Polymerase Chain Reaction Analysis Research Articles

Differential Expression of Phosphate Starvation-responsive Genes among Sweetpotato Cultivars during Establishment and Onset of Storage Root Formation

It has been documented that sweetpotato (Ipomoea batatas L.) cultivars vary in morphological and physiological adaptations to low phosphorus (P) availability but knowledge of the underlying molecular mechanisms is largely unknown. The objective of this research was to generate cultivar-specific information about phosphate starvation response (PSR) genes to variations in inorganic phosphate (Pi) availability at the onset of storage root formation among sweetpotato cultivars. Cultivars Bayou Belle (BB), Evangeline (EV), and Orleans (OR) were grown under varying Pi levels: 0 mg·L−1 (low Pi), 15 mg·L−1 (Pi-sufficient), and 31 mg·L−1 (high Pi). Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of PSR genes IbPHR1, IbSPX1, IbSPX3, IbPHT1;4, IbPHT1;5, and IbNLA was performed at 5 and 10 days after planting (DAP), corresponding to adventitious root system establishment and the onset of storage root formation, respectively. The cultivar BB grown under low Pi showed significantly higher expression of all genes except IbPHR1. The cultivar OR grown in both low and high Pi exhibited upregulation of IbPHR1. On the other hand, EV grown under low Pi revealed no response for all genes investigated. Exposure of BB to high Pi resulted in a decrease in expression for all genes except IbNLA. The cultivar EV grown in high Pi conditions showed increase in expression for IbSPX1, IbPHT1;5, and IbNLA. Expression pattern difference among cultivars relative to Pi levels corroborates field observations showing that BB, EV, and OR have different Pi requirements. The increased activity of BB PSR genes grown under low Pi supports the hypothesis that BB requires low P fertilizer input relative to OR and EV. Results from this study corroborate findings from well-characterized crop species and model systems and pave the way for the development of tools and practices to increase phosphorus use efficiency in sweetpotato.

Ubiquitin-Specific Protease 15 Plays an Important Role in Controlling the Tolerance to Salt, Drought and Abscisic Acid in Arabidopsis thaliana

Ubiquitin-specific proteases (UBPs), the largest subfamily of deubiquitinating enzymes (DUBs), are critical for plant growth and development as well as abiotic-stress responses. In this study, we discovered that the expression of the ubiquitin-specific protease 15 (UBP15) gene of the gene ubiquitin-specific protease 15 (UBP15) was induced by salt, mannitol and abscisic acid (ABA) treatments. Further research revealed that UBP15 is involved in modulation of salt, drought tolerance and ABA signaling during seed germination, early seedling development, post-germination root growth or adult-plant stage. Enrichment analysis showed that many genes related to abiotic stresses and metabolic pathways were altered in the ubp15-1 mutant. Through the joint analysis of the quantitative real-time polymerase chain reaction (qRT-PCR) and differentially-expressed gene relationship network, we found that UBP15 may mainly regulate salt-stress tolerance by modulating the dwarf and delayed flowering 1 (DDF1) pathway through a cascade reaction. In the regulation of drought-stress responses, ring domain ligase1 (RGLG1) may be a direct substrate of UBP15. Moreover, we cannot exclude the possibility that UBP15 acts in a feed-forward loop mechanism in the regulation of drought-stress responses via ethylene response factor 53 (ERF53) and its ubiquitin (Ub) ligase RGLG1. In ABA signal transduction, UBP15 may play a role in at least three aspects of the ABA signaling pathway: ABA synthesis, stomatal closure regulated by ABA signaling, and transcription factors in the ABA pathway. Taken together, our results suggest that UBP15 is involved in salt, osmotic, and drought-stress tolerance and the ABA signaling pathway by directly regulating the stability of key substrates or indirectly affecting the expression of genes related to abiotic stresses in Arabidopsis thaliana. Our research provides new germplasm resources for stress-resistant crops cultivation. These results demonstrate that UBP15 is a key regulator of salt, drought and ABA tolerance in Arabidopsis.

L-Securinine Induces ROS-Dependent Apoptosis on Pancreatic Cancer Cells via the PI3K/AKT/mTOR Signaling Pathway.

