PCR Analysis Research Articles

IL-32 aggravates metabolic disturbance in human nucleus pulposus cells by activating FAT4-mediated Hippo/YAP signaling

Extracellular matrix (ECM) metabolism disorders in the inflammatory microenvironment play a key role in the pathogenesis of intervertebral disc degeneration (IDD). Interleukin-32 (IL-32) has been reported to be involved in the progression of various inflammatory diseases; however, it remains unclear whether it participates in the matrix metabolism of nucleus pulposus (NP) cells. Therefore, this study aimed to investigate the mechanism of IL-32 on regulating the ECM metabolism in the inflammatory microenvironment. RNA-seq was used to identify aberrantly expressed genes in NP cells in the inflammatory microenvironment. Western blotting, real-time quantitative PCR, immunohistochemistry and immunofluorescence analysis were performed to measure the expression of IL-32 and metabolic markers in human NP tissues or NP cells treated with or without tumor necrosis factor-α (TNF-α). In vivo, an adeno-associated virus overexpressing IL-32 was injected into the caudal intervertebral discs of rats to assess its effect on IDD. Proteins interacting with IL-32 were identified via immunoprecipitation and mass spectrometry. Lentivirus overexpressing IL-32 or knocking down Fat atypical cadherin 4 (FAT4), yes-associated protein (YAP) inhibitor-Verteporfin (VP) were used to treat human NP cells, to explore the pathogenesis of IL-32. Hippo/YAP signaling activity was verified in human NP tissues. IL-32 expression was significantly upregulated in degenerative NP tissues, as indicated in the clinical samples. Furthermore, IL-32 was remarkably overexpressed in TNF-α-induced degenerative NP cells. IL-32 overexpression induced IDD progression in the rat model. Mechanistically, the elevation of IL-32 in the inflammatory microenvironment enhanced its interactions with FAT4 and mammalian sterile 20-like kinase1/2 (MST1/2) proteins, prompting MST1/2 phosphorylation, and activating the Hippo/YAP signaling pathway, causing matrix metabolism disorder in NP cells. Our results suggest that IL-32 mediates matrix metabolism disorders in NP cells in the inflammatory micro-environment via the FAT4/MST/YAP axis, providing a theoretical basis for the precise treatment of IDD.

Single-cell sequencing reveals that specnuezhenide protects against osteoporosis via activation of METTL3 in LEPR+ BMSCs

BackgroundOsteoporosis (OP) has garnered significant attention due to its substantial morbidity and mortality rates, imposing considerable health burdens on societies worldwide. However, the molecular mechanisms underlying osteoporosis pathogenesis remain largely elusive, and the available therapeutic interventions are limited. Therefore, there is an urgent need for innovative strategies in the treatment of osteoporosis. PurposeThe primary objective of this study was to elucidate the molecular mechanisms underlying osteoporosis pathogenesis using single-cell RNA sequencing (scRNA-seq), thereby proposing novel therapeutic agents. MethodsThe mice osteoporosis model was established through bilateral ovariectomy. Micro-computed tomography (μCT) and hematoxylin and eosin (H&E) staining were employed to assess the pathogenesis of osteoporosis. scRNA-seq was utilized to identify and analyze distinct molecular mechanisms and sub-clusters. Gradient dilution analysis was used to obtain specific sub-clusters, which were further validated by immunofluorescence staining and flow cytometry analysis. Molecular docking and cellular thermal shift assay (CETSA) were applied for screening potential agents in the TCMSPs database. Alkaline phosphatase (ALP) activity and alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Osteogenic organoids analysis was employed to assess the proliferation and sphere-forming ability of BMSCs. Quantitative real-time PCR (qRT-PCR) and western blot analysis were conducted to investigate signaling pathways. Wound healing assay and tube formation analysis were employed to evaluate the angiogenesis of endothelial cells. ResultsThe scRNA-seq analysis revealed the crucial role of LEPR+ BMSCs in the pathogenesis of osteoporosis, which was confirmed by immunofluorescence staining of the epiphysis. Subsequently, the LEPR+ BMSCs were obtained by gradient dilution analysis and identified by immunofluorescence staining and flow cytometry. Accordingly, specnuezhenide (Spe) was screened and identified as a potential compound targeting METTL3 from the TCMSPs database. Spe promoted bone formation as evidenced by μ-CT, and H&E analysis. Additionally, Spe enhanced the osteogenic capacity of LEPR+ BMSCs through ALP and ARS assay. Notably, METTL3 pharmacological inhibitors S-Adenosylhomocysteine (SAH) attenuated the aforementioned osteo-protective effects of Spe. Particularly, Spe enhanced the LEPR+ BMSCs-dependent angiogenesis through the secretion of SLIT3, which was abolished by SAH in LEPR+ BMSCs. ConclusionCollectively, these findings suggest that Spe could enhance the osteogenic potential of LEPR+ BMSCs and promote LEPR+ BMSCs-dependent angiogenesis by activating METTL3 in LEPR+ BMSCs, indicating its potential as an ideal therapeutic agent for clinical treatment of osteoporosis.

