Wild-type Research Articles

Molecular Events in Response to Triclosan-Induced Oxidative Stress in CRISPR/Cas9-Mediated p53-Targeted Mutants in Daphnia magna.

Triclosan (TCS), a widely used antimicrobial agent, has been implicated in the oxidative stress induction and disruption of cellular processes in aquatic organisms. As TCS is ubiquitous in the aquatic environment, many previous studies have documented the effects of exposure to TCS on aquatic organisms. Nevertheless, most of the research has concentrated on the molecular and physiological responses of TCS, but there are still limited studies on the function of specific genes and the consequences of their absence. In this study, we focused on p53, a gene that is crucial for molecular responses such as autophagy and apoptosis as a result of TCS exposure. In order to ascertain the role and impact of the p53 gene in TCS-induced molecular responses, we examined the molecular responses to TCS-induced oxidative stress in wild-type (WT) and CRISPR/Cas9-mediated p53 mutant (MT) water fleas. The result has been accomplished by examining changes in molecular mechanisms, including in vivo end points, enzyme activities, adenosine triphosphate release rate, and apoptosis, to determine the role and impact of the p53 gene on TCS-induced molecular responses. The results indicated that the sensitivity of MT water fleas to TCS was greater than that of WT water fleas; however, the difference in sensitivity was significant at short exposures within 48 h and decreased toward 48 h. Accordingly, when we confirmed the oxidative stress after 24 h of exposure, the oxidative stress to TCS exposure was stronger in the MT group, with an imbalance of redox. To identify the mechanisms of tolerance to TCS in WT and MT Daphnia magna, we checked mitochondrial and ER-stress-related biomarkers and found an increase in apoptosis and greater sensitivity to TCS exposure in the MT group than in the WT. Our results suggest that the absence of p53 caused alterations in molecular processes in response to TCS exposure, resulting in increased sensitivity to TCS, and that p53 plays a critical role in response to TCS exposure.

IL-10 deficiency aggravates cell senescence and accelerates BLM-induced pulmonary fibrosis in aged mice via PTEN/AKT/ERK pathway

BackgroundPulmonary fibrosis (PF) is an aging-related progressive lung disorder. The aged lung undergoes functional and structural changes termed immunosenescence and inflammaging, which facilitate the occurrence of fibrosis. Interleukin-10 (IL-10) is a potent anti-inflammatory and immunoregulatory cytokine, yet it remains unclear how IL-10 deficiency-induced immunosenescence participates in the development of PF.MethodsFirstly we evaluated the susceptibility to fibrosis and IL-10 expression in aged mice. Then 13-month-old wild-type (WT) and IL-10 knockout (KO) mice were subjected to bleomycin(BLM) and analyzed senescence-related markers by PCR, western blot and immunohistochemistry staining of p16, p21, p53, as well as DHE and SA-β-gal staining. We further compared 18-month-old WT mice with 13-month-old IL-10KO mice to assess aging-associated cell senescence and inflamation infiltration in both lung and BALF. Moreover, proliferation and apoptosis of alveolar type 2 cells(AT2) were evaluated by FCM, immunofluorescence, TUNEL staining, and TEM analysis. Recombinant IL-10 (rIL-10) was also administered intratracheally to evaluate its therapeutic potential and related mechanism. For the in vitro experiments, 10-week-old naïve pramily lung fibroblasts(PLFs) were treated with the culture medium of 13-month PLFs derived from WT, IL-10KO, or IL-10KO + rIL-10 respectively, and examined the secretion of senescence-associated secretory phenotype (SASP) factors and related pathways.ResultsThe aged mice displayed increased susceptibility to fibrosis and decreased IL-10 expression. The 13-month-old IL-10KO mice exhibited significant exacerbation of cell senescence compared to their contemporary WT mice, and even more severe epithelial-mesenchymal transition (EMT) than that of 18 month WT mice. These IL-10 deficient mice showed heightened inflammatory responses and accelerated PF progression. Intratracheal administration of rIL-10 reduced lung CD45 + cell infiltration by 15%, including a 6% reduction in granulocytes and a 10% reduction in macrophages, and increased the proportion of AT2 cells by approximately 8%. Additionally, rIL-10 significantly decreased α-SMA and collagen deposition, and reduced the expression of senescence proteins p16 and p21 by 50% in these mice. In vitro analysis revealed that conditioned media from IL-10 deficient mice promoted SASP secretion and upregulated senescence genes in naïve lung fibroblasts, which was mitigated by rIL-10 treatment. Mechanistically, rIL-10 inhibited TGF-β-Smad2/3 and PTEN/PI3K/AKT/ERK pathways, thereby suppressing senescence and fibrosis-related proteins.ConclusionsIL-10 deficiency in aged mice leads to accelerated cell senescence and exacerbated fibrosis, with IL-10KO-PLFs displaying increased SASP secretion. Recombinant IL-10 treatment effectively mitigates these effects, suggesting its potential as a therapeutic target for PF.Graphical