Accumulating evidence has shown that l-securinine can, in certain circumstances, suppress tumor development by elevating reactive oxygen species (ROS) levels. The current work set out to examine l-securinine's apoptotic effects on HuP-T3 cells as well as any potential underlying molecular mechanism(s) that could explain its action as an anticancer agent. In this study, we used 1.2B4 cells as a control human cell line to verify our findings. Hup-T3 and 1.2B4 cells were cultured with a medium containing the following dilutions of l-securinine: 1-10 μΜ for up to 72 h. We examined the viability and proliferation levels of cells in both cell lines. Then, we measured only 1.2B4 insulin levels and content. We also quantified cell apoptosis, cell cycle levels, and the intracellular reactive oxygen species on HuP-T3 and 1.2B4. Afterwards, we performed a real-time quantitative polymerase chain reaction and western blot analysis. Our results demonstrated that l-securinine inhibited both proliferation and growth of Hup-T3 cells, showing inhibitory and antiproliferative activity in comparison with the control group. In addition, l-securinine had no impact on the proliferation and growth of 1.2B4 cells, nor on their insulin levels and content. By boosting ROS production, and inhibiting the PI3K/AKT/mTOR signaling pathway, l-securinine induced apoptosis on HuP-T3 cells. Pancreatic cancer was successfully inhibited by l-securinine in vitro. l-securinine triggers ROS-dependent apoptosis on pancreatic cancer cells while inhibiting the PI3K/AKT/mTOR signaling pathway. These findings suggest that l-securinine holds promise as a potential lead for future drug development in the fight against pancreatic adenocarcinoma.

Steroid‐responsive granulomatous mural folliculitis associated with ovine herpesvirus‐2 in a Pygmy goat

AbstractThis case reports a Pygmy goat diagnosed with granulomatous mural folliculitis associated with ovine herpesvirus‐2 infection. Three biopsy samples were collected at different time points throughout the evolution of the disease, which were submitted to histopathology and polymerase chain reaction analyses. Despite the suspected viral aetiology, the goat showed significant clinical improvement with a tapering regimen of immunosuppressive corticosteroids, achieving clinical remission. A progressive reduction in the follicular granulomatous infiltrate and in apoptosis of follicular keratinocytes was observed, while the viral presence persisted. The disease remained controlled with a follow‐up period of 2 years, although a flare occurred after discontinuation of the treatment. The findings suggest that the granulomatous reaction affecting the follicles may be immune‐mediated rather than directly induced by the cytopathic effects of the virus.

Preventive effects of matrine on LPS-induced inflammation in RAW 264.7 cells and intestinal damage in mice through the TLR4/NF-κB/MAPK pathway

BackgroundMatrine is a tetracyclic quinolizidine alkaloid with diverse bioactive properties, including anti-inflammatory and neuroprotective properties. However, the underlying anti-inflammatory mechanisms remain unclear. PurposeThis study aimed to explore how matrine reduces Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and to assess its protective effects against LPS-induced intestinal damage. MethodsThe effect of matrine on cell viability was assessed using the cell counting kit-8 (CCK-8) assay. Additionally, its impact on inflammatory cytokines and macrophage polarization was assessed using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The effects on intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), nitric oxide (NO) production, and oxidative stress were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate staining and JC-1 and Griess assays. Immunofluorescence staining was used to observe the translocation of the NF-κB p65 subunit. Western blotting (WB) and qRT-PCR were employed to analyze the expression levels of proteins related to the toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathway. An LPS-induced mouse model was established to study the intestinal inflammation and barrier injury. Mouse feces characteristics, colon length, and disease activity index (DAI) were recorded. Hematoxylin-eosin (H&E) and alcian blue/periodic acid schiff (AB/PAS) staining were used to observe morphological changes and barrier damage in the duodenum, jejunum, ileum, and colon and to measure villus length, crypt depth, goblet cell count, and positive areas in the duodenum, jejunum, and ileum. The content of short-chain fatty acids (SCFAs) in the colon was determined using gas chromatography (GC). ResultsMatrine inhibited LPS-induced inflammatory cytokine levels, suppressed macrophage M1 polarization, and promoted M2 macrophage polarization. Matrine reduced LPS-induced increases in ROS and NO levels and regulates oxidative stress. Additionally, matrine inhibited the nuclear translocation of the NF-κB p65 subunit and exerted anti-inflammatory effects by suppressing the activation of the TLR4/NF-κB/MAPK pathway. In vivo experiments indicated that matrine significantly alleviated LPS-induced diarrhea, increased DAI, and shortened the colon. Matrine reduced the production of the pro-inflammatory cytokine interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α and the pro-inflammatory mediator NO in mouse intestinal tissues while promoting the content of the anti-inflammatory cytokine IL-10. Furthermore, it improved intestinal tissue structure and alleviated LPS-induced intestinal barrier damage. Finally, matrine increased the SCFA levels in the intestine. ConclusionMatrine exerted its anti-inflammatory effects and protects against intestinal injury through the TLR4/NF-κB/MAPK signaling pathway.

