Polymerase Chain Reaction Analysis Research Articles

Ascorbate peroxidase modulation confirms key role in Leishmania infantum oxidative defence

Abstract Background Ascorbate peroxidase (APX) has emerged as a promising target for chemotherapy because of its absence in humans and crucial role in the antioxidant defence of trypanosomatids. APXs, which are class I haeme-containing enzymes, reduces hydrogen peroxide using ascorbate to produce water and monodehydroascorbate, thereby preventing cell damage caused by H2O2. Methods We aimed to create an APX-knockout L. infantum line using CRISPR/Cas9. Despite unsuccessful attempts at full knockouts, we achieved deletion of chromosomal copies post-APX episomal insertion, yielding LiΔchrAPX::LbAPX parasites. We performed phenotypic characterization to assess the impact of these genetic modifications, which included the determination of APX transcript expression levels using quantitative PCR, drug sensitivity, infectivity, and parasite survival in macrophages. Results Quantitative polymerase chain reaction (PCR) analysis revealed a 10- to 13-fold reduction in APX transcript expression in LiΔchrAPX::LbAPX compared with wild-type (LiWT) and APX-overexpressing (Li::Cas9::LbAPX) parasites, respectively. The episomes in those knockdown parasites remained stable even after 20 drug-free passages in vitro. Li::Cas9::LbAPX parasites showed increased resistance to trivalent antimony (SbIII) and isoniazid, reduced tolerance to H2O2, and unchanged macrophage infectivity compared with LiWT. In contrast, LiΔchrAPX::LbAPX parasites were more sensitive to SbIII and isoniazid, exhibited greater susceptibility to H2O2-induced oxidative stress, and 72 h post-infection, showed fewer infected macrophages and intracellular amastigotes compared with LiWT parasites. Conclusions Our findings hint at the indispensability of APX in L. infantum and raise the possibility of its potential as a therapeutic target for leishmaniasis. Graphical Abstract

Clinical characteristics, progression patterns and treatment outcomes in microsporidial keratoconjunctivitis: a prospective study in Thailand.

To assess the clinical characteristics, progression patterns, and treatment outcomes of microbiologically confirmed microsporidial keratoconjunctivitis (MKC). This prospective cross-sectional study included patients with superficial punctate epithelial keratitis clinically suspected of MKC. Comprehensive slit-lamp examinations were conducted, and corneal scraping was performed for Gram-chromotrope staining and polymerase chain reaction (PCR) analysis. A standardized questionnaire gathered demographic data, clinical features, and risk behaviors. Treatment regimens and corneal findings, including medication and frequency, were documented at each visit. PCR confirmed the diagnosis of MKC in 96 out of 117 eyes (82.1%), identifying Vittaforma corneae in 93.7% of cases, Microsporidium sp. in 4.2%, and Encephalitozoon hellem in 2.1%. All cases exhibited similar characteristics and pattern of progression, including elevated epithelial lesions in diffused distribution (34.1%), typical target lesions (31.3%), and subepithelial infiltrations (41.7%). Treatment with topical moxifloxacin, with or without oral albendazole, followed by topical steroids for subepithelial infiltrates, led to clinical improvement within approximately two weeks, with 52% of patients achieving complete recovery. This study identifies key clinical features and progression patterns in MKC. Topical fluoroquinolone monotherapy, or its combination with topical steroids or oral albendazole, results in favorable visual outcomes without corneal scarring. These insights may inform and enhance clinical management of MKC.

Nuclear receptor subfamily 4 group a member 1 eases angiotensin II-arose oxidative stress in vascular smooth muscle cell by boosting nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 3 transcription