Genome sequencing analysis and validation of infestation-related functional genes of Vibrio parahaemolyticus LG2206 isolated from the hepatopancreas of diseased mud crab (Scylla paramamosain) in South China

Vibrio parahaemolyticus (V. parahaemolyticus) is a major bacterial pathogen found in brackish environments, leading to disease outbreaks and great economic losses in the mud crab industry. This study investigated the molecular mechanism of V. parahaemolyticus infecting mud crabs through genome sequencing analysis, survival experiments, and the expression patterns of related functional genes. A strain of V. parahaemolyticus with high pathogenicity and lethality was isolated from diseased mud crab in South China. The genome sequencing results showed that the genome size of V. parahaemolyticus was a circular chromosome of 3,357,271 bp, with a GC content of 45%, containing 2,985 protein-coding genes, denoted as V. parahaemolyticus LG2206. Genome analysis data revealed that a total of 113 adherence coding genes were obtained, including 120 virulence factor coding genes, 37 type III secretion system (T3SS) coding genes, and 277 sequences of T3SS effectors. Survival experiments showed that the mortality was 20% within 96 h in the 1 × 104 CFU/mL infection group, 90% in the 3.2 × 105 CFU/mL treatment group, and 100% in the 1 × 106 CFU/mL treatment group. The LD50 of V. parahaemolyticus LG2206 was determined as 4.6 × 104 CFU/mL. Six genes of znuA and fliD (flagellin encoding genes), yscE and yscR (T3SS encoding genes), and nfuA and htpX (virulence factor encoding genes) were selected and validated by quantitative real-time PCR analysis after infection with 4.6 × 104 CFU/mL of V. parahaemolyticus LG2206 for 96 h. The expression of the six genes exhibited a significant up-regulation trend at all tested time points. The results indicated that the infestation-related genes screened in the experiment play important roles in the infestation process. This study provides timely and effective information to further analyze the molecular mechanism of V. parahaemolyticus infection and develop comprehensive measures for disease prevention and control.

Prevalence and diversity of enteric Helicobacter spp. in healthy and diarrheic cats.

Helicobacters are gastric and enterohepatic and live in the gut. The role of enterohepatic Helicobacters as intestinal pathogens is uncertain, while stomach Helicobacters are well-known. The prevalence of Helicobacter species in cat feces helps us understand their impact on cat health and human disease transmission. This study used PCR to identify Helicobacter spp. in feces samples from healthy and diarrhoeic cats, independent of the reason. The study also compared intestinal and stomach Helicobacter species. PCR analysis was performed on fecal samples from 40 cats, with 20 cats having diarrhea and 20 cats showing no symptoms. The PCR analysis aimed to detect Helicobacter's presence using a method that identifies the bacteria through the 16S rRNA gene. The diarrhoeic group had a greater prevalence of infection (17:9 ratio), with an overall 65% infection rate detected. Cats that were older than 2 years showed a higher incidence of disease. H. canis had the highest occurrence rate (69.2%), followed by H. bilis, H. bizzozeronii, and H. salomonis. Significantly, H. pylori, H. felis, and H. heilmannii were not reported. H. canis was the predominant species found in both healthy and diarrheic cats, indicating the need for more investigation. The detection of the gastric species H. salomonis and H. bizzozeronii further complicates the classification. This highlights the complex nature of Helicobacter infections in cats, emphasizing the need for further investigation to guide the development of preventative measures and treatment techniques for both veterinary and public health purposes.

Identification of biallelic mutations in MCM3AP and comprehensive literature analysis.