Gentiopicroside Attenuates Lithium/Pilocarpine-Induced Epilepsy Seizures by Down-Regulating NR2B/CaMKII/CREB and TLR4/NF-κB Signaling Pathways in the Hippocampus of Mice

Background: Epilepsy is a prevalent and disabling neurological condition characterized by recurrent seizures. Approximately 50% of adults with active epilepsy have at least one comorbidity and they are at a greater risk of premature death than the general population. Gentiopicroside (Gent) is a primary component of Gentiana macrophylla Pall. that has been shown to have diverse pharmacological properties. However, its role in epileptic seizures in adult mice and its underlying mechanism of action remain obscure. We aimed to explore the anti-epileptic effect and mechanism of Gent on lithium/pilocarpine (Pilo)-induced epilepsy seizures in mice. Methods: In this study, we established a lithium/Pilo-induced epilepsy model, and Gent was first given to mice 30 min before Pilo administration. Then, we detected behavioral and histopathological changes through electrocorticographic (ECoG) measurements, Nissl staining, Fluoro-Jade B (FJB) staining, and immunohistochemical staining. We then used molecular biology techniques, such as Western blotting, quantitative polymerase chain reaction (qPCR) analysis, and the enzyme-linked immunosorbent assay (ELISA) to investigate the mechanisms of Gent in lithium/Pilo-induced epileptic seizures in mice and lipopolysaccharide (LPS)-induced inflammatory astrocytes. Results: We confirmed that Gent could prevent abnormal ECoG activity, behavioral changes, and neurodegeneration. Subsequently, we found Gent could downregulate the factors that could promote apoptosis (i.e., the NR2B/CaMKII/CREB signaling cascade) and neuroinflammatory-related factors (i.e., the TLR4/NF-κB signaling cascade). Conclusions: Gent could be a potential therapeutic agent for epilepsy, offering possibilities for both prevention and treatment. Our research establishes a preliminary experimental framework for ongoing studies into Gent’s efficacy as a treatment for epilepsy.

Global regulator AdpA directly binds to tunicamycin gene cluster and negatively regulates tunicamycin biosynthesis in Streptomyces clavuligerus.

Since a transcriptional regulator has yet to be identified within the tunicamycin biosynthetic gene cluster in Streptomyces clavuligerus, we conducted a comprehensive investigation by focusing on the possible function of the pleiotropic regulator AdpA on tunicamycin. The genes encoding early steps of tunicamycin biosynthesis were significantly upregulated in S. clavuligerus ΔadpA. At the same time, they were downregulated in adpA overexpressed strain as shown by RNA-sequencing (RNA-seq) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis. The tunicamycin gene cluster's co-transcription pattern was understood by reverse transcriptase polymerase chain reaction (RT-PCR). Our Electrophoretic Mobility Shift Assay (EMSA) data clearly showed AdpA's binding to the upstream sequence of the tunA gene, asserting its regulatory control. In addition to its direct negative regulation of tunicamycin biosynthesis, AdpA operates at a global level by orchestrating various regulatory genes in S. clavuligerus, such as wblA, whiB, bldM, arpA, brp, and adsA involved in morphological differentiation and secondary metabolite biosynthesis as depicted in RNA-seq data. This study represents a significant milestone by unveiling the AdpA regulator's pathway-specific and global regulatory effect in S. clavuligerus.