Revealing a hidden conducting state by manipulating the intracellular domains in KV10.1 exposes the coupling between two gating mechanisms.

The KCNH family of potassium channels serves relevant physiological functions in both excitable and non-excitable cells, reflected in the massive consequences of mutations or pharmacological manipulation of their function. This group of channels shares structural homology with other voltage-gated K+ channels, but the mechanisms of gating in this family show significant differences with respect to the canonical electromechanical coupling in these molecules. In particular, the large intracellular domains of KCNH channels play a crucial role in gating that is still only partly understood. Using KCNH1(KV10.1) as a model, we have characterized the behavior of a series of modified channels that could not be explained by the current models. With electrophysiological and biochemical methods combined with mathematical modeling, we show that the uncovering of an open state can explain the behavior of the mutants. This open state, which is not detectable in wild-type channels, appears to lack the rapid flicker block of the conventional open state. Because it is accessed from deep closed states, it elucidates intermediate gating events well ahead of channel opening in the wild type. This allowed us to study gating steps prior to opening, which, for example, explain the mechanism of gating inhibition by Ca2+-Calmodulin and generate a model that describes the characteristic features of KCNH channels gating.

SARS-CoV-2 Omicron XBB.1 variant outbreak in a defined cohort: an epidemiological investigation incorporating longitudinal assessment of humoral response

BackgroundWe describe an epidemiological investigation of a COVID-19 caused by SARS-CoV-2-XBB.1 outbreak among healthcare workers (HCWs) returning from a 5-days educational tour abroad. MethodsWe prospectively followed participants for symptoms and sampled blood for neutralization assays of four SARS-CoV-2 variants (wild type, XBB, EG.5.1, and BA.2.86) at 1, 3, and 6 months after their return. When available, samples from the 3 months preceding the outbreak were also tested. We compared geometric mean titers (GMT) of neutralizing antibody titers of infected vs uninfected HCWs and febrile vs afebrile infected HCWs. ResultsNineteen (10%) of 181 HCWs were infected, all had mild COVID-19, 90% (17/19) had symptoms, and 16% (3/19) reported fever. Infected individuals tended to have lower pre-exposure XBB-neutralizing antibody titers (GMT of 32 vs 107 ID50, p=0.248). Neutralization against XBB and newer sub-variants peaked at 3 months and was higher among infected individuals (GMT 702 vs 156 [p<0.001], 558 vs 163 [p=0.001], and 558 vs 182 [p=0.002], ID50 for XBB, EG.5.1., and BA.2.86, respectively). By six months, these differences were no longer observed. Fever was positively associated with XBB neutralization (GMT 3474 vs 485, ID50 p=0.005). ConclusionsRecently infected individuals are protected from reinfection with newer sub-variants. However, protection is likely short lived.