Minichromosome maintenance complex component 3 associated protein (MCM3AP) is a gene in which mutations can result in autosomal recessive peripheral neuropathy with or without impaired intellectual development. The MCM3AP genotype-phenotype correlation and prognosis remain unclear. The aim of this study was to explore the genotype-phenotype correlations pertaining to MCM3AP. Whole-exome sequencing (WES) combined with copy number variation sequencing (CNV-seq) were performed on the genomic DNA isolated from a Chinese family, and Sanger sequencing, quantitative PCR and cDNA analyses were performed to examine the mutations. The retrospective study was conducted on 28 individuals with biallelic MCM3AP mutation-related diseases, including features such as mutations, motor development impairment, intellectual disability, weakness/atrophy, and cerebral magnetic resonance imaging abnormalities. Sequencing identified novel compound heterozygous mutations in MCM3AP, namely, a paternal variant c.1_5426del (loss of exons 1-25) and a maternal splicing variant c.1858 + 3A>G. Functional studies revealed that the variant c.1858 + 3A>G resulted in the heterozygous deletion of exon 5, thereby affecting splicing functionality. Furthermore, the compound heterozygous mutation may affect the functionality of the protein domain. Retrospective analysis revealed different genotype-phenotype correlations for the pathogenic variants in biallelic MCM3AP: all individuals (100%) with mutations outside the Sac3 domain exhibited early-onset symptoms, motor developmental delays, and cognitive abnormalities, conversely, the proportions of individuals carrying mutations within the domain were 26.7% (motor delays) and 46.7% (cognitive abnormalities). Our findings further expand the genetic mutation spectrum of biallelic MCM3AP and highlight the genotype-phenotype associations. Additionally, we elaborate on the importance of rehabilitation intervention.

A lipid metabolism-related gene model reveals the prognosis and immune microenvironment of cutaneous melanoma

Abstract Objectives Lipid metabolic dysregulation plays a critical role in the biological behavior of skin cutaneous melanoma (SKCM). Hence, we aimed to identify lipid metabolism-related genes (LMGs) and possible prognostic models for SKCM, perform prognostic risk assessment, and predict possible effective therapies. Methods SKCM patient data were downloaded from The Cancer Genome Atlas (TCGA) and used as the training set; GSE65904 was used as the validation dataset. A prognostic risk model was established by multivariate Cox regression analysis and the LASSO algorithm. The samples in training and validation sets were grouped into high- and low-risk groups, respectively, in accordance with the risk model, and risk score (RS) distribution and survival ROC curve were obtained. The ‘limma’ package in R3.6.1 Version 3.34.7 was used to filter significant differentially expressed genes (DEGs) in the training set between the high- and low-risk groups. For DEGs, functional enrichment and immune infiltration analyses were used to reveal potential disease mechanisms and responses to immunotherapy. The expression level of LMGs involved in the prognostic risk was verified by diverse methods. Results A predictive model comprising four LMGs, including ADH4, ALDH7A1, HADH, and HADHA, was established to predict SKCM patient survival. Functional enrichment has revealed enriched immune-associated pathways. Different immune microenvironments were identified by immune infiltration analysis. HPA immunohistochemical analysis, Real-time PCR analysis, and Western blotting revealed the upregulation of HADH and HADHA and the downregulation of ADH4 and ALDH7A1 in melanoma tissues or cell lines compared to normal skin tissues and melanocyte cells. Conclusions LMGs, including ADH4, ALDH7A1, HADH, and HADHA involved in the predictive model may play a critical role in the biological behaviors and therapeutic response of melanoma. The model we constructed may serve as a prospective biological marker to predict the prognosis and therapeutic response of melanoma patients.

Examination of the roles of CgTH in tyrosine metabolism and pigmentation of Pacific oyster (Crassostrea gigas)