Objective: Hypertension significantly contributes to morbidity and mortality. Nuclear receptor subfamily 4 group a member 1 (Nur77) participates in regulating oxidative stress, but the mechanism in hypertension remains unclear. This study aimed to explore the function of Nur77 in oxidative stress induced by Angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs) in hypertension. Material and Methods: First, models of VSMC with Nur77, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 3 (NLRC3) and tumor necrosis factor receptor-associated factor 6 (TRAF6) knockdown or overexpression were constructed using Short Hairpin RNA (Nur77) or pcDNA3.1 vector, respectively. Next, the putative-binding motifs between Nur77 and NLRC3 promoters were detected by dual luciferase assay. We conducted reverse transcription quantitative polymerase chain reaction (qPCR) and Western blot (WB) analysis to detect Nur77, NLRC3, and TRAF6 levels in VSMCs. Then, cell counting kit-8 assay, 5-ethynyl-2’-deoxyuridine assay, wound-healing assay, enzyme-linked immunosorbent assay, and 2’,7’-dichlorofluorescin diacetate were employed to examine the impact of the knockdown or overexpression of Nur77, NLRC3, and TRAF6 on VSMCs treated with Ang II. The assays measured cell viability and proliferation, cell migration, malondialdehyde levels, and reactive oxygen species levels. Results: The overexpression of Nur77 repressed cell growth (P < 0.001), migration (P < 0.01), and oxidative stress (P < 0.01) induced by Ang II in VSMCs. Nur77 transcriptionally promoted the expression of NLRC3 (P < 0.001), and the upregulation of NLRC3 suppressed cell proliferation (P < 0.05) and oxidative stress (P < 0.001) mediated by Ang II. Furthermore, NLRC3 negatively regulated the TRAF6/nuclear factor-kappa B (NF-κB) axis activated by Ang II, which resulted in the repression of hyperproliferation of VSMCs (P < 0.01) and oxidative stress (P < 0.001). Conclusion: Nur77 suppressed growth and oxidative stress induced by Ang II in VSMCs by promoting NLRC3 transcription, which, further, repressed the TRAF6/NF-κB axis. This understanding provides novel insights into the pathogenesis of hypertension.

Regulatory Effects of Copper on Ghrelin Secretion in Rat Fundic Glands.

Copper (Cu) is an effective additive in feed for promoting growth. Growth dan axis comprising growth hormone (GH), somatostatin (SS) and GH-releasing hormone (GHRH), with ghrelin regulating their release. The growth-promoting effects of Cu are closely related to ghrelin, but the specific mechanism behind the relationship remains unknown. We investigated the adjustment of ghrelin synthesis and secretion by Cu. Sprague-Dawley rats were fed basal diets with an addition of 0, 120 or 240 mg/kg Cu sulfate for 28 day to establish a growth-promoting model. Signalling molecules relevant to ghrelin synthesis and secretion were detected and mechanistically explored using enzyme-linked immunosorbent assay, quantitative reverse-transcription polymerase chain reaction and Western blot analysis. The 120 mg/kg supplement improved growth performance; significantly increased the serum levels of ghrelin, ghrelin O-acyltransferase (GOAT), acylated ghrelin (AG), GH, and reactive oxygen species (ROS) and decreased those of SS; significantly increased the mRNA and protein expression of ghrelin, GOAT, ghrelin receptor (GHS-R1α), and activator protein 1 (AP-1); increased the phosphorylation ratio of JNK and p38 MAPK; and inhibited the mRNA and protein expression of SS and SS receptor subtype 2 (SSTR2) in gastric fundic gland tissues. Thus, Cu may affect gastric ghrelin synthesis at the transcriptional level by activating the JNK/p38 MAPK pathway through increased ROS levels and regulating the activation of the downstream redox-sensitive transcription factor AP-1. SS plays a crucial determinant role in ghrelin regulation via intragastric Cu. Cu promotes GOAT activity and ghrelin secretion by inhibiting SS secretion, affecting AG levels, and promoting ghrelin acylation through ghrelin/GOAT/GHS-R1α system, modulating ghrelin secretion.

Effect of zinc or copper supplementation on the efficacy and sustainability of botulinum toxin A "Botox" injection in masseter muscle of albino rats.