Outbreak investigation of food poisoning attributed to Omphalotus japonicus using polymerase chain reaction‐based sequencing

AbstractObjectiveThis study aims to report and analyse an outbreak of Omphalotus japonicus poisoning following mushroom ingestion in Taiwan, and to employ PCR methods for accurate identification of the toxic mushrooms involved.MethodsA descriptive observational study was conducted involving a family of four who ingested the mushrooms in the mountainous rural area of Yilan County, Taiwan. Data were collected through interviews with the affected individuals and a review of their medical records. Due to the destruction of the mushroom samples, Polymerase Chain Reaction (PCR) methods were utilized for identification. Genomic DNA was extracted from the raw fruiting bodies, and specific primers targeting the internal transcribed spacer region of the nuclear ribosomal DNA were designed for PCR detection.ResultsThree family members (mother, daughter, and son) experienced gastrointestinal symptoms, including nausea, vomiting, abdominal cramps, and diarrhea, approximately 30 min after consuming the mushrooms. Upon admission to the emergency room, they were hydrated and monitored, with laboratory results showing slightly elevated alanine transaminase levels. The affected individuals recovered within 12 h and were discharged in good condition on day 2. PCR analysis of the mushroom sample confirmed the identification of Omphalotus japonicus, known to produce Illudin S and Illudin M, which cause acute gastrointestinal symptoms. This case represents the first documentation of using PCR‐based sequencing to diagnose Omphalotus japonicus poisoning in the English literature.

Genome-Wide Identification of the Cyclic Nucleotide-Gated Ion Channel Gene Family and Expression Profiles Under Low-Temperature Stress in Luffa cylindrica L.

Cyclic nucleotide-gated ion channels (CNGCs) are cell membrane channel proteins for calcium ions. They have been reported to play important roles in survival and in the responses to environmental factors in various plants. However, little is known about the CNGC family and its functions in luffa (Luffa cylindrica L.). In this study, a bioinformatics-based method was used to identify members of the CNGC gene family in L. cylindrica. In total, 20 LcCNGCs were detected, and they were grouped into five subfamilies (I, II, Ⅲ, IV-a, and IV-b) in a phylogenetic analysis with CNGCs from Arabidopsis thaliana (20 AtCNGCs) and Momordica charantia (17 McCNGCs). The 20 LcCNGC genes were unevenly distributed on 11 of the 13 chromosomes in luffa, with none on Chromosomes 1 and 5. The members of each subfamily encoded proteins with highly conserved functional domains. An evolutionary analysis of CNGCs in luffa revealed three gene losses and a motif deletion. An examination of gene replication events during evolution indicated that two tandemly duplicated gene pairs were the primary driving force behind the evolution of the LcCNGC gene family. PlantCARE analyses of the LcCNGC promoter regions revealed various cis-regulatory elements, including those responsive to plant hormones (abscisic acid, methyl jasmonate, and salicylic acid) and abiotic stresses (light, drought, and low temperature). The presence of these cis-acting elements suggested that the encoded CNGC proteins may be involved in stress responses, as well as growth and development. Transcriptome sequencing (RNA-seq) analyses revealed tissue-specific expression patterns of LcCNGCs in various plant parts (roots, stems, leaves, flowers, and fruit) and the upregulation of some LcCNGCs under low-temperature stress. To confirm the accuracy of the RNA-seq data, 10 cold-responsive LcCNGC genes were selected for verification by quantitative real-time polymerase chain reaction (RT-qPCR) analysis. Under cold conditions, LcCNGC4 was highly upregulated (>50-fold increase in its transcript levels), and LcCNGC3, LcCNGC6, and LcCNGC13 were upregulated approximately 10-fold. Our findings provide new information about the evolution of the CNGC family in L. cylindrica and provide insights into the functions of the encoded CNGC proteins.

RGS10 Deficiency Alleviated Intestinal Mucosal Inflammation Through Suppression of Th1/Th17 Cell Immune Responses in Ulcerative Colitis.