The visually appealing shell colours and intricate patterns of shellfish not only attract consumers but also enhance their economic value. Understanding the inheritance pattern of shell colour and the molecular mechanism of pigmentation is highly important for enhancing breeding efficiency. This study involved the identification of a tyrosine hydroxylase gene (CgTH) and the investigation of its function in the metabolism of tyrosine and pigmentation in Pacific oysters (Crassostrea gigas). CgTH contains a conserved Biopterin_H domain that consists of a binding site for iron atoms and a binding site for tetrahydrobiopterin (BH4). Quantitative PCR (qPCR) and immunofluorescence analysis revealed that the expression of CgTH was higher in the mantle of black shell oysters in comparison to the white strain. The in vitro and in vivo experiments confirmed that CgTH is involved in tyrosine metabolism through the catalysis of the conversion of L-tyrosine to L-DOPA. In order to examine its functions in melanin synthesis in oysters, 3-Iodo-L-tyrosine (3-IT) treatment, RNA interference (RNAi), and L-DOPA treatment assays were conducted. The results of our study showed that 3-IT effectively inhibited CgTH activity, leading to a suppression of tyrosine metabolism. Knockdown of CgTH hindered the cAMP-CREM-MITF axis and decreased pigmentation. On the other hand, L-DOPA stimulated melanin production through cAMP signaling and counteracted the inhibitory effects of 3-IT on the metabolism of tyrosine. These data indicated that there may be a feedback loop involving CgTH and the cAMP-CREM-MITF axis that regulates tyrosine metabolism and melanogenesis in Pacific oysters. This study enhances our understanding of the molecular mechanism that governs oyster pigmentation and offers a possible molecular indicator for the targeted breeding of particular strains of Pacific oysters.

Noncanonical formation of SNX5 gene-derived circular RNA regulates cancer growth

Oral squamous cell carcinoma (OSCC) is a prevalent cancer worldwide, exhibiting unique regional prevalence. Despite advancements in diagnostics and therapy, the 5-year survival rate for patients has seen limited improvement. A deeper understanding of OSCC pathogenesis, especially its molecular underpinnings, is essential for improving detection, prevention, and treatment. In this context, noncoding RNAs, such as circular RNAs (circRNAs), have gained recognition as crucial regulators and potential biomarkers in OSCC progression. Our study highlights the discovery of previously uncharacterized circRNAs, including a SNX5 gene-derived circRNA, circSNX5, through deep sequencing of OSCC patient tissue transcriptomes. We established circSNX5’s tumor-specific expression and its strong correlation with patient survival using structure-specific and quantitative PCR analyses. In vitro and in vivo experiments underscored circSNX5 RNA’s regulatory role in cancer growth and metastasis. Further, our omics profiling and functional assays revealed that ADAM10 is a critical effector in circSNX5-mediated cancer progression, with circSNX5 maintaining ADAM10 expression by sponging miR-323. This novel circRNA-miRNA-mRNA regulatory axis significantly contributes to oral cancer progression and malignancy. Moreover, we discovered that circSNX5 RNA is produced via noncanonical sequential back-splicing of pre-mRNA, a process negatively regulated by the RNA-binding protein STAU1. This finding adds a new dimension to our understanding of exonic circRNA biogenesis in the eukaryotic transcriptome. Collectively, our findings offer a detailed mechanistic dissection and functional interpretation of a novel circRNA, shedding light on the role of the noncoding transcriptome in cancer biology and potentially paving the way for innovative therapeutic strategies.

Carboxypeptidase Inhibitor LXN Expression in Endometrial Tissue Is Menstrual Cycle Phase-Dependent and Is Upregulated in Endometriotic Lesions.

Endometriosis is a chronic hormone-dependent disease characterized by the spread of endometrial cells outside the uterus, which form endometriotic lesions and disrupt the functions of the affected organs. The etiopathogenesis of endometriosis is still unclear, and thus it is important to examine the genes that may contribute to the establishment of endometriotic lesions. The aim of this study was to investigate the expression of new potential candidate gene latexin (LXN), an inhibitor of carboxypeptidases, in endometrium and endometriotic lesions to elucidate its possible role in endometriosis development. LXN expression in tissues was assessed using quantitative reverse transcription PCR (qRT-PCR) analysis and immunohistochemical staining (IHC). The functions of LXN were examined using Transwell and MTT assays. qRT-PCR analysis revealed that LXN expression in endometrium was menstrual cycle-dependent, being lowest in the early-secretory phase and highest in the late-secretory phase and was significantly upregulated in endometriotic lesions. IHC confirmed LXN expression in endometrial stromal cells, and in vitro assays demonstrated that knockdown of LXN effectively reduced the migratory capacity of endometrial stromal cells while promoting cell viability. In conclusion, our results showed that LXN can be involved in the pathogenesis of endometriosis by regulating the proliferation and migration activity of endometriotic stromal cells.

Smart Delivery of Biomolecules Interfering with Peri-Implant Repair in Osteoporotic Rats.