Methicillin resistant Staphylococcus aureus mazEF expression promotes infections by influencing cellular growth, antibiotic sensitivity, and formation of biofilms

Staphylococcus aureus infections are hard to treat due to the emergence of antibiotic resistant strains, as well as their ability to form biofilms. The MazEF toxin–antitoxin system is thought play a role in bacterial biofilm phenotype as well as antibiotic resistance. In S. aureus, the physiologic function of the mazEF gene in the disease transition from acute to chronic infection is not well understood. In methicillin resistant S. aureus (MRSA), loss of mazF expression results in loss of resistance to first generation cephalosporins. mazF::tn displayed sensitivity while the isogenic wild type (WT) remained resistant. mazF::tn displayed significantly increased growth of biofilms on metal implants over 48 h compared to WT and the complemented transposon mutant. mazF::tn biofilms displayed significantly decreased antibiotic tolerance to vancomycin and cefazolin in comparison to WT and complement biofilms. Mice given mazF::tn in a sepsis model displayed less abscess burden and increased survival (100%) when treated with cefazolin compared to WT bacteremia treated with cefazolin (20%). mazF::tn periprosthetic joint infections displayed increased biofilm burden at acute time points and decreased biofilm burden at chronic time points. Our data suggests MazEF in MRSA is responsible for controlling growth of biofilms, antibiotic tolerance, and influence chronic infections in vivo.

Insights into epithelial-mesenchymal transition from cystic fibrosis rat models

BackgroundMolecular pathways contributing to Cystic Fibrosis pathogenesis remain poorly understood. Epithelial-mesenchymal transition (EMT) has been recently observed in CF lungs and certain CFTR mutation classes may be more susceptible than others. No investigations of EMT processes in CF animal models have been reported. AimThe aim of this study was to assess the expression of EMT-related markers in Phe508del and knockout (CFTR-KO) rat lung tissue and tracheal-derived basal epithelial stem cells, to determine whether CFTR dysfunction can produce an EMT state. MethodThe expression of EMT-related markers in lung tissue and cultured tracheal basal epithelial stem cells from wildtype (WT), Phe508del, and CFTR-KO rats were assessed using qPCR and Western blots. Cell responses were evaluated in the presence of Rho-associated protein kinase (ROCK) inhibitor Y27632, which blocks EMT-pathways, or after treatment with TGFβ1 to stimulate EMT. ResultsDifferent gene expression profiles were observed between Phe508del and CFTR-KO rat models compared to wild type. There was lower expression of type 1 collagen in KO lungs and primary cell cultures, while Phe508del lungs and cells had higher expression, particularly when treated with TGFβ1. The addition of Y27632 rescued changes in EMT related genes in Phe508del cells but not in KO cells. ConclusionOur findings show the first evidence of upregulated EMT pathways in the lungs and airway cells of any CF animal model. Differences in the regulation of the EMT genes and proteins in the Phe508del and CFTR-KO cells suggest that the signalling pathways underlying EMT are CFTR mutation dependent.

USP11 deubiquitinates E-cadherin and maintains luminal fate of mammary tumor cells to suppress breast cancer

Basal-like breast cancer may originate from luminal epithelial or cancerous cells. Inadequately repaired DNA damage impairs luminal differentiation and promotes aberrant luminal to basal trans-differentiation in mammary epithelial cells (MECs). Ubiquitin-specific peptidase 11 (USP11), a deubiquitinase, plays a critical role in DNA damage repair. The role of USP11 in controlling mammary cell differentiation and tumorigenesis remains poorly understood. We generated Usp11 knockout mice and breast cancer cell lines expressing wild-type (WT) and mutant form of USP11. By using these mutant mice, cell lines, and human USP11-deficient and -proficient breast cancer tissues, we tested how USP11 controls mammary cell fate. We generated Usp11 knock-out mice and found that deletion of Usp11 reduced the expression of E-cadherin and promoted DNA damage in MECs. Overexpression of WT USP11, but not a deubiquitinase-inactive mutant form of USP11, promoted luminal differentiation, enhanced DNA damage repair, and suppressed tumorigenesis in mice. Mechanistically, we found that USP11 enhanced the protein expression of E-cadherin dependent on its deubiquitinase activity, and that USP11 deubiquitinated E-cadherin at K738. We discovered that USP11 bound to E-cadherin through its C-terminal region. In human breast cancers, expression of USP11 was positively correlated with that of E-cadherin, and high USP11 predicted better recurrence-free survival. Our findings provide compelling genetic and biochemical evidence that USP11 not only promotes DNA damage repair but also deubiquitinates E-cadherin and maintains the luminal feature of mammary tumor cells, thereby suppressing luminal breast cancer.

