Search Results (786)

Microbial protein production from acetate represents a promising route for sustainable protein supply, yet its efficiency is constrained by limited understanding of carbon–nitrogen metabolic coordination. In this study, nitrogen availability was systematically varied to investigate its role in regulating biomass composition and protein biosynthesis in Yarrowia lipolytica. Nitrogen limitation markedly reduced cell growth and protein accumulation (19.56% of dry cell weight) while increasing lipid content (up to 34.16%), indicating a altered protein and lipid accumulation under different nitrogen conditions. Transcriptomic analysis revealed a global downregulation of anabolic pathways under nitrogen limitation, accompanied by a shift in nitrogen assimilation from the glutamate dehydrogenase (GDH) pathway to the glutamine synthetase/glutamate synthase (GS–GOGAT) pathway, as well as significant upregulation of genes related to ammonium and amino acid transport. Guided by these findings, metabolic engineering of key nitrogen assimilation pathways was performed. Strains harboring additional copies of GDH and GS expression cassettes showed increased protein content from 48.52% to 55.77% and improved amino acid composition, whereas strains with an additional copy of the GOGAT gene exhibited reduced growth and protein accumulation. These results demonstrate that nitrogen availability regulates biomass composition through coordinated control of nitrogen transport and assimilation, and that balanced upregulation of GDH and GS genes is an effective strategy to improve microbial protein production from acetate, supporting the development of efficient fermentation processes using low-cost carbon sources. Full article

DNA vaccines have emerged as a promising immunization platform, offering key advantages over conventional vaccine approaches, including superior stability, a favorable safety profile, rapid and flexible antigen design, and scalable manufacturing. However, their clinical efficacy has remained limited, primarily due to inefficient cellular uptake, poor endosomal escape, and degradation of the plasmid DNA within host cells. Recent advances have highlighted minicircle DNA (mcDNA) as a next-generation alternative to conventional plasmid vectors. mcDNA constructs are compact, backbone-free episomal vectors containing only the expression cassette, including the promoter, transgene, and polyadenylation signal, while lacking bacterial sequences such as antibiotic resistance genes and origins of replication. This reduced vector size reduced vector-driven innate immune activation and susceptibility to epigenetic silencing, thereby improving transfection efficiency and supporting more sustained transgene expression in both dividing and non-dividing cells. This review provides a comprehensive overview of mcDNA technology in the context of vaccine development, discussing its structural design and production principles, mechanistic advantages over conventional plasmid DNA, and current applications across infectious disease and cancer vaccine platforms. In addition, we explore recent delivery strategies to enhance mcDNA transfection and immunogenicity, summarize existing limitations that hinder translation into applications, and outline future directions to optimize mcDNA-based vaccine technologies. Full article

Chromosomal rearrangements that lead to the formation of oncogenic gene fusions, such as EML4-ALK, are thought to arise from incorrect repair of double-strand breaks in DNA. However, the mechanisms and factors driving rearrangement formation remain poorly understood, and analysis of these processes is limited by detection methods that are labor-intensive, low-throughput, and not readily quantitative at single-cell resolution. Here, we developed a genetically encoded ALK reporter based on A549 lung adenocarcinoma cells, created by inserting an ALK-P2A-mCherry cassette into the endogenous ALK locus, so that induced EML4-ALK fusion activated mCherry fluorescence. Reporter activation yielded a readily quantifiable mCherry-positive subpopulation that could be measured and enriched by flow cytometry and correlated with EML4-ALK levels. Using this platform, we combined CRISPR-mediated rearrangement induction with knockdown of DNA repair factors using RNA interference. Of the factors involved in base excision repair, homologous recombination-related pathways and canonical non-homologous end joining, knockdown of the APEX1 gene encoding apurinic endonuclease 1 (APE1) selectively increased EML4-ALK levels both in the reporter cell line and in parental A549 cells. Together, this work provides a sensitive, single-cell A549-based ALK reporter platform and a framework for future studies aimed at identifying cellular and environmental factors that modulate oncogenic EML4-ALK rearrangement formation. Full article