Bisphosphonates are widely used for the treatment of postmenopausal osteoporosis; however, they cause several long-term side effects, necessitating the investigation of local ways to improve osseointegration in compromised bone tissue. The purpose of this study was to evaluate peri-implant bone repair using implants functionalized with zoledronic acid alone (OVX ZOL group, n = 11), zoledronic acid + teriparatide (OVX ZOL + TERI group, n = 11), and zoledronic acid + ruterpy (OVX ZOL + TERPY group, n = 11) compared to the control group (OVX CONV, n = 11). Analyses included computer-assisted microtomography, qualitative histologic analysis, and real-time PCR analysis. Histologically, all functionalized surfaces improved peri-implant repair, with the OVX ZOL + TERI group standing out. Similar results were found in computerized microtomography analysis. In real-time PCR analysis, however, the OVX ZOL and OVX ZOL + TERPY groups showed better results for bone formation, with the OVX ZOL + TERPY group standing out, while there were no statistical differences between the OVX CONV and OVX ZOL + TERI groups for the genes studied at 28 postoperative days. Nevertheless, all functionalized groups showed a reduced rate of bone resorption. In short, all surface functionalization groups outperformed the control group, with overall better results for the OVX ZOL + TERI group.

High-fiber diet reduces bone formation but does not affect bone microarchitecture in type 2 diabetes individuals.

Bone fragility is a recognized complication of type 2 diabetes mellitus (T2DM), increasing patient morbidity. Thus, the development of an effective intervention to prevent diabetic bone fragility is urgently needed. As lifestyle intervention represents an effective option for diabetes management, it may have an impact on bone health. While studies have shown a beneficial effect of dietary fiber in T2DM management, its effect on bone health is still unclear. Thus, we investigated the impact of a high-fiber diet on bone and glucose control in men and women with T2DM. Forty-five T2DM patients (HbA1c: 6.5% ± 0.49%, age: 74 ± 7.29yr) scheduled for hip arthroplasty were randomly assigned to follow a high-fiber diet (38g/day) or to make no diet changes for 12wk. Interestingly, BMI decreased by 4% (p <.0001) and HbA1c by 3.4% (p <.0001) in the high-fiber diet group, but did not decrease in the control group. However, serum concentration of the bone formation marker procollagen type 1 amino-terminal propeptide (P1NP) decreased by 8.6 % in the high-fiber diet group (p =.0004), whereas it remained unchanged in the control group. In contrast, similar to the control group, serum concentration of the bone resorption marker C-terminal telopeptide of type I collagen (CTX) concentrations did not change in the high-fiber diet group. Bone microCT analysis revealed no changes in trabecular and cortical bone parameters between the high-fiber diet and control groups. Similarly, real-time (RT)-PCR analysis in bone tissue showed no changes in the gene expression of Wnt pathway-related genes (Sost, Dkk-1, Wnt10b, and Lef-1), bone formation markers (Runx2, Col1a1, and Ocn), and inflammatory cytokines (IL-6, IL-8, TNF-α, and IL-10) between the two groups. Our findings suggest that 12-wk high-fiber diet intervention improves metabolic outcomes in patients with T2DM. However, it may reduce bone formation without affecting bone microarchitecture or Wnt pathway regulation.

Genetic Characterization, Antibiotic Resistance, and Virulence Genes Profiling of Bacillus cereus Strains from Various Foods in Japan.

Bacillus cereus sensu stricto is a foodborne pathogen that causes food poisoning. Their spore and biofilm-forming abilities persist in various environments and foods. This study investigated the prevalence, virulence, antibiotic resistance, and genetic diversity of B. cereus s. s. strains isolated from various food samples. Of 179 samples, 22.34% were positive for B. cereus s. s., with significantly high detection rates in milk products and raw chicken meat. Forty strains were isolated from positive samples. Matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis revealed nine distinct clusters and multi-locus sequence typing revealed 34 sequence types including 23 novel sequences, demonstrating high genetic diversity among the isolates. PCR analysis revealed that all the strains contained at least one toxin gene, but none contained the cytK gene. Antibiotic resistance tests revealed that all isolates were classified as multidrug-resistant, with high resistance levels, particularly to β-lactam antibiotics and vancomycin, but were susceptible to gentamicin. All isolates showed variations in biofilm formation. This study highlights the significant public health risk due to B. cereus s. s. and underscores the need for stringent monitoring and control measures in food production to manage antimicrobial resistance and ensure food safety.