Regulator of G-protein signalling (RGS) 10 plays critical roles in several immune related diseases. However, whether RGS10 is involved in colonic inflammation of ulcerative colitis (UC) is still obscure. This study aimed to investigate the role of RGS10 in UC. In this study, RGS10 expression was examined by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, immunohistochemistry, and immunofluorescent analysis. Single-cell RNA sequencing of intestinal mucosa was performed to identify key immune cells with differentially expressed RGS10. RGS10 knockout mice were generated and established dextran sulphate sodium (DSS)-induced colitis. Expression of inflammatory cytokines on mRNA and protein levels was detected by qRT-PCR, enzyme-linked immunosorbent assay, and flow cytometry. We found that RGS10 expression was significantly elevated in UC patients, especially in CD4+ T cells, compared with healthy subjects. Intriguingly, RGS10 deficiency markedly alleviated DSS-induced colitis and decreased the proportion of Th1 and Th17 cells in lamina propria mononuclear cells (LPMCs), peripheral blood (PB), spleens, and mesenteric lymph nodes (mLNs). Mechanistically, RGS10 deficiency blocked the differentiation of Th1 and Th17 cells by inhibiting the phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3. The co-immunoprecipitation analysis further showed that RGS10 could interact with protein tyrosine phosphatase non-receptor type 2 (PTPN2), and further regulated Th1 and Th17 cells differentiation of CD4+ T cells. In conclusion, RGS10 deficiency alleviated intestinal mucosal inflammation through inhibition of Th1/Th17 cell-mediated immune responses via interaction with PTPN2 in CD4+ T cells. Therefore, targeting RGS10 may represent a novel therapeutic approach for UC treatment.

Fine mapping and identification of the downy mildew resistance gene BoDMR2 in Cabbage (Brassica oleracea L. var. capitata).

Cabbage (Brassica oleracea L. var. capitata) is an important crop within the Brassica oleracea species and is extensively cultivated worldwide. In recent years, outbreaks of downy mildew caused by Hyaloperonospora parasitica have resulted in substantial losses in cabbage production. Despite this, there have been limited studies on genes associated with resistance to downy mildew in cabbage. This study identified sister lines exhibiting significant differences in disease resistance and susceptibility. Using bulked segregant analysis followed by sequencing (BSA-seq) and linkage analysis, the cabbage resistance locus BoDMR2 was accurately mapped to an approximately 300kb interval on chromosome 7. Among the candidate genes identified, several single nucleotide polymorphisms (SNPs) and a 3-bp insertion were found within the conserved domain of the Bo7g117810 gene, encoding a leucine-rich repeat domain protein, in susceptible genotypes. Additionally, real-time quantitative polymerase chain reaction (RT‒qPCR) analysis revealed that the expression level of Bo7g117810 in resistant specimens was 2.5-fold higher than that in susceptible specimens. An insertion‒deletion (InDel) marker was designed based on the identified insertion in susceptible materials, facilitating the identification and selection of downy mildew-resistant cabbage cultivars. This study identifies Bo7g117810 as a potential candidate gene associated with adult-stage resistance to downy mildew in cabbage, supported by observed differences in gene sequence and expression levels. Furthermore, the development of an InDel marker I1-3, based on its mutation, provides valuable resources for breeding resistant cabbage cultivars.

Potential role of heteroplasmic mitochondrial DNA mutations in modulating the subtype-specific adaptation of oral squamous cell carcinoma to cisplatin therapy

Cancer cells are constantly evolving to adapt to environmental changes, particularly during exposure to drug treatment. In this work, we aimed to characterize genetic and epigenetic changes in mitochondrial DNA (mtDNA) that may increase the resistance of oral squamous cell carcinoma (OSCC) to cisplatin. We first derived drug-resistant cells from two human OSCC cell lines, namely SAS and H103, by continual cisplatin treatments for about 4 months. To determine mtDNA changes induced by cisplatin, we performed nanopore sequencing and quantitative polymerase chain reaction analysis of mtDNA extracted from the cells pre- and post-treatment. We also assessed the mitochondrial functions of the cells and their capacity to generate intracellular reactive oxygen species (ROS). We found that in the cisplatin-resistant cells derived from SAS, there was a reduction in mtDNA content and significant enrichment of a m.3910G > C mutation in the MT-ND1 gene. However, such changes were not detected in cisplatin-resistant H103 cells. The cisplatin treatment also altered methylation patterns in both SAS and H103 cells and decreased their sensitivity to ROS-induced cytotoxicity. We suggest that the sequence alterations and epigenetic changes in mtDNA and the reduction in mtDNA content could be key drivers of cisplatin resistance in OSCC. These mtDNA alterations may participate in cellular adaptation that serves as a response to adverse changes in the environment, particularly exposure to cytotoxic agents. Importantly, the observed mtDNA changes may be influenced by the distinct genetic landscapes of various cancer subtypes. Overall, this study reveals significant insights into cisplatin resistance driven by complex mtDNA dynamics, particularly in OSCC. This underscores the need for targeted therapies tailored to the genetic profiles of individual OSCC patients to improve disease prognosis.