This study aimed to evaluate whether oral zinc or copper supplementation affected the efficacy and sustainability of botulinum toxin-A (BTX-A) injection in masseter muscle of albino rats. 32 adult male albino rats were allocated equally into four groups: group I (control), group II received 10U BTX-A injection, group III received 10U BTX-A injection + zinc (1 mg, 4 days pre-injection), and group IV received 10U BTX-A injection + copper (0.04 mg, 7 days post-injection). Rats were euthanized at 2 and 12 weeks (4 rats per subgroup) after injection. The masseter muscle was examined via histological, histochemical, histomorphometrical and real-time polymerase chain reaction (qRT-PCR) analyses. The histopathological results of the BTX-A group showed atrophied muscle fibers with increased atrophy with time compared to the control group. The BTX-A + zinc group displayed more atrophy compared to BTX-A group. Conversely, the BTX-A + copper group demonstrated improved histology of muscle fibers compared to BTX-A and BTX-A + zinc groups. Histomorphometric analysis of Masson trichrome staining at 2 and 12 weeks revealed that collagen area percentage was the highest in the BTX-A + copper and control groups, followed by BTX-A and BTX-A + zinc groups. At 12 weeks, the nuclear factor kappa beta (NF-κB) mRNA expression was significantly higher in BTX-A + zinc and BTX-A groups compared to BTX-A + copper group and relative to the control group. Zinc supplementation significantly improved the effectiveness and durability of BTX-A, whereas copper supplementation reduced its efficacy.

The LINC01094/miR-545-3p/SLC7A11 Signaling Axis Promotes the Development of Gastric Cancer by Regulating Cell Growth and Ferroptosis.

This study aimed to investigate the role and mechanism of action of LINC01094 in the development of gastric cancer (GC). The expression levels of LINC01094 in GC patients and healthy individuals were analyzed online using the Cancer Genome Atlas database. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to determine the expression of LINC01094/miR-545-3p/SLC7A11 in GC tissues and cells. Functional experiments (MTT assay, colony formation assay, and flow cytometry) were conducted to assess the effect of LINC01094 and miR-545-3p on cell proliferation, viability, apoptosis, cell cycle, and reactive oxygen species. Correlations between LINC01094 and miR-545-3p, as well as SLC7A11, were analyzed and validated using the dual-luciferase reporter assay and RNA immunoprecipitation. The levels of Fe2+, malondialdehyde, and glutathione in the cells were measured biochemically, and the protein expression levels of Bcl-2, cleaved caspase3, Cyclin D1, and p21 were detected by Western blotting. LINC01094 was significantly upregulated in the GC tissues and cells with a targeting relationship with miR-545-3p; the expression levels of LINC01094 and miR-545-3p were negatively correlated. Knockdown of LINC01094 notably inhibited the proliferation and viability of GC cells and promoted cell ferroptosis, which, however, was abrogated by the silencing of miR-545-3p. These findings indicate that miR-545-3p could target and positively correlate with SLC7A11 expression. Additionally, LINC01094 could promote GC cell progression and affect cellular ferroptosis by regulating the miR-545-3p/SLC7A11 signaling axis.

The role of the KEAP1-NRF2 signaling pathway in form deprivation myopia guinea pigs.

In recent years, the global prevalence of myopia has reached an unprecedented level, especially‌ in East Asia. Multitude of studies has shown that the etiology of myopia is complex. Some researchers have suggested that oxidative stress (OS) may contribute to myopia, although there are limited reports on the alterations of related signaling pathways. Notably, the Kelch-like ECH-associated protein 1 (KEAP1) -nuclear factor erythroid 2-related factor 2 (NRF2), which plays a significant role in regulating OS and the mechanism, has not been explored in myopia. To investigate the modulation of KEAP1-NRF2 signaling pathway and its downstream superoxide dismutase (SOD) during the development of form-deprivation myopia, three-week-old guinea pigs were randomly assigned to four groups: negative control (NC), self-control (SC), form-deprivation myopia (FDM), and FDM group treated with tert-butylhydroquinone (TBHQ). Spherical equivalent (SE) and axial length (AL) were measured by retinoscopy and A-scan ultrasound, respectively. The results revealed that TBHQ treatment decelerated the progression in SE and AL changes. Immunohistochemistry (IHC) assessed the distribution and expression of KEAP1, NRF2, and SOD. The results shown that they located in the retinal ganglion cells (RGC). Subsequently, retinal mRNA and protein expression levels of KEAP1, NRF2, and SOD were quantified using real-time polymerase chain reaction (RT-PCR) and Western blot (WB) analysis. The RT-PCR and WB results demonstrated that TBHQ could activate NRF2, induce KEAP1 degradation, and enhance the expression of the antioxidant SOD. In summary, the modulation of KEAP1-NRF2 and it downstream SOD expression could alter the retinal antioxidant capacity and influence the development of myopia.