Monitoring of pathogenic Leptospira infection in wolves (Canis lupus) from Spain and Italy

Leptospirosis is a bacterial disease of worldwide distribution with relevant implications for animal and human health. Different large wild carnivore species can act as reservoirs of this zoonotic pathogen. This study aimed to evaluate the circulation of Leptospira spp. in free-ranging wolves (Canis lupus) from southern Europe. A total of 281 kidney samples of wolves from Spain and Italy were collected between 2017 and 2023. The presence of Leptospira DNA was analysed by real-time PCR and phylogenetic analyses were carried out using a Bayesian approach. The overall prevalence was 3.2 % (9/281; 95 %CI: 1.1–5.3). Leptospira DNA was detected in nine of the 180 wolves from Spain (5.0 %; 95 %CI: 1.8–8.2), but not in the Italian wolf population (0 %; 0/101). Molecular analyses revealed high homology between the sequences obtained in the present study and isolates of Leptospira interrogans and Leptospira borgpetersenii from different rodent and domestic ungulate species. Our results provide evidence of a low and spatially heterogeneous circulation of this pathogen in wolf populations of southern Europe. The detection of zoonotic Leptospira species in this survey supports the need to consider wolf populations in monitoring programs for leptospirosis with a One Health approach.

Dihydroartemisinin inhibited vasculogenic mimicry in gastric cancer through the FGF2/FGFR1 signaling pathway

Vasculogenic mimicry (VM) is a novel model for supplying blood to multiple tumors, including gastric cancer (GC), and is a potential target for its treatment. Dihydroartemisinin (DHA) is a potential natural antitumor substance that inhibits the progression of tumors in many ways. The research aimed to evaluate the impact of DHA on VM formation and its mechanisms. The IC50 of DHA, DHA's effect on proliferation, invasion, and migration in GC cells and VM formation in both cell and animal models were determined through wound healing, MTT, EdU, colony formation, and Transwell assays. Genomics was employed to identify genes related to DHA inhibition of VM formation, and to analyze their relationship to VM formation. qRT‒PCR and western blot (WB) analysis were carried out to analyze the changes in protein and mRNA levels after DHA treatment and the changes in VM-associated protein biomarkers after blocking target gene-related pathways. The mechanism by which DHA inhibits VM in GC was elucidated in vivo. DHA reduced the invasion, proliferation, and migration of GC cells and inhibited VM in cells and in vivo. A total of 220 DEGs were identified in the DHA-treated HGC-27 cells. Among the 146 downregulated genes, fibroblast growth Factor 2 (FGF2) was most closely associated with angiogenesis and VM. The level of FGF2 in GC tissues with VM was markedly greater than in VM lacking tissues. Treatment with DHA or FGFR1 blockade suppressed VM formation and reduced VM-related biomarker proteins. DHA suppressed tumor progression and VM formation by reducing FGF2 in xenograft mouse models. Per our knowledge, this is the first study to demonstrate the inhibitory effect of DHA on VM, providing a novel strategy for the treatment of GC.

PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with the occurrence of pear fruit russeting

Non-specific lipid-transfer proteins (nsLTPs) are a group of small, cysteine-rich proteins that are involved in the transport of cuticular wax and other lipid compounds. Accumulating evidence suggests that dynamic changes in cuticular waxes are strongly associated with fruit russeting, an undesirable visual quality that negatively affects consumer appeal in pears. Currently, the regulatory role of nsLTPs in cuticular wax deposition and pear fruit skin russeting remains unclear. Here, we characterized the variations of cuticular waxes in non-treated (russeted) and preharvest bagging treated (non-russeted) pear fruits throughout fruit development and confirmed that the contents of cuticular waxes were significantly negatively correlated with the occurrence of pear fruit russeting. Based on RNA-Sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) analyses, two nsLTP genes (PpyLTP36 and PpyLTP39) were identified, which exhibited high expression levels in non-russeted pear fruit skins and were significantly repressed during fruit skin russeting. Subcellular localization analysis demonstrated that PpyLTP36 and PpyLTP39 were localized to the plasma membrane (PM). Further, transient Virus-Induced Gene Silencing (VIGS) analyses of PpyLTP36 and PpyLTP39 in pear fruits significantly reduced cuticular wax deposition. In conclusion, PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with pear fruit skin russeting.

