Search Results (59)

Background/Objectives: Protein kinases play crucial roles in signal transduction pathways that regulate growth and differentiation in Trypanosoma cruzi. These protein kinases are attractive targets to develop new drugs to treat Chagas disease. Methods: We used several protein kinase inhibitors targeting the p38 MAPK, MEK, and ERK pathways to evaluate their effects on the in vivo phosphorylation status of T. cruzi proteins, particularly DNA polymerase beta (TcPolβ). We also used Genistein, a protein tyrosine kinase inhibitor, to assess its effects on global protein phosphorylation and TcPolβ phosphorylation. Also, we investigated the effect of oxidative stress on global tyrosine phosphorylation. Finally, we determined the phosphorylation sites on TcPolβ by the protein kinases TcPKC2 and TcWee570 in vitro. Results: p38 MAPK and MEK protein kinase inhibitors inhibited approximately 50% of the Ser/Thr phosphorylation of TcPolβ. Genistein inhibited both Ser/Thr and Tyr phosphorylation of several polypeptides in epimastigotes. Oxidative stress increases global Tyr phosphorylation by about twofold and also TcPolβ phosphorylation. TcPKC2 and TcWee570 were able to phosphorylate TcPolβ at both Ser/Thr and Tyr residues. Conclusions: Small-molecule protein kinase inhibitors can affect the phosphorylation status of TcPolβ in vivo. Since Genistein can inhibit both Ser/Thr and Tyr protein phosphorylation, and TcPKC2 and TcWee570 can phosphorylate both Ser/Thr and Tyr residues, it suggests the existence of dual protein kinases in T. cruzi. However, this possibility must be further studied. Full article

DNA polymerase beta (Polβ) is a 39 kDa, single polypeptide enzyme that possesses both gap tailoring and nucleotidyl transferase activity and is the key polymerase involved in base excision repair (BER) and the final steps of active gene demethylation. We demonstrated that residues in the mouse Polβ protein, L301 and V303, are critical for Polβ’s interaction with the BER scaffolding protein X-ray repair cross-complementing 1 (XRCC1), and mutation of these residues impairs Polβ’s ability to bind to XRCC1, negatively impacting BER complex assembly. We developed Polβ^{L301R-V303R/L301R-V303R} knock-in mice to explore how defects with this essential protein complex impact genome stability in the mouse. We found these mice to be viable and fertile yet exhibited a modest reduction in body weight. Here, we examined the protein and mRNA levels in tissues from wild-type (WT), heterozygous (HET), and homozygous (HOM) Polβ^{L301R-V303R/L301R-V303R} mice and the derived fibroblast cell lines. We show that HOM mice have significantly diminished Polβ protein levels, as compared to WT mice, in several tissues, yet Polβ mRNA levels were not significantly different, suggesting the decreased levels of Polβ protein could not be attributed to lower gene expression. Upon examination of Polβ stability in mouse ear fibroblasts derived from WT and HOM mice, results are consistent with human cell studies that the Polβ^L301R-V303R protein is unstable and undergoes proteasome-mediated degradation. Finally, we evaluated WT, and HOM, liver and brain genomic DNA samples for 5-methylcytosine/5-hydroxymethylcytosine (5mC/5hmC) levels by nanopore sequencing to investigate the impact of suppressed Polβ protein levels on active gene demethylation. As expected, we found tissue-specific trends in methylation, when comparing the brain and liver. However, we were unable to discern substantial differences in methylation levels between WT and HOM mice, suggesting that in the absence of external stressors, low Polβ levels do not impact methylation patterns. Full article