Abstract 4125528: Interleukin-6-Modulated Inflammation Augments Deep Vein Thrombosis Organization in Mice

Background: A disorganized thrombus jeopardizes its stability, leading to an augmented risk of fatal acute complications following deep vein thrombosis (DVT). One of its plausible causes is impaired immune cell responses. While the importance of the cytokine interleukin-6 (IL-6) in immune responses is well established, its action towards DVT organization is poorly elucidated. Accordingly, we aimed to investigate the role of IL-6 in the acute immunoinflammatory orchestration of DVT organization in vivo. Methods and Results: We performed total ligation of the infrarenal inferior vena cava (IVC) in 8–12-week-old male C57BL/6J mice a day after starting continuous, subcutaneous mini-osmotic pump infusion of recombinant IL-6 (rIL-6; 1.2 µg/day, n = 21) or vehicle (n = 21), after which DVT pathology was examined. Exogenous IL-6 administration significantly decreased DVT mass at day 2 (16.6±1.9 vs. 23.0±0.9 mg/cm; p<0.01) and day 4 (16.2±1.6 vs. 21.4±0.8 mg/cm; p<0.05) compared to vehicle-treated mice. Intriguingly, rIL-6-treated mice’s DVT exhibited distal tropism of a macroscopically white lesion with abundant neutrophils and platelet accumulation, indicating enhanced DVT organization (Figures A–B). Quantitative polymerase chain reaction (qPCR) analysis revealed the distal edge upregulation of leukocyte chemoattractants, leukocyte-platelet interaction markers, and extracellular matrix regulator genes, confirming that the IL-6-driven inflammatory response was linked to augmented DVT organization. Spatiotemporally, real-time intravital imaging on stasis- and irradiation-induced saphenous vein thrombosis among intraperitoneal rIL-6 (0.3 µg/mouse)-treated mice demonstrated marked acute leukocyte and platelet recruitment with subsequent aggregation to the distal edge of the thrombus (Figures C–D), which consequently yielded an enhanced thrombus resolution at the distal edge. Conclusions: Our findings suggest that IL-6-driven inflammation results in distal tropism of leukocyte-platelet infiltration, which is important for modulating thrombus organization and subsequent DVT amelioration. This study presents a novel insight into the immunothrombosis process, with potential clinical implications for DVT.

M6A-modified circCacna1c regulates necroptosis and ischemic myocardial injury by inhibiting Hnrnpf entry into the nucleus.

Circular RNAs (circRNAs) are differentially expressed in various cardiovascular diseases, including myocardial infarction (MI) injury. However, their functional role in necroptosis-induced loss of cardiomyocytes remains unclear. We identified a cardiac necroptosis-associated circRNA transcribed from the Cacna1c gene (circCacna1c) to investigate the involvement of circRNAs in cardiomyocyte necroptosis. To investigate the role of circCacna1c during oxidative stress, H9c2 cells and neonatal rat cardiomyocytes were treated with hydrogen peroxide (H2O2) to induce reactive oxygen species (ROS)-induced cardiomyocyte death. The N6-methyladenosine (m6A) modification level of circCacna1c was determined by methylated RNA immunoprecipitation quantitative polymerase chain reaction (MeRIP-qPCR) analysis. Additionally, an RNA pull-down assay was performed to identify interacting proteins of circCacna1c in cardiomyocytes, and the regulatory role of circCacna1c in target protein expression was tested using a western blotting assay. Furthermore, theMI mouse model was constructed to analyze the effect of circCacna1c on heart function and cardiomyocyte necroptosis. The expression of circCacna1c was found to be reduced in cardiomyocytes exposed to oxidative stress and in mouse hearts injured by MI. Overexpression of circCacna1c inhibited necroptosis of cardiomyocytes induced by hydrogen peroxide and MI injury, resulting in a significant reduction in myocardial infarction size and improved cardiac function. Mechanistically, circCacna1c directly interacts with heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in the cytoplasm, preventing its nuclear translocation and leading to reduced Hnrnpf levels within the nucleus. This subsequently suppresses Hnrnpf-dependent receptor-interacting protein kinase 1 (RIPK1) expression. Furthermore, fat mass and obesity-associated protein (FTO) mediates demethylation of m6A modification on circCacna1c during necrosis and facilitates degradation of circCacna1c. Our study demonstrates that circCacna1c can improve cardiac function following MI-induced heart injury by inhibiting the Hnrnpf/RIPK1-mediated cardiomyocyte necroptosis. Therefore, the FTO/circCacna1c/Hnrnpf/RIPK1 axis holds great potential as an effective target for attenuating cardiac injury caused by necroptosis in ischemic heart disease.