The improvement of sucrose yield in sugarcane is impeded by the crop’s complex polyploid genome and slow progress in breeding. To clarify how arginine (Arg) regulates sugar metabolism and identify key genes associated with sucrose transport and accumulation in sugarcane, a screening experiment was performed by spraying L-arginine hydrochloride on the leaves and leaf sheaths of three sugarcane varieties (YZ05-51, YZ08-1609, and YT93-159), which differ in growth vigor, leaf morphology and other phenotypic traits. YZ05-51 exhibited the most prominent sugar-increasing effect, and subsequent optimization experiments on its leaf sheaths revealed that 20 g/mu L-arginine hydrochloride at pH 7.0 was optimal, significantly enhancing stem sucrose content. Transcriptomic analysis revealed the upregulation of genes related to sucrose synthesis and transport, with candidate genes enriched in pathways such as starch-sucrose metabolism, glycolysis/gluconeogenesis, and ATP-binding cassette (ABC) transporters. Metabolomic analysis detected 32 sugar metabolites across three categories, of which 24 were differentially abundant (e.g., glucose, galactose, fructose, and mannose). Integrated multi-omics analysis identified key regulatory genes, including SBEs and TPS1 (sucrose synthesis and carbon flux regulation), RBSK, α-amylases, GH28 (starch breakdown, glycolysis, and sugar mobilization), ABC transporters, GTs, and TIM10/TIM12 (sucrose transporter). Collectively, these analyses demonstrate enhanced activity of genes and metabolites involved in sucrose synthesis/transport in leaf sheaths, accompanied by reduced synthesis of other monosaccharides and oligosaccharides. Vigorously metabolizing leaf sheaths is more conducive to sucrose transport. This study provides valuable insights into the molecular mechanisms underlying Arg-mediated sucrose accumulation specifically in the sugarcane YZ05-51 sugarcane, highlighting its critical regulatory roles. Full article

Background/Objectives: Banana (Musa spp.) production is severely threatened by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), highlighting the need to identify genetic determinants of resistance. Methods: We performed a genome-wide analysis of NBS genes in Musa acuminata ssp. malaccensis, including phylogenetic, chromosomal, and microsynteny analyses. The genomic context and promoter regions of MamRGA2 were characterized, its response to defense-related phytohormones was evaluated by RT-qPCR, and its protein structure was predicted by homology modeling. Results: A total of 118 NBS genes were identified. Notably, we report for the first time in banana two NBS genes encoding proteins with integrated domains, corresponding to an ATP-binding cassette (ABC) transporter and a Nuclear Factor Y subunit A (NF-YA) transcription factor. Chromosomal mapping revealed a marked enrichment of NBS genes on chromosome 3, which harbors MamRGA2, an NBS gene associated with resistance to Foc TR4. RT-qPCR analyses showed that MamRGA2 is strongly induced by exogenous methyl jasmonate (MeJA) in the resistant wild genotype but not in a susceptible Cavendish cultivar, a pattern associated with divergence in promoter sequences between the two genotypes. Structural modeling suggested that the MamRGA2 protein possesses features consistent with a resistosome-like architecture. Conclusions: Overall, these findings expand current knowledge of NBS gene diversity in banana and provide a framework for future studies aimed at elucidating the molecular mechanisms underlying resistance to Foc TR4. Full article