The fruiting stage of soybean (Glycine max L.) is critical for determining both its yield and quality, thereby influencing global production. While some studies have provided partial explanations for the occurrence of Fusarium species on soybean seeds and pods, the fungal diversity affecting soybean pods in Sichuan Province, a major soybean cultivation region in Southwestern China, remains inadequately understood. In this study, 182 infected pods were collected from a maize–soybean relay strip intercropping system. A total of 10 distinct pod-infecting fungal genera (132 isolates) were identified, and their pathogenic potential on soybean seeds and pods was evaluated. Using morphological characteristics and DNA barcode markers, we identified 43 Fusarium isolates belonging to 8 species, including F. verticillioides, F. incarnatum, F. equiseti, F. proliferatum, F. fujikuroi, F. oxysporum, F. chlamydosporum, and F. acutatum through the analysis of the translation elongation factor gene (EF1-α) and RNA polymerases II second largest subunit (RPB2) gene. Multi-locus phylogenetic analysis, incorporating the Internal Transcribed Spacer (rDNA ITS), β-tubulin (β-tubulin), Glyceraldehyde 3-phosphate dehydrogenase (GADPH), Chitin Synthase 1 (CHS-1), Actin (ACT), Beta-tubulin II (TUB2), and Calmodulin (CAL) genes distinguished 37 isolates as 6 Colletotrichum species, including C. truncatum, C. karstii, C. cliviicola, C. plurivorum, C. boninense, and C. fructicola. Among these, F. proliferatum and C. fructicola were the most dominant species, representing 20.93% and 21.62% of the isolation frequency, respectively. Pathogenicity assays revealed significant damage from both Fusarium and Colletotrichum isolates on soybean pods and seeds, with varying isolation frequencies. Of these, F. proliferatum, F. acutatum, and F. verticillioides caused the most severe symptoms. Similarly, within Colletotrichum genus, C. fructicola was the most pathogenic, followed by C. truncatum, C. karstii, C. cliviicola, C. plurivorum, and C. boninense. Notably, F. acutatum, C. cliviicola, C. boninense, and C. fructicola were identified for the first time as pathogens of soybean pods under the maize–soybean strip intercropping system in Southwestern China. These findings highlight emerging virulent pathogens responsible for soybean pod decay and provide a valuable foundation for understanding the pathogen population during the later growth stages of soybean. Full article

Chlorpyrifos (CPF), an organophosphate pesticide, is known to induce pulmonary toxicity through oxidative stress, mitochondrial dysfunction, and inflammation. Astaxanthin (ASX), a xanthophyll carotenoid derived primarily from marine microalgae (Haematococcus pluvialis), possesses strong antioxidant properties and has demonstrated cellular protective effects in numerous oxidative stress studies. However, its efficacy against CPF-induced lung cell damage remains uncharacterized. This study revealed the protective role of ASX, as a pretreatment and co-treatment, against CPF-induced cytotoxicity in human A549 lung adenocarcinoma cells by assessing cell viability, intracellular reactive oxygen species (IROS), total oxidative status (TOS), total antioxidant capacity (TAC), mitochondrial membrane potential (MMP), intracellular calcium ions (Ca²⁺), lactate dehydrogenase (LDH) release, malondialdehyde (MDA) levels, glutathione peroxidase (GPx) activity, superoxide dismutase (SOD) activity, DNA fragmentation, and apoptosis/inflammation-associated gene expression. CPF treatment significantly decreased cell viability and TAC, while elevating IROS, TOS, MMP, intracellular Ca²⁺, and LDH release. CPF also increased MDA levels and suppressed GPx and SOD activities. DNA fragmentation and quantitative polymerase chain reaction (qPCR) analysis revealed upregulation of pro-apoptotic and inflammatory markers such as BCL2-associated X protein (BAX), caspase-3 (CASP3), tumor protein p53 (TP53), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), nuclear factor kappa B (NFκB), and voltage-dependent anion-selective channel protein 1 (VDAC1) and suppression of anti-apoptotic B-cell lymphoma 2 (BCL2) and antioxidant defense genes nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). ASX treatment, particularly when administered as a pretreatment, significantly reversed CPF-induced oxidative and inflammatory responses by restoring SOD, GPx, and TAC levels, reducing IROS, TOS, MDA, and LDH release, and downregulating apoptotic and inflammatory gene expressions. ASX pretreatment notably decreased MMP and intracellular Ca²⁺ levels, indicating protection against mitochondrial dysfunction and calcium dysregulation. ASX upregulated Nrf2 and HO-1 expression and restored the BCL2/BAX balance, suggesting inhibition of mitochondrial-mediated apoptosis. Additionally, ASX significantly attenuated CPF-induced anti-angiogenic effects in the in ovo Hen’s Egg Test Chorioallantoic Membrane (HET-CAM) assay. These findings demonstrate, for the first time, that ASX exerts a broad spectrum of protective effects against CPF-induced cytotoxicity in lung cells, mainly through the stabilization of mitochondrial redox status and modulation of apoptosis- and inflammation-related gene pathways, highlighting ASX as a promising candidate for further therapeutic development. Furthermore, the pronounced efficacy observed in the pretreatment regimen suggests that ASX can be evaluated as a potential nutritional preventive strategy in high-risk populations with occupational or environmental CPF exposure. Full article