Effect of gedunin on cell proliferation and apoptosis in skin melanoma cells A431 via the PI3K/JNK signaling pathway.

Melanoma is an aggressive skin malignancy with rapid metastasis and high morbidity. Gedunin (GN) is a tetranortriterpenoid belonging to the Meliaceae family, described for its anticancer, antiproliferative and apoptotic properties. In the present study, we investigated the effect of GN on A431 melanoma cell proliferation and apoptosis. The inflammatory proteins (tumor necrosis factor alpha (TNF-α), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), cycloxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), and interleukin 6 (IL-6)) apoptosis-related proteins, such as Bax, Bcl-2 and caspase-3, and alterations in the PI3K/JNK and p38 pathways in A431 cells after GN treatment were examined. The cytotoxicity assay and cell apoptosis of GN activity on A431 cells were assessed using MTT assay, acridine orange/ethidium bromide (AO/EB), DAPI (4',6-diamidino-2-phenylindole), propidium iodide (PI), enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR) and western blot analyses. The findings demonstrated that GN (10 and 15 μM/mL) inhibits the growth of melanoma cells, triggers apoptosis by enhancing Bax and caspase, and reduces Bcl-2, cyclin-D1, c-myc, and survivin in a concentration-reliant manner. Additionally, GN attenuated the protein expression of inflammatory proteins (TNF-α, NF-κB, COX-2, iNOS, and IL-6) and the cell proliferative PI3K/JNK/p38 signaling pathway. Due to the imbalance in the Bax/Bcl-2 ratio, apoptosis is promoted, and the caspase cascade and Cyt-c are activated. This led us to conclude that GN treatment inhibited Bcl-2, cyclin-D1, c-myc, and survivin activity through the TNF-α/NF-κB and PI3K/JNK/p38 signaling pathways, further preventing the proliferation and stimulation of apoptosis, which contributes to growth arrest in melanoma cells. Gedunin has been shown to promote melanoma cell death in vitro, suggesting that it could be used as a future treatment for malignant melanoma. Our findings suggested that GN might be applied as a preventative measure in the management of skin melanoma cells.

PINK1-deficiency facilitates mitochondrial iron accumulation and colon tumorigenesis

ABSTRACT Mitophagy, the process by which cells eliminate damaged mitochondria, is mediated by PINK1 (PTEN induced kinase 1). Our recent research indicates that PINK1 functions as a tumor suppressor in colorectal cancer by regulating cellular metabolism. Interestingly, PINK1 ablation activated the NLRP3 (NLR family pyrin domain containing 3) inflammasome, releasing IL1B (interleukin 1 beta). However, inhibiting the NLRP3-IL1B signaling pathway with an IL1R (interleukin 1 receptor) antagonist or NLRP3 inhibitor did not hinder colon tumor growth after PINK1 loss. To identify druggable targets in PINK1-deficient tumors, ribonucleic acid sequencing analysis was performed on colon tumors from pink1 knockout and wild-type mice. Gene Set Enrichment Analysis highlighted the enrichment of iron ion transmembrane transporter activity. Subsequent qualitative polymerase chain reaction and western blot analysis revealed an increase in mitochondrial iron transporters, including mitochondrial calcium uniporter, in PINK1-deficient colon tumor cells and tissues. Live-cell iron staining demonstrated elevated cellular and mitochondrial iron levels in PINK1-deficient cells. Clinically used drugs deferiprone and minocycline reduced mitochondrial iron and superoxide levels, resulting in decreased colon tumor cell growth in vitro and in vivo. Manipulating the mitochondrial iron uptake protein MCU (mitochondrial calcium uniporter) also affected cell and xenograft tumor growth. This study suggests that therapies aimed at reducing mitochondrial iron levels may effectively inhibit colon tumor growth, particularly in patients with low PINK1 expression.