Synergisitic anti-colorectal cancer effects of WNT974 combined with artesunate via cooperative regulation of p21, p27, and AKT

ObjectiveTo evaluate the synergistic effects and underlying mechanisms of WNT974 and artesunate (ART) combination therapy in the treatment of colorectal cancer (CRC). MethodsWe conducted an initial assessment of the synergistic impact of WNT974 and ART on cell viability using the MTT assay across eight CRC cell lines with diverse mutation statuses.Subsequently, a 3D in vitro model was utilized to confirm the synergistic effect of the combination. Flow cytometry was employed to analyze cell cycle distribution. The molecular mechanisms were further investigated by examining the expression levels of cell cycle-related proteins (p21, p27, and CDK2) and AKT activity through PCR and Western blot analyses. The in vivo efficacy of the combination therapy was evaluated using a xenograft mouse model of CRC. ResultsThe combination of WNT974 and ART significantly reduced cell viability in all eight CRC cell lines, demonstrating a synergistic effect. Additionally, the combination exhibited synergy in inhibiting 3D spheroid growth. Cell cycle analysis revealed that the combination therapy induced a marked G1/G0 arrest through CDK2 inhibition, surpassing the efficacy of individual treatments. Molecular analysis indicated that the combination therapy upregulated p21 and p27 expression and reduced AKT activity. In vivo, the combination therapy significantly inhibited tumor growth in the xenograft mouse model, notably outperforming 5FU, the standard first-line drug for CRC. ConclusionThe study highlights the synergistic anti-CRC effects of combining WNT974 and ART. This combination induces G1/G0 phase cell cycle arrest by cooperatively regulating p21, p27, and AKT, presenting a promising alternative to current CRC treatments. These findings elucidate the molecular basis of this interaction and provide a scientific foundation for advancing ART combined with WNT94 as a novel therapy for CRC, offering a potential new avenue.

Enhancing neurite growth and neural functions on polymeric nerve conduit with BMSC-derived ECM coating

The therapy of large defects in peripheral nerve injury (PNI) suffers from several drawbacks, especially the lack of autologous nerve donors. Nerve conduits are considered as a solution for nerve injury treatment, but biocompatibility improvements is still required for conduits prepared with synthetic materials. Cell-derived extracellular matrix (ECM) has drawn attention due to its lower risk of immunogenic response and independence from donor availability. The goal of this study is to coat bone mesenchymal stem cell-derived ECMs on poly(lactic-co-glycolic) acid (PLGA) conduits to enhance their ability to support neural growth and neurite extensions. The ECM-coated conduits have better hydrophilic properties than the pure PLGA conduits. A marked increase on PC12 and RSC96 cells' viability, proliferation and dorsal root ganglion neurite extension was observed. Quantitative PCR analysis exhibited a significant increase in markers for cell proliferation (GAP43), neurite extension (NF-H, MAP2, andβIII-tubulin) and neural function (TREK-1). These results show the potential of ECM-coated PLGA conduits in PNI therapy.

Unraveling novel mRNA transcripts of the human DNA N-glycosylase 1 (NTHL1) gene with the implementation of an innovative targeted DNA-seq assay

The human NTHL1 gene encodes a DNA glycosylase that plays a key role in the base excision repair (BER) pathway, repairing oxidative DNA damage and maintaining genome integrity. The physiological activity of NTHL1 is crucial in preventing genetic alterations that can lead to cancer. In this study, we employed an innovative targeted DNA sequencing (DNA-seq) methodology to explore the transcriptional landscape of the NTHL1 gene, revealing previously uncharacterized alternative splicing events and novel exons. Our designed approach provided significantly improved sequencing depth and coverage, enabling the identification of novel NTHL1 mRNA transcripts. Bioinformatics analysis confirmed all annotated splice junctions of the main NTHL1 transcripts (v.1 – v.3) and revealed novel mRNA transcripts (NTHL1 v.4 – v.9) derived from splicing events between annotated exons as well as mRNAs containing previously uncharacterized exons (NTHL1 v.10 – v.14). Quantitative PCR analysis highlighted a diverse expression pattern of these novel transcripts across different human cell lines, suggesting cell-specific roles and regulatory mechanisms. Notably, NTHL1 v.5 was overexpressed in luminal A breast cancer cells (MCF-7), while v.13 was prominent in triple negative (BT-20), HER2 + breast cancer (SK-BR-3), prostate, colorectal cancer cells and HEK-293 cells. Our findings suggest that specific novel NTHL1 transcripts may encode protein isoforms with distinct structural features, as indicated by ribosome profiling datasets, while others containing premature termination codons could function as long non-coding RNAs. These insights enhance our understanding of NTHL1 regulatory role and its potential as a biomarker and therapeutic target in human malignancies. This study underscores the importance of exploring the transcriptional diversity of NTHL1 to fully elucidate its role in cancer pathobiology.