All beta- and gamma-herpesviruses utilize a set of six viral proteins to facilitate transcription from specific promoters that become active late in the viral life cycle. These proteins form a complex that interacts with a TA-rich sequence upstream of the late transcription start sites and recruits RNA polymerase II (Pol II). The structure of any of the late transcription factors (LTFs) alone or in complexes has not been solved by standard means yet, but a fair amount is known about how the proteins interact and where the complex is positioned over the late promoters. Here, AlphaFold3 was used to predict and analyze the LTF complex using proteins from the beta-herpesviruses HCMV, MCMV, HHV6, and HHV7, and from the gamma-herpesviruses EBV and KSHV. The predicted structures had high levels of confidence and were remarkably similar even though there is little sequence conservation in the LTFs across the viruses. The results are consistent with most of the previously determined information concerning the interaction of the factors with each other and with DNA. A conserved threonine phosphorylation in one of the subunits that is critical to the function of the LTFs is predicted to be at the junction of five subunits. AlphaFold 3 predicts seven metal ion binding sites in each of the four beta-herpesviruses and either five or six in the gamma-herpesviruses created by conserved residues in three of the subunits. The structures also provide insights into the function of the subunits and which host general transcription factors (GTFs) may or may not be utilized during initiation. Full article

Diabetic kidney disease (DKD) displays a high prevalence of cardiovascular and cerebrovascular disease. Both the kidney and the brain share common pathogenic mechanisms, such as inflammation, endothelial dysfunction, oxidative stress, and mitochondrial dysfunction. The aim of this study was to establish a potential association of cerebral vessel remodeling and its related functional impairment with biomarkers of inflammation, oxidative stress, and mitochondrial dysfunction in the early stages of DKD in type 2 diabetes mellitus (DM) patients. A cohort of 184 patients and 39 healthy controls was assessed concerning serum and urinary stromal cell-derived factor-1 (SDF-1), P-selectin, advanced oxidation protein products (AOPPs), urinary synaptopodin, podocalyxin, kidney injury molecule-1 (KIM-1), and N-acetyl-β-(D)-glucosaminidase (NAG). The quantification of the mitochondrial DNA copy number (mtDNA-CN) and nuclear DNA (nDNA) in urine and peripheral blood was conducted using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Using TaqMan tests, the beta-2 microglobulin nuclear gene (B2M) and the cytochrome b (CYTB) gene, which encodes subunit 2 of NADH dehydrogenase (ND2), were evaluated. The MtDNA-CN is the ratio of mitochondrial DNA to nuclear DNA copies, ascertained through the examination of the CYTB/B2M and ND2/B2M ratios. The intima-media thickness (IMT) measurements of the common carotid arteries (CCAs), along with the pulsatility index (PI) and resistivity index (RI) of the internal carotid arteries (ICAs) and middle cerebral arteries (MCAs), were obtained through cerebral Doppler ultrasonography (US). Additionally, the breath-holding index (BHI) was also measured by cerebral Doppler US. PI-ICAs, PI-MCAs, CCAs-IMT, RI-MCAs, and RI-ICAs demonstrated direct relationships with SDF-1, P-selectin, AOPPs, urine mtDNA, podocalyxin, synaptopodin, NAG, and KIM-1 while showing indirect correlations with serum mtDNA and the eGFR. In contrast, the BHI had negative correlations with SDF-1, P-selectin, AOPPs, urine mtDNA, synaptopodin, podocalyxin, KIM-1, and NAG while showing direct associations with serum mtDNA and the eGFR. In conclusion, a causative association exists among SDF-1, P-selectin, and AOPPs, as well as mitochondrial dysfunction, in early diabetic kidney disease (DKD) and significant cerebrovascular alterations in patients with type 2 diabetes mellitus and normoalbuminuric DKD, with no neurological symptoms. Full article