Systems biology of electrogenic Pseudomonas putida - multi-omics insights and metabolic engineering for enhanced 2-ketogluconate production

BackgroundPseudomonas putida KT2440 has emerged as a promising host for industrial bioproduction. However, its strictly aerobic nature limits the scope of applications. Remarkably, this microbe exhibits high bioconversion efficiency when cultured in an anoxic bio-electrochemical system (BES), where the anode serves as the terminal electron acceptor instead of oxygen. This environment facilitates the synthesis of commercially attractive chemicals, including 2-ketogluconate (2KG). To better understand this interesting electrogenic phenotype, we studied the BES-cultured strain on a systems level through multi-omics analysis. Inspired by our findings, we constructed novel mutants aimed at improving 2KG production.ResultsWhen incubated on glucose, P. putida KT2440 did not grow but produced significant amounts of 2KG, along with minor amounts of gluconate, acetate, pyruvate, succinate, and lactate. 13C tracer studies demonstrated that these products are partially derived from biomass carbon, involving proteins and lipids. Over time, the cells exhibited global changes on both the transcriptomic and proteomic levels, including the shutdown of translation and cell motility, likely to conserve energy. These adaptations enabled the cells to maintain significant metabolic activity for several weeks. Acetate formation was shown to contribute to energy supply. Mutants deficient in acetate production demonstrated superior 2KG production in terms of titer, yield, and productivity. The ∆aldBI ∆aldBII double deletion mutant performed best, accumulating 2KG at twice the rate of the wild type and with an increased yield (0.96 mol/mol).ConclusionsBy integrating transcriptomic, proteomic, and metabolomic analyses, this work provides the first systems biology insight into the electrogenic phenotype of P. putida KT2440. Adaptation to anoxic-electrogenic conditions involved coordinated changes in energy metabolism, enabling cells to sustain metabolic activity for extended periods. The metabolically engineered mutants are promising for enhanced 2KG production under these conditions. The attenuation of acetate synthesis represents the first systems biology-informed metabolic engineering strategy for enhanced 2KG production in P. putida. This non-growth anoxic-electrogenic mode expands our understanding of the interplay between growth, glucose phosphorylation, and glucose oxidation into gluconate and 2KG in P. putida.

Elasticity of the HIV-1 core facilitates nuclear entry and infection.

HIV-1 infection requires passage of the viral core through the nuclear pore of the cell, a process that depends on functions of the viral capsid. Recent studies have shown that HIV-1 cores enter the nucleus prior to capsid disassembly. Interactions of the viral capsid with the nuclear pore complex are necessary but not sufficient for nuclear entry, and the mechanism by which the viral core traverses the comparably sized nuclear pore is unknown. Here we show that the HIV-1 core is highly elastic and that this property is linked to nuclear entry and infectivity. Using atomic force microscopy-based approaches, we found that purified wild type cores rapidly returned to their normal conical morphology following a severe compression. Results from independently performed molecular dynamic simulations of the mature HIV-1 capsid also revealed its elastic property. Analysis of four HIV-1 capsid mutants that exhibit impaired nuclear entry revealed that the mutant viral cores are brittle. Adaptation of two of the mutant viruses in cell culture resulted in additional substitutions that restored elasticity and rescued infectivity and nuclear entry. We also show that capsid-targeting compound PF74 and the antiviral drug Lenacepavir reduce core elasticity and block HIV-1 nuclear entry at concentrations that preserve interactions between the viral core and the nuclear envelope. Our results indicate that elasticity is a fundamental property of the HIV-1 core that enables nuclear entry, thereby facilitating infection. These results provide new insights into the role of the capsid in HIV-1 nuclear entry and the antiviral mechanisms of HIV-1 capsid inhibitors.

FHOD-1 and profilin protect sarcomeres against contraction-induced deformation in C. elegans.