At the mercy of the AI algorithm - discrepant PCR results.

As pathogen molecular diagnostics technology develops, the software analysis of polymerase chain reaction (PCR) sigmoidal curves, crossing threshold (Ct) values and the limit of nucleic acid detection for that assay, is not always available when reporting test results. These are critical components in interpreting results and can affect the management of patients. Our view is that all commercial assay providers must ensure users, as they can with laboratory developed tests, are able to see the curve and Ct value for all samples. Some examples demonstrating this principle are included. Regulatory bodies have a key role to play.

The Effects of Model Insoluble Copper Compounds in a Sedimentary Environment on Denitrifying Anaerobic Methane Oxidation (DAMO) Enrichment

The contribution of denitrifying anaerobic methane oxidation (DAMO) as a methane sink across different habitats, especially those affected by anthropogenic activities, remains unclear. Mining and industrial and domestic use of metals/metal-containing compounds can all cause metal contamination in freshwater ecosystems. Precipitation of metal ions often limits their toxicity to local microorganisms, yet microbial activity may also cause the redissolution of various precipitates. In contrast to most other studies that apply soluble metal compounds, this study investigated the responses of enriched DAMO culture to model insoluble copper compounds, malachite and covellite, in simulated sedimentary environments. Copper ≤ 0.22 µm from covellite appeared to cause immediate inhibition in 10 h. Long-term tests (54 days) showed that apparent methane consumption was less impacted by various levels of malachite and covellite than soluble copper. However, the medium-/high-level malachite and covellite caused a 46.6–77.4% decline in denitrification and also induced significant death of the representative DAMO microorganisms. Some enriched species, such as Methylobacter tundripaludum, may have conducted DAMO or they may have oxidized methane aerobically using oxygen released by DAMO bacteria. Quantitative polymerase chain reaction analysis suggests that Candidatus Methanoperedens spp. were less affected by covellite as compared to malachite while Candidatus Methylomirabilis spp. responded similarly to the two compounds. Under the stress induced by copper, DAMO archaea, Planctomycetes spp. or Phenylobacterium spp. synthesized PHA/PHB-like compounds, rendering incomplete methane oxidation. Overall, the findings suggest that while DAMO activity may persist in ecosystems previously exposed to copper pollution, long-term methane abatement capability may be impaired due to a shift of the microbial community or the inhibition of representative DAMO microorganisms.

Expression Analysis of the Extensive Regulation of Mitogen-Activated Protein Kinase (MAPK) Family Genes in Buckwheat (Fagopyrum tataricum) During Organ Differentiation and Stress Response

The mitogen-activated protein kinase (MAPK) signaling cascade is a unique and relatively conserved signaling pathway in eukaryotes, transmitting extracellular signals into cells through successive phosphorylation and eliciting appropriate responses from the organism. While its mechanism in plant immune response has been partially elucidated in Arabidopsis, it has been rarely examined in Tartary buckwheat (Fagopyrum tataricum). Based on the conserved MAPK domain, we identified 16 MAPK family genes in Tartary buckwheat. The FtMAPKs have similar structures and motif compositions, indicating that this gene family is conserved yet functionally diverse. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, we observed significant expression variation in 15 genes across different organs, except for FtMAPK12. FtMAPK9 showed specific expression in vegetative organs, FtMAPK4 in reproductive organs, and FtMAPK1 and FtMAPK10 in leaves and flowers, respectively, indicating their regulatory roles in Tartary buckwheat development. Following drought and salt stress treatments, 12 and 14 FtMAPKs, respectively, showed significantly altered expression in leaves exhibiting notable biological oxidation. Among these, FtMAPK3, FtMAPK4, and FtMAPK8 demonstrated highly significant changes across both treatments. Transcriptome analysis confirmed these findings, suggesting that these three genes play pivotal roles in Tartary buckwheat’s response to abiotic stress and hold potential for molecular breeding improvements.

Endophytic Bacillus velezensis GsB01 controls Gleditsia sinensis wilt by secreting antifungal metabolites and modulates symbiotic microbiota within trees.