The bla_OXA-1 gene encodes an oxacillin-hydrolyzing beta-lactamase of extended-spectrum beta-lactamase (ESBL)-producing microorganisms. The bla_OXA-1 gene is found in the resistomes of some Enterobacteriaceae, Morganellaceae, Pasteurellaceae, Moraxellaceae, Aeromonadaceae, Pseudomonadaceae, Yersiniaceae, and Vibrionaceae. Most ESBL detection methods, including those to detect OXA-1-producing microorganisms, are time-consuming, and require specialized equipment and qualified personnel. Here, we report a new CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas12a-based detection assay coupled with polymerase chain reaction (PCR) to sensitively detect OXA-1-bearing microorganisms. The PCR-coupled CRISPR/Cas12a-based fluorescence assay includes (i) a pre-amplification step and (ii) a nucleic acid detection step. The pre-amplification step is based on a commonly used PCR, and the detection step is based on the CRISPR/Cas12a property to nonspecifically hydrolyze single-stranded DNA fluorescent reporter molecules. The pre-amplification step takes 65 min, and the detection step is shortened and takes only 5 min. The developed assay can easily detect single (1.25) copies of the bla_OXA-1 gene in a reaction and is efficient not only in the detection of a bla_OXA-1 model matrix but also in the detection of bla_OXA-1-positive microorganisms. We hope that our assay has the potential to improve the monitoring of OXA-1-producing microorganisms and therefore contribute to mitigating the deadly global threat of antibiotic-resistant microorganisms. Full article

Background: Variability in recanalization success during endovascular treatment for acute ischemic stroke (AIS) has led to increased interests in thrombus composition and associated cellular materials. While evidence suggests that bacteria may influence thrombus characteristics, limited data exist on microbiological profiles of thrombi in stroke patients. Objectives: Characterization of bacterial communities present in thrombi of AIS patients undergoing mechanical thrombectomy, providing insights into microbial contributions to stroke pathogenesis and treatment outcomes. Methods: Thrombi were collected from 20 AIS patients. After extracting metagenome, 16S rDNA sequencing was performed. Bioinformatic analysis included taxonomy and diversity assessments. The presence of bacterial DNA and viable bacteria in thrombi was validated using polymerase chain reaction (PCR) and bacterial culturing followed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis, respectively. Results: 16S rDNA was amplified in 19/20 thrombi (95%). Analysis identified a diverse microbial community, with Corynebacterium spp. as the most prevalent genus, followed by Staphylococcus spp., Bifidobacterium spp., Methylobacterium spp., and Anaerococcus spp. Alpha diversity analyses (Shannon index: 4.0–6.0 and Simpson index: 0.8–1.0) revealed moderate to high microbial diversity across samples; beta diversity demonstrated distinct clustering, indicating inter-patient variability in microbial profiles. PCR confirmed the presence of DNA specific to dominant bacterial taxa identified through sequencing. Culturing showed the presence of Staphylococcus epidermidis and Enterococcus faecalis in some clots as identified through MALDI analysis. Conclusions: This study shows bacterial communities present in AIS patients’ thrombi, suggesting a potential link between microbial signatures and thrombus characteristics. Full article