Formin HOmology Domain 2-containing (FHOD) proteins are a subfamily of actin-organizing formins important for striated muscle development in many animals. We showed previously that absence of the sole FHOD protein, FHOD-1, from Caenorhabditis elegans results in thin body wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that mutations predicted to specifically disrupt actin polymerization by FHOD-1 similarly disrupt muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and with profilins PFN-2 and PFN-3 to promote body wall muscle growth. We further demonstrate that dense bodies in worms lacking FHOD-1 or PFN-2/PFN-3 are less stable than in wild type animals, having a higher proportion of dynamic protein, and becoming distorted by prolonged muscle contraction. We also observe accumulation of actin and actin depolymerization factor/cofilin homolog UNC-60B in body wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, possibly accounting for the abnormally slow growth and reduced body wall muscle strength in fhod-1 mutants. Overall, these results implicate FHOD protein-mediated actin assembly in forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development.

The Impact of YabG Mutations on Clostridioides difficile Spore Germination and Processing of Spore Substrates.

YabG is a sporulation-specific protease that is conserved among sporulating bacteria. Clostridioides difficile YabG processes the cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations in yabG were found which altered the co-germinants required to initiate spore germination. To understand how these mutations in the yabG locus contribute to C. difficile spore germination, we introduced these mutations into an isogenic background. Spores derived from C. difficile yabGC207A (a catalytically inactive allele), C. difficile yabGA46D, C. difficile yabGG37E, and C. difficile yabGP153L strains germinated in response to taurocholic acid alone. Recombinantly expressed and purified preproSleC incubated with E. coli lysate expressing wild type YabG resulted in the removal of the presequence from preproSleC. Interestingly, only YabGA46D showed any activity toward purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in yabG expression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.

A conditional protein diffusion model generates artificial programmable endonuclease sequences with enhanced activity

Deep learning-based methods for generating functional proteins address the growing need for novel biocatalysts, allowing for precise tailoring of functionalities to meet specific requirements. This advancement leads to the development of highly efficient and specialized proteins with diverse applications across scientific, technological, and biomedical fields. This study establishes a pipeline for protein sequence generation with a conditional protein diffusion model, namely CPDiffusion, to create diverse sequences of proteins with enhanced functions. CPDiffusion accommodates protein-specific conditions, such as secondary structures and highly conserved amino acids. Without relying on extensive training data, CPDiffusion effectively captures highly conserved residues and sequence features for specific protein families. We applied CPDiffusion to generate artificial sequences of Argonaute (Ago) proteins based on the backbone structures of wild-type (WT) Kurthia massiliensis Ago (KmAgo) and Pyrococcus furiosus Ago (PfAgo), which are complex multi-domain programmable endonucleases. The generated sequences deviate by up to nearly 400 amino acids from their WT templates. Experimental tests demonstrated that the majority of the generated proteins for both KmAgo and PfAgo show unambiguous activity in DNA cleavage, with many of them exhibiting superior activity as compared to the WT. These findings underscore CPDiffusion’s remarkable success rate in generating novel sequences for proteins with complex structures and functions in a single step, leading to enhanced activity. This approach facilitates the design of enzymes with multi-domain molecular structures and intricate functions through in silico generation and screening, all accomplished without the need for supervision from labeled data.

Determination of the combined effect of grape seed extract and cold atmospheric plasma on foodborne pathogens and their environmental stress knockout mutants.