Background/Objectives: Respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and parainfluenza virus type 3 (PIV3) are leading causes of acute respiratory infections in children and the elderly, yet no licensed T-cell vaccines are available. This study aimed to develop multivalent T-cell vaccine candidates against these pathogens using a live attenuated influenza virus (LAIV) vector platform. Methods: Conserved F, N, and M proteins of RSV, hMPV, and PIV3 were identified through multiple sequence alignments. Fragments enriched with experimentally confirmed and predicted T-cell epitopes were selected using the IEDB and NetMHCpan servers. These fragments were assembled into polyepitope immunogenic cassettes, and their selected order was determined by thermodynamic analysis of mRNA secondary structures using the RNAfold Web Server. The selected cassettes were cloned into the neuraminidase (NA) gene of a cold-adapted LAIV vector. Recombinant viruses were rescued by reverse genetics and assessed for replicative fitness in embryonated chicken eggs and MDCK cells, NA enzymatic activity and genetic stability upon serial passaging. Results: Four cassettes were designed for RSV, three for hMPV, and one for PIV3, all containing fragments with multiple T-cell epitopes. Three recombinant viruses of LAIV/RSV type and three of LAIV/hMPV type were successfully rescued, while attempts to recover the remaining recombinant viruses, i.e., LAIV/RSV and LAIV/PIV3, were not successful. All rescued recombinant viruses replicated to titers comparable to the parental LAIV strain and retained the full-length insert for at least eight passages in eggs. Importantly, NA enzymatic activity of the LAIV vector was not compromised by the insertion of the polyepitope T-cell cassettes. Conclusions: We developed a panel of recombinant T cell-based vaccine candidates against RSV and hMPV using the LAIV vector platform. These recombinant viruses encode conserved T-cell epitopes of the target viruses while retaining the biological properties of LAIV strains. Taken together, these characteristics warrant further evaluation of these recombinant viruses in appropriate relevant in vitro models to directly assess their immunogenicity in terms of stimulating a T-cell response against target pathogens. Full article

The rice stem borer (RSB), Chilo suppressalis, is one of the most destructive rice pests in China and chlorantraniliprole has been extensively used for its control over the past decade. However, the continuous and intensive application of chlorantraniliprole has accelerated the development of resistant RSB populations in field, thereby threatening sustainable rice production. In this study, a field resistant strain of RSB exhibited a 181.76-fold resistance level to chlorantraniliprole compared to a susceptible strain. To explore the potential involvement of ATP-binding cassette (ABC) transporters in chlorantraniliprole resistance, four candidate ABC transporter genes (CsABCC1, CsABCC3, CsABCA3 and CsABCD2) were analyzed in resistant and susceptible strains. Compared to the susceptible strain, the expressional levels of CsABCC1 and CsABCC3 were significantly upregulated by 1.58- and 1.38-fold, respectively, whereas of CsABCA3 and CsABCD2 showed non-significant differences in the resistant strain. RNA interference assays demonstrated that naked dsRNA induced only limited gene silencing, while chitosan-mediated dsRNA delivery significantly improved RNAi efficiency. Following feeding with chitosan-coated dsCsABCC1 and dsCsABCC3, the expression levels of both genes were reduced by 44.63% and 38.49%, respectively, relative to the control and the larval mortality increased following chlorantraniliprole treatment to 63.33% and 56.67%, respectively. In addition, silencing CsABCC1 caused a greater reduction in larval weight after insecticide treatment. These findings indicated that CsABCC1 and CsABCC3 are involved in chlorantraniliprole detoxification and may contribute to resistance. Overall, this study provides evidence for the functional involvement of ABC transporters in chlorantraniliprole resistance and highlights chitosan-mediated RNAi as a promising complementary approach for resistance management within integrated pest management programs. Full article