Reverse transcription polymerase chain reaction (RT-PCR) is an important tool for the detection of target RNA molecules and the assay of RNA pathogens. Coupled RT-PCR is performed with an enzyme mixture containing a reverse transcriptase and a thermostable DNA polymerase. To date, several biotechnological companies offer artificial thermostable DNA polymerases with a built-in reverse transcriptase activity for use in the coupled RT-PCR instead of the enzyme mixtures. Here, we compared the artificial DNA polymerases and conventional enzyme mixtures for the RT-PCR by performing end-point and real-time RT-PCR assays using severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV2) RNA and endogenous mRNA molecules as templates. We found that the artificial enzymes were suitable for different RT-PCR applications, including SARS-CoV2 RNA detection but not for long-fragment RT-PCR amplification. Full article

Fungal biota represents important constituents of phyllosphere microorganisms. It is taxonomically highly diverse and influences plant physiology, metabolism and health. Members of the order Diaporthales are distributed worldwide and include devastating plant pathogens as well as endophytes and saprophytes. However, many phyllosphere Diaporthales species remain uncharacterized, with studies examining their diversity needed. Here, we report on the identification of several diaporthalean taxa samples collected from diseased leaves of Cinnamomum camphora (Lauraceae), Castanopsis fordii (Fagaceae) and Schima superba (Theaceae) in Fujian province, China. Based on morphological features coupled to multigene phylogenetic analyses of the internal transcribed spacer (ITS) region, the large subunit of nuclear ribosomal RNA (LSU), the partial beta-tubulin (tub2), histone H3 (his3), DNA-directed RNA polymerase II subunit (rpb2), translation elongation factor 1-α (tef1) and calmodulin (cal) genes, three new species of Diaporthales are introduced, namely, Diaporthe wuyishanensis, Gnomoniopsis wuyishanensis and Paratubakia schimae. This study contributes to our understanding on the biodiversity of diaporthalean fungi that are inhabitants of the phyllosphere of trees native to Asia. Full article

The nervous system is susceptible to DNA damage and DNA repair defects, and if DNA damage is not repaired, neuronal cells can die, causing neurodegenerative diseases in humans. The overall picture of what is known about DNA repair mechanisms in the nervous system is still unclear. The current challenge is to use the accumulated knowledge of basic science on DNA repair to improve the treatment of neurodegenerative disorders. In this review, we summarize the current understanding of the function of DNA damage repair, in particular, the base excision repair and double-strand break repair pathways as being the most important in nervous system cells. We summarize recent data on the proteins involved in DNA repair associated with neurodegenerative diseases, with particular emphasis on PARP1 and ND-associated proteins, which are involved in DNA repair and have the ability to undergo liquid–liquid phase separation. Full article

In an extensive fungal investigation conducted in southern China, a large number of fungal strains were isolated by collecting and treating diseased and decayed leaves. Using internal transcribed spacer regions (ITSs) sequence data for a BLAST search to screen for suspected strains of Daldinia, followed by phylogenetic analysis using internal transcribed spacer regions, partial sequences of the large subunit of the rDNA (LSU), RNA polymerase II (rpb2), and beta tubulin (tub2) sequence data, combined with morphological characteristics of anamorphic species, ninety-four strains of Daldinia were identified. Furthermore, their geographical distribution and host specificity of the genus were thoroughly analyzed and summarized. Additionally, seven new anamorphic species of the genus Daldinia were also detected, Daldinia ehretiae sp. nov., D. jianfengensis sp. nov., D. ledongensis sp. nov., D. menghaiensis sp. nov., D. rhododendri sp. nov., D. spatholobi sp. nov., and D. thunbergiae sp. nov. Full article