The study aimed to explore the antimicrobial efficacy of grape seed extract (GSE) and cold atmospheric plasma (CAP) individually or in combination against L. monocytogenes and E. coli wild type (WT) and their isogenic mutants in environmental stress genes. More specifically, we examined the effects of 1% (wt/vol) GSE, 4 min of CAP treatment, and their combined effect on L. monocytogenes 10403S WT and its isogenic mutants ΔsigB, ΔgadD1, ΔgadD2, ΔgadD3, as well as E. coli K12 and its isogenic mutants ΔrpoS, ΔoxyR, and ΔdnaK. In addition, the sequence of the combined treatments was tested. A synergistic effect was achieved for all L. monocytogenes strains when exposure to GSE was followed by CAP treatment. However, the same effect was observed against E. coli strains, only for the reversed treatment sequence. Additionally, L. monocytogenes ΔsigB was more sensitive to the individual GSE and the combined GSE/CAP treatment, whereas ΔgadD2 was more sensitive to CAP, as compared to the rest of the mutants under study. Individual GSE exposure was unable to inhibit E. coli strains, and individual CAP treatment resulted in higher inactivation of E. coli in comparison to L. monocytogenes with the strain ΔrpoS appearing the most sensitive among all studied strains. Our findings provide a step toward a better understanding of the mechanisms playing a role in the tolerance/sensitivity of our model Gram-positive and Gram-negative bacteria toward GSE, CAP, and their combination. Therefore, our results contribute to the development of more effective and targeted antimicrobial strategies for sustainable decontamination.IMPORTANCEAlternative approaches to conventional sterilization are gaining interest from the food industry, driven by (i) the consumer demand for minimally processed products and (ii) the need for sustainable, environmentally friendly processing interventions. However, as such alternative approaches are milder than conventional heat sterilization, bacterial pathogens might not be entirely killed by them, which means that they could survive and grow, causing food contamination and health hazards. In this manuscript, we performed a systematic study of the impact of antimicrobials derived from fruit industry waste (grape seed extract) and cold atmospheric plasma on the inactivation/killing as well as the damage of bacterial pathogens and their genetically modified counterparts, for genes linked to the response to environmental stress. Our work provides insights into genes that could be responsible for the bacterial capability to resist/survive those novel treatments, therefore, contributing to the development of more effective and targeted antimicrobial strategies for sustainable decontamination.

Altered Endoplasmic Reticulum Integrity and Organelle Interactions in Living Cells Expressing INF2 Variants

The cytoskeleton mediates fundamental cellular processes by organizing inter-organelle interactions. Pathogenic variants of inverted formin 2 (INF2) CAAX isoform, an actin assembly factor that is predominantly expressed in the endoplasmic reticulum (ER), are linked to focal segmental glomerulosclerosis (FSGS) and Charcot–Marie–Tooth (CMT) neuropathy. To investigate how pathogenic INF2 variants alter ER integrity, we used high-resolution live imaging of HeLa cells. Cells expressing wild-type (WT) INF2 showed a predominant tubular ER with perinuclear clustering. Cells expressing INF2 FSGS variants that cause mild and intermediate disease induced more sheet-like ER, a pattern similar to that seen for cells expressing WT-INF2 that were treated with actin and microtubule (MT) inhibitors. Dual CMT-FSGS INF2 variants led to more severe ER dysmorphism, with a diffuse, fragmented ER and coarse INF2 aggregates. Proper organization of both F-actin and MT was needed to modulate the tubule vs. sheet conformation balance, while MT arrays regulated spatial expansion of tubular ER in the cell periphery. Pathogenic INF2 variants also induced mitochondria fragmentation and dysregulated mitochondria distribution. Such mitochondrial abnormalities were more prominent for cells expressing CMT-FSGS compared to those with FSGS variants, indicating that the severity of the dysfunction is linked to the degree of cytoskeletal disorganization. Our observations suggest that pathogenic INF2 variants disrupt ER continuity by altering interactions between the ER and the cytoskeleton that in turn impairs inter-organelle communication, especially at ER–mitochondria contact sites. ER continuity defects may be a common disease mechanism involved in both peripheral neuropathy and glomerulopathy.

FABP4 deficiency ameliorates alcoholic steatohepatitis in mice via inhibition of p53 signaling pathway