Identifying effective biological control agents against fungal pathogens and determining their mechanisms of action are important in the control of plant diseases. In this study, we isolated an endophytic bacterial strain, GsB01, from the branches of asymptomatic Gleditsia sinensis. Multi-locus sequence analysis identified the strain as Bacillus velezensis. GsB01 exhibited significant antifungal activity against Thyronectria austroamericana, the causative agent of G. sinensis wilt. Liquid chromatography-mass spectrometry identified four consistently present antimicrobial compounds in GsB01 metabolite fractions with high antifungal activity: macrolactin A, bacillaene A, surfactin, and iturin. GsB01's active metabolite fractions altered the metabolic profiles of T. austroamericana, disrupting seven pathways, including arginine biosynthesis, nucleotide metabolism, purine metabolism, and the pentose phosphate pathway. Furthermore, absolute quantitative polymerase chain reaction analysis suggested that GsB01 may increase the abundance of endophytic bacteria in G. sinensis. The 16S rRNA amplicon sequencing revealed changes in the endophytic landscape in stems and roots following GsB01 introduction, particularly with significant variation in the dominant bacterial genera within the stems. The study highlights GsB01's potential against plant wilt and suggests that its antifungal activity is achieved by secreting antifungal metabolites. The study also recorded changes in the symbiotic microbiota within trees that had been infected with a pathogenic fungus and subsequently treated with an endophytic antagonistic bacterial strain. © 2024 Society of Chemical Industry.

METTL3 alters AK3 RNA expression in an m6A-dependent manner to affect the proliferation and metastasis of hepatocellular carcinoma

In recent years, the significance of N6-methyladenosine (m6A) modification in the process of tumorigenesis has garnered substantial attention from researchers in the field. Among the myriad of factors involved, METTL3, which functions as an m6A methyltransferase, has emerged as a critical player in this context. This enzyme has been shown to participate actively in the regulation of RNA stability and expression across a diverse array of tumors. To achieve this, we employed quantitative polymerase chain reaction (qPCR) and Western blot analysis techniques to measure the expression levels of both METTL3 and AK3 in various hepatocellular carcinoma cell lines. We utilized small interfering RNA (siRNA) technology to inhibit the expression of METTL3, subsequently observing the resulting changes in AK3 expression. We analyzed the levels of m6A modification using the MeRIP-Seq method to obtain a comprehensive understanding of the underlying molecular interactions. Through MeRIP-Seq analysis, we discovered that METTL3 directly modulates the stability of AK3, thereby promoting its expression via m6A modification. This finding is pivotal as it underscores the regulatory role that METTL3 plays in AK3 RNA expression, facilitated through the M6A modification pathway.

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.

Perspective: Transcriptomics of Cryopreserved Cells

Cryopreservation is a well-known strategy to conserve genetic resources at ultra-low temperature. However, there is still limited knowledge on the cellular processes and molecular adjustments that allow cells to withstand the multiple stresses to which they are exposed during cryopreservation. To evaluate these processes, transcriptomics, the sub-discipline of omics that simultaneously examines mRNA transcripts formed by transcription from the genome, has been recently used. This article reviews recent scientific studies which use the basic principles of cryopreservation practices together with transcriptomics approaches, within the conceptual framework of cryobiomics. Moreover, the connections between factors that may be useful to optimize and validate approaches for mammalian or plant cell cryopreservation are also assessed. Transcriptomic applications are mainly performed with methods such as reverse transcriptase polymerase chain reaction (RT-PCR), simultaneous polymerase chain reaction (real-time PCR), northern blot, microarray/biochip and gene expression analysis (SAGE). Transcriptomic technologies allow a global view of gene expression profiles of different mammalian or plant cell types to be obtained before and after cryopreservation under multiple stress conditions. For these processes, small amounts of RNA enable efficient transcriptomics analysis. Transcriptomic analysis of cryopreserved mammalian and plant cells provides a conceptual way to identify the genes and their relative alterations in transcriptional abundances together with non-coding RNAs involved in important pathways related to cell viability and proliferation during and after cryopreservation. Moreover, it greatly contributes to understanding of non-fatal cryodamage and related developmental disorders in cryopreserved mammalian oocytes and sperm. In addition, single cell transcriptomics has the potential to non-invasely monitor immune actions and to diagnose the stage of the inflammatory process in kidney. Finally, qRT-PCR and RNA-seq studies have also revealed that some transcription factors are effective at inducing cold tolerance in many plants by elevating the levels of soluble sugars, proline and unsaturated fatty acids in cells. Hence transcriptomics studies may also aid investigations of the main mechanisms behind the so-called 'cryo-recalcitrance' that is observed mostly in plant cells.