In mammals, 17-beta hydroxysteroid dehydrogenase 2 (Hsd17b2) enzyme specifically catalyzes the oxidation of the C17 hydroxyl group and efficiently regulates the activities of estrogens and androgens to prevent diseases induced by hormone disorders. However, the functions of the hsd17b2 gene involved in animal sex differentiation are still largely unclear. The ricefield eel (Monopterus albus), a protogynous hermaphroditic fish with a small genome size (2n = 24), is usually used as an ideal model to study the mechanism of sex differentiation in vertebrates. Therefore, in this study, hsd17b2 gene cDNA was cloned and its mRNA expression profiles were determined in the ricefield eel. The cloned cDNA fragment of hsd17b2 was 1230 bp, including an open reading frame of 1107 bp, encoding 368 amino acid residues with conserved catalytic subunits. Moreover, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis showed that hsd17b2 mRNA expressed strongly in the ovaries at early developmental stages, weakly in liver and intestine, and barely in testis and other tissues. In particular, hsd17b2 mRNA expression was found to peak in ovaries of young fish and ovotestis at the early stage, and eventually declined in gonads from the late ovotestis to testis. Likewise, chemical in situ hybridization results indicated that the hsd17b2 mRNA signals were primarily detected in the cytoplasm of oogonia and oocytes at stage I–II, subsequently concentrated in the granulosa cells around the oocytes at stage Ⅲ–Ⅳ, but undetectable in mature oocytes and male germ cells. Intriguingly, in ricefield eel ovaries, hsd17b2 mRNA expression could be significantly reduced by 17β-estradiol (E2) or tamoxifen (17β-estradiol inhibitor, E2I) induction at a low concentration (10 ng/mL) and increased by E2I induction at a high concentration (100 ng/mL). On the other hand, both the melatonin (MT) and flutamide (androgen inhibitor, AI) induction could significantly decrease hsd17b2 mRNA expression in the ovary of ricefield eel. This study provides a clue for demonstrating the mechanism of sexual differentiation in fish. The findings of our study imply that the hsd17b2 gene could be a key regulator in sexual differentiation and modulate sex reversal in the ricefield eel and other hermaphroditic fishes. Full article

Eclipta alba (Linn.) Hassk. (Asteraceae) is a high value medicinal plant which possesses diverse medicinal properties. It is an important herb for the treatment of various disorders, and is primarily used as a hepatoprotectant. Its major biochemical constituents include wedelolactone and dimethyl-wedelolactone (coumestans), which possess anti-hepatotoxic properties. Due to its numerous medicinal properties, it is in high demand by the pharmaceutical industry and therefore requires urgent biotechnological interventions for its improvement. Therefore, the present study was constructed with the aim of developing an efficient genetic transformation protocol for E. alba, which will help in the mass production of the active compounds found in E. alba. Agrobacterium tumefaciens strain LBA 4404, containing vector pBI121, was used to genetically transform the plant, and the effect of various factors such as infection type, light cycle effect, effect of pH, among others, on the genetic transformation efficiency was analyzed. Regenerated transformed shoots were confirmed using the standard Polymerase Chain Reaction PCR method. Kanamycin-resistant and beta- glucurosidaseGUS-positive shoots indicated the development of transgenic shoots in E. alba. Amplification of nptll and uidA genes confirmed the integration of t-DNA transgenic shoots. In conclusion, various factors affecting the transformation efficiency were analyzed, and a reliable A. tumefaciens-mediated genetic transformation protocol was developed. Full article

In the cell, DNA polymerase β (Polβ) is involved in many processes aimed at maintaining genome stability and is considered the main repair DNA polymerase participating in base excision repair (BER). Polβ can fill DNA gaps formed by other DNA repair enzymes. Single-nucleotide polymorphisms (SNPs) in the POLB gene can affect the enzymatic properties of the resulting protein, owing to possible amino acid substitutions. For many SNP-associated Polβ variants, an association with cancer, owing to changes in polymerase activity and fidelity, has been shown. In this work, kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring polymorphic variants G274R, G290C, and R333W. Previously, the amino acid substitutions at these positions have been found in various types of tumors, implying a specific role of Gly-274, Gly-290, and Arg-333 in Polβ functioning. All three polymorphic variants had reduced polymerase activity. Two substitutions—G274R and R333W—led to the almost complete disappearance of gap-filling and primer elongation activities, a decrease in the deoxynucleotide triphosphate–binding ability, and a lower polymerization constant, due to alterations of local contacts near the replaced amino acid residues. Thus, variants G274R, G290C, and R333W may be implicated in an elevated level of unrepaired DNA damage. Full article