Fatty acid-binding protein 4 (FABP4) plays an essential role in metabolism and inflammation. However, the role of FABP4 in alcoholic steatohepatitis (ASH) remains unclear. This study aimed to investigate the function and underlying mechanisms of FABP4 in the progression of ASH. We first obtained alcoholic hepatitis (AH) datasets from the National Center for Biotechnology Information–Gene Expression Omnibus database and conducted bioinformatics analysis to identify critical genes in the FABP family. We then established ASH models of the wild-type (WT) and Fabp4-deficient (Fabp4−/−) mice to investigate the role of FABP4 in ASH. Additionally, we performed transcriptional profiling of mouse liver tissue and analyzed the results using integrative bioinformatics. The FABP4-associated signaling pathway was further verified. FABP4 was upregulated in two AH datasets and was thus identified as a critical biomarker for AH. FABP4 expression was higher in the liver tissues of patients with alcoholic liver disease and ASH mice than in the corresponding control samples. Furthermore, the Fabp4−/− ASH mice showed reduced hepatic lipid deposition and inflammation compared with the WT ASH mice. Mechanistically, Fabp4 may be involved in regulating the p53 and sirtuin-1 signaling pathways, subsequently affecting lipid metabolism and macrophage polarization in the liver of ASH mice. Our results demonstrate that Fabp4 is involved in the progression of ASH and that Fabp4 deficiency may ameliorate ASH. Therefore, FABP4 may be a potential therapeutic target for ASH treatment.

Insights into the functional mechanism of the non-specific lipid transfer protein nsLTP in Kalanchoe fedtschenkoi (Lavender scallops)

Plant non-specific lipid transfer protein (nsLTP) is able to bind and transport lipids and essential oils, as well as engage in various physiological processes, including defense against phytopathogens. Kalanchoe fedtschenkoi (Lavender Scallops) is an attractive and versatile succulent. To investigate the functional mechanism of Kalanchoe fedtschenkoi nsLTP (Ka-nsLTP), we expressed, purified and successfully obtained monomeric Ka-nsLTP. Mutational experiments revealed that the C6A variant retained the same activity as the wild-type (WT) Ka-nsLTP. Ka-nsLTP showed weak antiphytopathogenic bacterial activity, but inhibited fungal growth. Ka-nsLTP possessed a hydrophobic cavity effectively binding lauric acid. Our results offer novel molecular insights into the functional mechanism of nsLTP, which broadens our knowledge of the biological function of nsLTP in crops and provides a useful locus for genetic improvement of plants.

Overexpression of rice lectin receptor-like kinase, OsLec-RLK, confers salinity stress tolerance and increases seed yield in pigeon pea (Cajanus cajan (L.) Millsp.).

OsLec-RLK overexpression enhances cell signalling and salt stress tolerance in pigeon pea, enhancing seed yield and harvest index and thus, enabling marginal lands to increase food and nutritional security. Lectin Receptor-like kinases (Lec-RLKs) are highly effective cell signaling molecules that counteract various stresses, including salt stress. We engineered pigeon pea by overexpressing OsLec-RLK gene for enhancing salt tolerance. The OsLec-RLK overexpression lines demonstrated superior performance under salt stress, from vegetative to reproductive phase, compared to wild types (WT). The overexpression lines had significantly higher K+/Na+ratio than WT exposed to 100mM NaCl. Under salt stress, transgenic lines showed higher levels of chlorophyll, proline, total soluble sugars, relative water content, and peroxidase and catalase activity than WT plants. Membrane injury index and lipid peroxidation were significantly reduced in transgenic lines. Analysis of phenological and yield attributes confirmed that the OsLec-RLK pigeon pea lines maintain plant vigor, with 10.34-fold increase in seed yield (per plant) and 4-5-fold increase in harvest index of overexpression lines, compared to wild type. Meanwhile, the overexpression ofOsLec-RLK up-regulated the expression levels ofhistone deacetylase1, acyl CoA, ascorbate peroxidase, peroxidase, glutathione reductase and catalase, which were involved in the K+/Na+homeostasis pathway. This study showed the potential of OsLec-RLK gene for increasing crop productivity and yields under salt stress and enabling the crops to be grown on marginal lands for increasing food and nutritional security.

Overexpression of dehydroascorbate reductase gene IbDHAR1 improves the tolerance to abiotic stress in sweet potato.