Background: Colistin- and carbapenem-resistant Acinetobacter baumannii (CRAB) represent a critical threat in intensive care unit (ICU) settings. This study aimed to provide a comprehensive molecular epidemiological characterization of extensively drug-resistant (XDR) A. baumannii clinical isolates from a tertiary-care hospital in Kırşehir, Central Anatolia, a region previously absent from the national surveillance literature. Methods: A total of 43 non-duplicate XDR A. baumannii isolates recovered from ICU patients between November 2021 and December 2023 were included. Antimicrobial susceptibility testing was performed by automated systems and broth microdilution for colistin. Resistance genes, including OXA-type carbapenemases, extended-spectrum β-lactamases (ESBLs), metallo-β-lactamases, plasmid-mediated colistin resistance (mcr-1 to mcr-5), plasmid-mediated quinolone resistance genes (qnr, qepA, oqxAB, aac(6′)-Ib-cr), and class 1 and 2 integrons, were screened by PCR. Integron gene cassettes were characterized by sequencing. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) using ApaI digestion. Results: All 43 isolates exhibited the XDR phenotype with universal resistance to carbapenems, colistin, fluoroquinolones, aminoglycosides (except amikacin), piperacillin, cephalosporins, and tobramycin. Amikacin susceptibility was retained in 58.1% of isolates. blaOXA-51 was detected in all isolates (100%), and blaOXA-23 was the predominant acquired carbapenemase (90.7%). Notably, blaOXA-48, a carbapenemase typically associated with Enterobacteriaceae, was identified in 3 isolates (7.0%), each belonging to a distinct pulsotype. No blaOXA-24/40, blaOXA-58, or class B metallo-β-lactamase genes were detected. ESBL genes were found in a subset of isolates, with blaCTX-M group 1 being the most prevalent (20.9%). The aac(6′)-Ib-cr gene was detected in 81.4% of isolates, and oqxA/oqxB in 60.5% and 39.5%, respectively. No mcr or classical qnr genes were identified. Class 1 and 2 integrons were detected in 4.7% and 7.0% of isolates, respectively, carrying dfrA12-DUF1010-aadA2 (class 1) and dfrA1-sat-1 (class 2) gene cassettes. PFGE identified 12 pulsotypes among the typeable isolates; PT4 (n = 20, 47.6%) and PT11 (n = 8, 19.0%) were the dominant clonal clusters, together accounting for 65.1% of typeable isolates. Conclusions: This study presents one of the first comprehensive molecular epidemiological analyses of XDR A. baumannii from Central Anatolia. The dominance of OXA-23-carrying clonal lineages, the detection of blaOXA-48 in A. baumannii distributed across three distinct pulsotypes, the high prevalence of aac(6′)-Ib-cr, and the concurrent distribution of resistance determinants across genetically diverse clonal backgrounds indicate that both clonal expansion and possible horizontal gene transfer contribute to resistance dissemination in this setting. These findings underscore the need for systematic molecular surveillance and reinforced infection control strategies in ICU settings, at both the regional and national levels. Full article

Indole-3-acetic acid (IAA) is the main natural auxin and a key regulator of plant growth. However, most commercial auxins are synthetically produced from non-renewable resources. Here, we present a minimal synthetic biology platform for microbial IAA production that also serves as a teaching model for genetic circuit design and bioprocess development. We developed codon-optimized versions of the iaaM and iaaH genes, which encode tryptophan 2-monooxygenase and indole-3-acetamide hydrolase, and assembled them into a compact expression cassette in Escherichia coli TOP10. Correct expression of both enzymes was confirmed by SDS-PAGE. The engineered strain was cultivated in a low-cost medium made from avocado seed hydrolysate, an agro-industrial waste, supplemented with tryptophan as a precursor. IAA was quantified using the Salkowski colorimetric assay and further validated by HPLC, reaching approximately 303–313 µg/mL at 48 h, with the medium costing approximately fivefold cheaper locally than traditional LB. The supernatants containing biosynthetic IAA induced root formation in 100% of tobacco leaf explants, outperforming the commercial standard at the same concentration and confirming biological activity. Since this workflow follows the Design–Build–Test–Learn (DBTL) cycle, Design (pathway selection and codon optimization), Build (plasmid assembly), Test (protein expression, metabolite quantification, plant bioassays), and Learn (medium and process optimization), it provides a sustainable production method and an accessible educational platform for synthetic biology. Full article