Molecular identification and functional characterization of a C-type lectin gene in Meretrix meretrix

C-type lectins (CTLs) are a kind of Ca2+-dependent immunoreactive factors, which participated in pathogens recognition and defense. The present study identified a new CTL from hard clam Meretrix meretrix (designated as MmCTL4). The full-length of MmCTL4 cDNA was 608 bp, encoding a presumed signal peptide of 19 bp and a carbohydrate recognition domain (CRD) of 131 bp. The tertiary structure of recombinant MmCTL4 protein (rMmCTL4) was the typical long double-ring structure with three conserved disulfide bonds, and the motifs in Ca2+-binding sites of MmCTL4 were QPN and WSD. The SYBR Green real-time PCR analysis indicated that MmCTL4 was widely expressed in the hemocytes, hepatopancreas and mantle of healthy clams. After Vibrio splendidus stimulation, the temporal expression profile of MmCTL4 mRNA in hemocytes and hepatopancreas increased by 7.8-fold at 6 hpi and 3.9-fold at 12 hpi, respectively. The cDNA fragments encoding MmCTL4 were recombined into pET-32a (+) vectors, and transformed into Escherichia coli BL21 (DE3). The rMmCTL4 with the presence of Ca2+ performed obvious hemagglutination activity, and could agglutinate E. coli, Bacillus subtilis, and Staphylococcus aureus, while it only weakly agglutinate Vibrio parahaemolyticus and fungi P. pastoris. The agglutination activity of rMmCTL4 were significantly inhibited by D-mannose, D-xylose, D-lactose, maltose and lipopolysaccharides. These results indicated that MmCTL4, as a class of typical pattern recognition receptors (PRRs), could protect the host against pathogen invasion in the innate immunity of clams.

Efficacy of cordycepin against Neospora caninum infection in vitro and in vivo

Neospora caninum (N. caninum) is an obligate intracellular Apicomplexa parasite that causes abortions in dairy cows and incurs substantial to significant economic losses in the global dairy farming industry. Cordycepin, a nucleoside antibiotic derived from Chinese medicine Cordyceps militaries, exhibits diverse biological activities. However, it remains unclear whether cordycepin possesses inhibitory effects against N. caninum infection. Therefore, this study aimed to establish both in vivo and in vitro models of N. caninum to investigate the potential impact of cordycepin against N. caninum infection. We successfully established an in vitro model of N. caninum infection in RAW264.7 cells, followed by qRT- PCR analysis to detect the content of N. caninum DNA within the cells. The effects of cordycepin on N. caninum was observed using the Giemsa method on RAW264.7, and the rate of cell infection was calculated. Cordycepin exhibited inhibitory effects on N. caninum tachyzoites in vitro, preserving cellular integrity and reducing the rate of cell infection. In mice, we established an in vivo model of N. caninum infection and detected N. caninum presence in tissues using. Real-time fluorescence quantitative PCR. Histopathological changes were observed through Hematoxylin-eosin staining. Liver function was assessed by using glutamic acid aminotransferase (ALT) and aspartic acid aminotransferase (AST) kits. Oxidative stress status was measured using catalase (CAT), malondialdehyde (MDA), and glutathione (GSH) kits. Compared with the model group, mice treated with cordycepin showed reduced clinical symptoms, increased food intake, and their body weight (P=0.0143, P=0.0068) was significantly higher than those in the model group. Furthermore, cordycepin treatment significantly alleviated hepatic cord disorders, hepatocellular swelling, detachment, and vacuolization; duodenal epithelial detachment and shortening of villi caused by N. caninum infection. Cordycepin administration reduced the increase in ALT (P=0.01, P=0.008) and AST (P<0.001) levels caused by N. caninum infection, while ameliorating hepatocyte swelling, necrosis, and detachment as well as inflammatory cell infiltration within mice liver; it also led to shortened or even disappeared duodenal villi along with and oedema of the submucosa. Analysis of oxidative stress showed that cordycepin ameliorated the damage caused by N. caninum by reducing MDA (P=0.03, P=0.02, P=0.005) and increasing CAT (P=0.004, P<0.001) and GSH (P=0.004, P<0.001) levels. In conclusion, this study reports for the first time on cordycepin's efficacy against N. caninum infection providing a potential candidate drug for neosporosis treatment.