Functional and transcriptional regulation of the anthocyanidin acyl modifier gene Gs5AT of Gentiana sino-ornata.

The Chinese gentian, Gentiana sino-ornata produces brilliant blue flowers. To investigate the biological function and transcriptional regulation mechanism of the anthocyanin 5-O-acyltransferase gene (Gs5AT ) in the corolla, it is beneficial to analyse the mechanism of blue flower colour presentation. In this investigation, we obtained the CDS and promoter sequences of the gene Gs5AT . Yeast one-hybrid experiments were used to identify the transcription factor GsbHLH7 that activates the gene Gs5AT . According to quantitive reverse transcription polymerase chain reaction analysis, the expression of the gene Gs5AT was significantly and positively correlated with the gene GsbHLH7 . The colour phenotype of the flowers was significantly altered by the virus-induced gene silencing transduction of Gs5AT and GsbHLH7 , with GsbHLH7 silencing producing more pronounced changes in the corolla colour than Gs5AT . The expression of GsF3'5'H , GsDFR , GsANS , Gs3GT , and Gs5GT all fell to varying degrees after GsbHLH7 silencing, indicating that GsbHLH7 may regulate transcription of these genes as well as Gs5AT . The results of this study indicate that Gs5AT was positively regulated by the GsbHLH7 , and thus affects the colour presentation of the blue corolla.

Exploring the Role of miR-132 in Rat Bladders and Human Urothelial Cells during Wound Healing

Urinary bladder wound healing shares many features with skin healing, involving several molecular players, including microRNAs (miRs). This study investigated the role of miR-132 in urothelial cells. We analyzed miR-132 expression in rat bladder using in situ hybridization and conducted gain and loss of miR-132 function assays in primary human urothelial cells (HUCs). These assays included cell proliferation and migration studies. To explore the regulation of miR-132 expression, cells were treated with wound-healing-related factors such as interleukin 6 (IL-6), interleukin 10 (IL-10), and transforming growth factor beta-1 (TGF-β1). Predictive bioinformatics and a literature review identified potential miR-132 targets, which were validated through real-time polymerase chain reaction (RT-PCR) and Western blot analysis. miR-132 was found to promote cellular proliferation and migration during the early stages of urothelial wound repair. Its expression was modulated by key cytokines such as IL-6, IL-10, and TGF-β1. miR-132 played a crucial role in urothelial wound healing by enhancing cell proliferation and migration, regulated by cytokines, suggesting its action within a complex regulatory network. These findings highlight the therapeutic potential of targeting miR-132 in bladder injury repair, offering new insights into bladder repair mechanisms.

Promotion of bone osteogenic and regeneration by resveratrol-derived carbon dots in inflammatory environments via a one-step hydrothermal method

In the context of bone fractures, local inflammation complicates the healing process, adversely affecting the recovery of bone defects. This inflammatory response impedes the regenerative potential of stem cells and induces the differentiation of macrophages into the M1 type, thereby inhibiting bone regeneration. To address this issue, we synthesized carbon dots (CDs) using resveratrol and citric acid. These CDs demonstrated excellent water solubility and retained the anti-inflammatory properties of resveratrol. In an inflammatory environment co-cultured with resveratrol carbon dots (RES-CDs), we observed a reduction in inflammatory factors such as iNOS and an increase in anti-inflammatory factors like CD206. Alkaline phosphatase (ALP) activity, Alizarin Red staining, and immunofluorescence assays indicated that MC3T3 cells produced more calcium nodules after co-culture with RES-CDs, along with enhanced expression of bone formation factors BMP-2 and ALP. Additionally, real-time polymerase chain reaction (qRT-PCR) and immunofluorescence analysis demonstrated that RES-CDs could induce bone tissue proliferation and differentiation by upregulating ALP, BMP-2, OCN, and OPN. In a rat cranial defect model, RES-CDs promoted tissue healing and bone proliferation at weeks 1 and 4 post-surgery. These findings underscore the potential of resveratrol carbon dots in promoting bone tissue healing while mitigating inflammation, thereby offering a promising approach for nanocomposite materials in tissue repair.