Background/Objectives: Antimicrobial resistance (AMR) is a global health threat, with raw milk serving as a potential reservoir for antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). This study characterized the resistome and mobilome of raw milk from cows (Bos taurus) and water buffalo (Bubalus bubalis) in the Brazilian Amazon, a region where unpasteurized dairy consumption is culturally ingrained. Methods: Using shotgun metagenomic sequencing, we analyzed 32 pooled milk samples from extensive and semi-intensive farms in the Manaus Metropolitan Region. Results: Sequencing yielded over 3.1 million contigs. While cow milk showed a higher prevalence of positive samples (80%), buffalo milk exhibited a significantly higher abundance and diversity of ARG-associated contigs (301 contigs vs. 85 in cows). Clinically relevant genes were identified, including AbaQ, ArnT, and KpnF, alongside complex multi-AMR cassettes co-occurring with plasmids and widespread viral sequences (dominated by Caudoviricetes). Integrons were ubiquitous in cattle and highly prevalent in buffalo samples. Conclusions: These findings indicate that raw milk in the Amazon harbors a rich reservoir of resistance determinants and MGEs, likely driven by farm-level antibiotic usage. This underscores a critical food safety risk and highlights the need for One Health-based surveillance in the region. Full article

Yeast harbour more than ten different multiple drug resistance (MDR) genes encoding transporters that extrude xenobiotics from the cytoplasm into the environment. These transporters, belonging to the ATP-binding cassette (ABC) or major facilitator superfamily (MFS), exhibit broad and significantly overlapping substrate specificities, though the precise boundaries of their individual substrate ranges remain undefined. During evolution, genes with overlapping functions tend either to specialize or to degenerate into pseudogenes. Here, we propose several explanations for how this apparent redundancy of MDR efflux pumps benefits cells, and we discuss the potential individual roles of the full MDR efflux pump repertoire in the model organism Saccharomyces cerevisiae. We posit that individual MDR transporters may vary in stability under challenging environmental conditions, in the energetic cost of their synthesis and maintenance, and in their degree of specialization toward particular classes of xenobiotics. Furthermore, given that ABC transporters and MFS transporters exploit distinct driving forces for xenobiotic efflux, each class may have its own vulnerabilities. We argue that deciphering the distinct roles of MDR proteins will reveal critical weaknesses in the MDR system and guide the development of strategies to overcome multidrug resistance in pathogenic fungi. Full article

The aim of this study was to assess the effect of sertraline on the gene expression of placental transporters for hormones, folates, nutrients and drugs over the course of pregnancy in rats. The studies were conducted on gestational days (GDs) 16 and 20 following oral treatment with 10 mg/kg/day sertraline or the vehicle, administered from weaning onward. The weight and area of the fetuses and placentas were analyzed, and maternal plasma sertraline concentrations were measured. Gene expression of ATP-binding cassette transporter b1a and b1b (Abcb1a and Abcb1b), organic anion-transporting polypeptide 4a1(Slco4A1/Oatp4a1), folate receptor-α (Folr1), reduced folate carrier (Slc19A1/Rfc), and L-type amino acid transporter (Slc7A5/Lat1) was evaluated in the placenta. Sertraline reduced fetal weight (p < 0.001) and fetal area (p < 0.01) at GD 16, while no significant differences were observed in placental weight or area between exposed and unexposed groups. Sertraline concentration was significantly lower at GD20 than at GD16 (p < 0.001). At GD 16, sertraline reduced the expression of Abcb1a (p = 0.027), Abcb1b (p < 0.01), and Oatp4a1 (p = 0.037) compared with controls. Conversely, sertraline induced Folr1 expression in both GDs and increased Rfc expression at GD 20, while Lat1 was not affected. These findings indicate that sertraline alters placental drug transporter gene expression and may impair nutrient transfer to the fetus. Full article

