptor interaction, olfactory transduction, olfactory signaling, extracellular matrix receptor interaction, calcium signaling, and focal adhesion pathways. Furthermore, NAT1 expression was found to significantly correlate with infiltration levels of various immune cells. The findings reveal NAT1's potential as a valuable prognostic biomarker for COAD. Moreover, its associated mechanisms offer insights that might pave the way for therapeutic interventions for COAD patients.

Elucidating the Anticancer Mechanisms of Cinnamoyl Sulfonamide Hydroxamate: Insights From DNA Content Analysis and Gene Expression Profiling in Squamous Cell Carcinoma.

Oral cancer is a major public health concern worldwide, with oral squamous cell carcinoma (OSCC) being one of its most common subtypes. Despite advances in diagnosis and management of this disease, there remains a need to develop new therapeutic approaches for better outcomes. This study aimed to investigate the molecular mechanisms through which cinnamoyl sulfonamide hydroxamate derivatives exert their anticancer effects on OSCC. The derivatives were synthesized via multi-step processes and then characterized at the molecular level. Flow cytometry assay for DNA content and cell cycle distribution, anisidine/toluidine double staining for apoptosis detection, as well as reverse transcription polymerase chain reaction (RT-PCR) gene expression analysis, were performed on OSCC cell lines exposed to cinnamoyl sulfonamide hydroxamate derivatives. Flow cytometry unveiled remarkable changes in the distribution of cells throughout the OSCC cell line upon treatment with cinnamoyl sulfonamide hydroxamate derivatives. Consequently, it led to a noticeable decrease in cells at the G0/G1 phase, together with an increase at the S phase, thereby indicating a retardation at various points of the cycle. In addition, apoptotic morphological alterations have been observed by anisidine/toluidine double staining after some treatments with the compounds. RT-PCR analysis showed a marked increase in p21 gene expression levels, further supporting the compounds' ability to induce cell cycle arrest and apoptosis. The research highlighted the potential of cinnamoyl sulfonamide hydroxamate derivatives as candidates for oral cancer, particularly OSCC treatment, shedding light on their operation at the molecular level and paving the way for the development of targeted therapies that could aid in the cure of oral cancer.