Higher Frequency of SARS-CoV-2 RNA Shedding by Cats than Dogs in Households with Owners Recently Diagnosed with COVID-19

Studies have demonstrated the susceptibility of companion animals to natural infection with SARS-CoV-2. Using quantitative reverse transcription polymerase chain reaction and sequencing analyses, this study investigated SARS-CoV-2 RNA excretion in pets in households with infected owners. Oropharyngeal and rectal swabs were collected from dogs and cats in Parana, Southern Brazil, between October 2020 and April 2021. Viral RNA was detected in 25% of cats and 0.98% of dog oropharyngeal swabs; however, systemic, respiratory, and gastrointestinal signs were absent. Complete viral genomes belonged to the Gamma lineage. Phylogenetic analyses indicated that pet samples were probably derived from human-positive cases in Parana. Viral excretion in the oropharynx was more frequent in cats than in dogs. Mutations in the S protein characteristic of Gamma strains were present in all sequenced SARS-CoV-2 strains. The receptor-binding domain of these Brazilian strains did not show any additional mutations not reported in the Gamma strains. Mutations in NSP6, NSP12, and N proteins previously mapped to strains that infect deer or minks were detected. This study highlights the importance of actively monitoring the SARS-CoV-2 strains that infect pets with continued viral exposure. Monitoring genetic changes is crucial because new variants adapted to animals may pose human health risks.

Insights into Clematis cirrhosa L. Ethanol Extract: Cytotoxic Effects, LC-ESI-QTOF-MS/MS Chemical Profiling, Molecular Docking, and Acute Toxicity Study

Background: In Jordanian traditional medicine, Clematis cirrhosa is commonly employed for the management of different diseases. Numerous investigations have documented the cytotoxic properties of different Clematis species against numerous types of cancer. Previously, we demonstrated the potential cytotoxicity of Clematis cirrhosa against HT-29 colorectal cancer cells. Extending our work, the current research aimed to explore the possible mechanisms underlying its antiproliferative activity with a plant safety evaluation. Methods: This study evaluates the extract’s impact on the cell cycle, apoptosis, and cell migration through in vitro assays, LC-ESI-QTOF-MS/MS analysis, docking studies, and an acute toxicity evaluation. Results: The Clematis cirrhosa ethanol extract (CEE) induced G2/M phase cell cycle arrest (19.63%), triggered significant apoptosis (41.99%), and inhibited cell migration/wound healing by 28.15%. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed increased expression of the proapoptotic markers BAX (6.03-fold) and caspase-3 (6.59-fold), along with the reduced expression of the antiapoptotic BCL-2, in CEE-treated cells. Moreover, CEE significantly restrained angiogenesis by reducing VEGF mRNA expression by 63.9%. High-resolution LC-ESI-QTOF-MS/MS studies identified 26 metabolites, including phenolic compounds, fatty acids, and triterpenoids. Docking studies suggested that manghaslin had the highest binding affinity for VEGFR-2, followed by calceolarioside B, quercetin 7-O-rhamnopyranoside, luteolin, and quercetin-3,7-O-diglucoside. On the other hand, salvadoraside exhibited the highest binding affinity for the inhibition of caspase-3, followed by quercetin-3,7-O-diglucoside, kaempferol-3,7-O-α-L-dirhamnoside, manghaslin, and tectoridin, supporting the observed apoptotic effects. Interestingly, the outcomes further indicate that a single oral administration of up to 5000 mg/kg CEE is safe for consumption. Conclusions: These outcomes point to the potential of Clematis cirrhosa as a promising candidate for further exploration in cancer therapy.

An undergraduate research experience in CRISPR-Cas9 mediated eukaryotic genome editing to teach fundamental biochemistry techniques.