Background: Pinellia ternata is a major medicinal herb widely utilized in traditional medicine, but is sensitive to high temperature, which often triggers a severe “sprout tumble” phenomenon. Methods: To elucidate the molecular mechanisms of heat tolerance in P. ternata, we screened two contrasting germplasms: the heat-tolerant JBX1 and the heat-sensitive XBX4. In the present study, a combined analysis of physiology, transcriptome, and metabolome was performed on JBX1 and XBX4 under heat stress at 40 °C. Results: JBX1 exhibited significantly greater leaf thickness, higher basal chlorophyll content, more stable antioxidant enzyme activities, and lower oxidative damage than XBX4 under heat stress. Transcriptomically, JBX1 maintained elevated basal expression of genes encoding key enzymes in carbon fixation, amino acid metabolism, and phenylpropanoid biosynthesis, as well as those encoding heat shock transcription factors (HSFs), heat shock proteins (HSPs), and the thermosensor Thermo-With ABA-Response 1 (TWA1). Metabolomically, JBX1 accumulated higher levels of key primary metabolites, antioxidants, and protective phenylpropanoids under both control and heat conditions. Notably, a “polarity reversal” emerged in nitrogen metabolism, where core amino acids accumulated in JBX1 but were depleted in XBX4. Integrated analysis revealed a more coordinated gene–metabolite network in JBX1 involving the phenylpropanoid, ATP-binding cassette (ABC) transporter, and glutathione pathways. Conclusions: Our findings demonstrate that JBX1 possessed stronger basal thermotolerance, which is derived from coordinated establishment of higher constitutive metabolic reserves and efficient dynamic metabolic reprogramming. This study provides insights into the molecular mechanisms of heat stress in P. ternata. Full article

Although the ATP-binding cassette (ABC) transporter superfamily of proteins typically facilitates the movement of compounds across cellular membranes, the ABC E subfamily (ABCE) influences protein synthesis via non-transporter roles in ribosome biogenesis. Despite this essential role, our understanding of the impact that ABCE proteins have on insect physiology is limited. Here, we identified and characterized the ABCE gene from Lygus hesperus, a major agricultural pest of crops in North America. LhABCE transcripts were constitutively expressed throughout development and were present in all adult tissues tested. RNA interference (RNAi)-mediated knockdown of LhABCE transcripts in fifth instar nymphs resulted in high nymphal mortality and an incomplete molt. LhABCE knockdown in adults disrupted gametogenesis and reduced longevity. In females, oogenesis was impaired and oocytes did not progress beyond the pre-vitellogenic phase. In males, LhABCE knockdown reduced both spermatozoa abundance and male fertility. LhABCE knockdown, however, had little to no impact on hemolymph protein levels or the levels of circulating vitellogenin. Taken together, the results indicate that LhABCE is critical for the normal progression of processes like molting and gametogenesis that require coordinated bursts of protein synthesis and suggest that ABCE may play an important role in the mechanisms underlying those bursts. Full article

Background: Epilepsy is a chronic disorder with a higher prevalence in low- and middle-income countries. ATP-binding cassette superfamily B1 (ABCB1) not only has a potential influence on the resistance to antiepileptic drugs but also plays a possible role in the occurrence of epilepsy. Purpose: To evaluate the association of ABCB1 polymorphisms, c.1236C>T (rs1128503), c.2677G>T (rs2032582), and c.3435C>T (rs1045642), with epilepsy susceptibility in a Jordanian cohort. Subjects and methods: Eighty-six cases of patients with epilepsy were analyzed using polymerase chain reaction (PCR) for ABCB1 c.1236C>T, c.2677G>T, and c.3435C>T gene variants. The proportions of genotypes and alleles in the epilepsy group were compared with one hundred healthy controls who were previously also analyzed by PCR. Results: The C alleles of the ABCB1 polymorphisms c.1236C>T and c.3435C>T were more prevalent in the epilepsy group than in controls. The patients with epilepsy were less likely to have the TT genotype compared with controls (concerning ABCB1 c.1236C>T) (OR_{TT vs. CC} = 0.42; 95% CI = [0.19–0.91]; p = 0.019). The CC genotype of ABCB1 c.3435C>T was more frequent in epileptics than healthy people (OR_{CC vs. TT} = 4.3; 95% CI = [1.8–9.95]; p = 0.0007). No significant difference in ABCB1 c.2677G>T allelic and genotypic frequencies was observed between epileptic cases and healthy volunteers. Conclusion: Our findings suggest that ABCB1 c.1236C>T and c.3435C>T variants were associated with epilepsy susceptibility in this Jordanian cohort, whereas no significant association was observed for c.2677G>T. These findings should be interpreted cautiously because of the modest sample size and require validation in larger, independent studies. Full article