Diosgenin attenuates nonalcoholic fatty liver disease through mTOR-mediated inhibition of lipid accumulation and inflammation

Excessive hepatic lipid accumulation and inflammatory injury are significant pathological manifestations of nonalcoholic fatty liver disease (NAFLD). Our previous research discovered that diosgenin, a natural steroidal saponin derived from Chinese herbs, can reduce hepatic lipid accumulation and steatosis; however, the exact mechanism remains unclear. This study aimed to investigate the protective mechanisms of diosgenin against NAFLD. We utilized network pharmacology and molecular docking approaches to identify the pathways through which diosgenin improves NAFLD. In high-fat diet (HFD)-fed rats, we measured biochemical markers in the serum and liver. Liver histopathology was assessed using HE and oil-red O staining. In free fatty acids (FFAs)-induced HepG2 cells, we employed the cell transfection overexpression method to verify the regulatory relationship of the identified pathways. The mechanisms in vitro and in vivo were examined using quantitative polymerase chain reaction and western blot analyses. Bioinformatics analysis indicated that the mTOR-FASN/HIF-1α/RELA/VEGFA pathway may be the target pathway for diosgenin in alleviating NAFLD. Diosgenin inhibited hepatic lipid accumulation and pro-inflammatory cytokines in HFD-fed rats, and reduced intracellular lipid accumulation as well as TG, TC, IL-1β, and TNF-α levels in FFAs-induced HepG2 cells. Mechanistically, diosgenin downregulated the expression of p-mTOR, FASN, HIF-1α, RELA, and VEGFA, which are associated with lipid synthesis and inflammation. Overexpression of mTOR abolished the beneficial effects of diosgenin on lipid reduction and inflammation, as well as its inhibitory effects on the expression of FASN, HIF-1α, RELA, and VEGFA. In conclusion, diosgenin alleviates NAFLD through mTOR-mediated inhibition of lipid accumulation and inflammation.

Investigation of Carbapenemase-Producing Pseudomonas aeruginosa at Secondary Care Hospital in Bolu, Turkey.

The global increase in carbapenem resistance poses a significant public health threat due to the potential emergence of multidrug-resistant pathogens and limited treatment options. To learn more about this issue and offer potential solutions, we conducted a study of carbapenem-resistant Pseudomonas aeruginosa (CRPA) infections in a secondary care hospital setting. The study utilized the carbapenem inactivation method (CIM), a leading phenotypic analysis, to determine carbapenemase activity in 63 CRPA isolates. Additionally, polymerase chain reaction (PCR) analysis was conducted to test for the presence of carbapenemase genes associated with the production or expression of various carbapenemase enzymes, including blaKPC, blaNDM, blaVIM, blaOXA-48, blaIMP, and blaGES. Arbitrary primed PCR (AP-PCR) was performed to assess the clonal relationship between different isolates. The isolates were also classified as either health care-associated infections or community-acquired infections, and their clonal relationship and gene positivity were evaluated. A total of 63 CRPA samples underwent evaluation, with 14 isolates determined to be carbapenemase producers via CIM tests. PCR assays revealed that 14 isolates carried carbapenemase genes, with 9 carrying blaNDM, 2 carrying blaGES, 2 carrying blaVIM, and 1 carrying blaIMP. CRPA exhibited a 22% prevalence of carbapenemase genes, of which 64% were attributed to the NDM gene responsible for multidrug resistance. AP-PCR revealed high clonal diversity among the isolates. Molecular epidemiological evaluation also showed no dominant outbreak strain among PA isolates. This study presents significant data on the prevalence and distribution of carbapenemase-producing CRPA strains isolated from secondary health care facilities. Typically, the literature focuses on resistance rates in tertiary care public hospitals. These findings may aid in understanding resistance and its mechanisms, as well as in developing effective treatment strategies and infection control measures.