CRISPR-Cas9 technology is an established, powerful tool for genome editing through the ability to target specific DNA sequences of interest for introduction of desired genetic modifications. CRISPR-Cas9 is utilized for a variety of purposes, ranging from a research molecular biology tool to treatment for human diseases. Due to its prominence across a variety of applications, it is critical that undergraduates in the life sciences are educated on CRISPR-Cas9 technology. To this end, we created an intensive eight-week long course-based undergraduate research experience (CURE) designed for students to understand CRISPR-Cas9 genome editing and perform it in Saccharomyces cerevisiae. Students enrolled in the CURE participate in 2, 3-h sessions a week and are engaged in the entire process of CRISPR-Cas9 genome editing, from preparation of genome editing reagents to characterization of mutant yeast strains. During the process, students master fundamental techniques in the life sciences, including sterile technique, Polymerase Chain Reaction (PCR), primer design, sequencing requirements, and data analysis. The course is developed with flexibility in the schedule for repetition of techniques in the event of a failed experiment, providing an authentic research experience for the students. Additionally, we have developed the course to be easily modified for the editing of any yeast gene, offering the potential to expand the course in research-driven classroom or laboratory settings.

CRISPR/Cas9-Mediated fech Knockout Zebrafish: Unraveling the Pathogenesis of Erythropoietic Protoporphyria and Facilitating Drug Screening

Erythropoietic protoporphyria (EPP1) results in painful photosensitivity and severe liver damage in humans due to the accumulation of fluorescent protoporphyrin IX (PPIX). While zebrafish (Danio rerio) models for porphyria exist, the utility of ferrochelatase (fech) knockout zebrafish, which exhibit EPP, for therapeutic screening and biological studies remains unexplored. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated fech-knockout zebrafish larvae as a model of EPP1 for drug screening. CRISPR/Cas9 was employed to generate fech-knockout zebrafish larvae exhibiting morphological defects without lethality prior to 9 days post-fertilization (dpf). To assess the suitability of this model for drug screening, ursodeoxycholic acid (UDCA), a common treatment for cholestatic liver disease, was employed. This treatment significantly reduced PPIX fluorescence and enhanced bile-secretion-related gene expression (abcb11a and abcc2), indicating the release of PPIX. Acridine orange staining and quantitative reverse transcription polymerase chain reaction analysis of the bax/bcl2 ratio revealed apoptosis in fech−/− larvae, and this was reduced by UDCA treatment, indicating suppression of the intrinsic apoptosis pathway. Neutral red and Sudan black staining revealed increased macrophage and neutrophil production, potentially in response to PPIX-induced cell damage. UDCA treatment effectively reduced macrophage and neutrophil production, suggesting its potential to alleviate cell damage and liver injury in EPP1. In conclusion, CRISPR/Cas9-mediated fech−/− zebrafish larvae represent a promising model for screening drugs against EPP1.

Detection of human papillomavirus in plantar warts and its impact on outcome.

Cutaneous warts are caused by human papillomavirus (HPV) infection. Distinguishing plantar warts from clavus and tylosis can be difficult. A less-invasive method of examining these lesions is necessary. Previously, we collected data on 90 patients with warts and related diseases to explore differentiation methods using HPV typing of tissue from the wart surface. In that study, 21 patients were diagnosed as cases with plantar warts, however, 10 of those 21 cases showed HPV-negative by polymerase chain reaction analysis, causing some ambiguity, thus their outcomes should be confirmed. To assess the role of HPV typing in clinical practice, we followed up these 21 cases (11 HPV-positive and 10 HPV-negative) and analyzed their outcomes. The HPV-positive group included HPV1a (one case), HPV27 (four cases), HPV57 (three cases), and HPV65 (three cases). The median age of the 21 patients was 43 years, that of the 11 HPV-positive cases was 37 years, and that of the 10 HPV-negative cases was 44 years. The sex ratios (male:female) of the HPV-positive and HPV-negative groups were 6:5 and 2:8, respectively. All 21 patients were treated with liquid nitrogen after surface keratin removal, concomitant with salicylic acid topical plaster or oral administration of Yokuinin. The longest follow-up period was 548 days. Kaplan-Meier analysis was performed to assess the healing rate according to HPV-positivity. The healing rate in HPV-positive cases was significantly higher than in HPV-negative cases (P = 0.001). Although the sample size was small, the results suggest HPV typing using non-invasive surface materials facilitates accurate diagnosis and prevents prolonged treatment of plantar warts.