Dehydroascorbate reductase (DHAR), an indispensable enzyme in the production of ascorbic acid (AsA) in plants, is vital for plant tolerance to various stresses. However, there is limited research on the stress tolerance functions of DHAR genes in sweet potato (Ipomoea batatas [L.] Lam). In this study, the full-length IbDHAR1 gene was cloned from the leaves of sweet potato cultivar Xu 18. The IbDHAR1 protein is speculated to be located in both the cytoplasm and the nucleus. As revealed by qRT-PCR, the relative expression level of IbDHAR1 in the proximal storage roots was much greater than in the other tissues, and could be upregulated by high-temperature, salinity, drought, and abscisic acid (ABA) stress. The results of pot experiments indicated that under high salinity and drought stress conditions, transgenic Arabidopsis and sweet potato plants exhibited decreases in H2O2 and MDA levels. Conversely, the levels of antioxidant enzymes APX, SOD, POD, and ACT, and the content of DHAR increased. Additionally, the ratio of AsA/DHA was greater in transgenic lines than in the wild type. The results showed that overexpression of IbDHAR1 intensified the ascorbic acid-glutathione cycle (AsA-GSH) and promoted the activity of the related antioxidant enzyme systems to improve plant stress tolerance and productivity.

CHCHD10P80L knock-in zebrafish display a mild ALS-like phenotype

Mutations in the nuclear-encoded mitochondrial gene CHCHD10 have been observed in patients with a spectrum of diseases that include amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathogenic nature of disease-associated variants of CHCHD10 we generated a zebrafish knock-in (KI) model expressing the orthologous ALS-associated CHCHD10P80L variant (zebrafish: Chchd10P83L). Larval chchd10P83L/P83L fish displayed reduced Chchd10 protein expression levels, motor impairment, reduced survival and abnormal neuromuscular junctions (NMJ). These deficits were not accompanied by changes in transcripts involved in the integrated stress response (ISR), phenocopying previous findings in our knockout (chchd10−/−). Adult, 11-month old chchd10P83L/P83L zebrafish, displayed smaller slow- and fast-twitch muscle cell cross-sectional areas compared to wild type zebrafish muscle cells. Motoneurons in the spinal cord of chchd10P83L/P83L zebrafish displayed similar cross-sectional areas to that of wild type motor neurons and significantly fewer motor neurons were observed when compared to chchd2−/− adult spinal cords. Bulk RNA sequencing using whole spinal cords of 7-month old fish revealed transcriptional changes associated with neuroinflammation, apoptosis, amino acid metabolism and mt-DNA inflammatory response in our chchd10P83L/P83L model. The findings presented here, suggest that the CHCHD10P80L variant confers an ALS-like phenotype when expressed in zebrafish.

Transcription factor CpWRKY50 enhances anthracnose resistance by promoting jasmonic acid signaling in papaya

Abstract Colletotrichum brevisporum is an important fungal pathogen that causes anthracnose and has led to serious postharvest losses of papaya (Carica papaya L.) fruit in recent years. WRKY transcription factors play vital roles in regulating plant resistance to pathogens, but their functions in papaya anthracnose resistance need further exploration. In this study, we identified a WRKY transcription factor, CpWRKY50, which belongs to the WRKY IIc subfamily. During infection with C. brevisporum, expression of CpWRKY50 in anthracnose-resistant papaya cultivars was significantly higher than that in susceptible cultivars. CpWRKY50 was induced by methyl jasmonate, and CpWRKY50 localized in the nucleus. In yeast, full-length CpWRKY50 had transactivation activity, but CpWRKY50 variants truncated at the N or C termini did not. CpWRKY50 positively regulated papaya resistance to C. brevisporum, as demonstrated by transient overexpression of CpWRKY50 in papaya and heterologous expression of CpWRKY50 in tomato. Moreover, endogenous jasmonic acid (JA) and JA-isoleucine levels in the fruits of transgenic tomato OE lines were higher than in wild type both before and after inoculation with C. brevisporum, indicating that increased CpWRKY50 expression promotes JA accumulation. Furthermore, our results revealed CpWRKY50 directly binds to W-box motifs (TTGACC) in the promoters of two JA signaling-related genes, CpMYC2 and pathogenesis-related 4 CpPR4, thereby activating their expression. Our data support that CpWRKY50 positively regulates anthracnose resistance in papaya by promoting JA signaling. These results broaden our understanding of papaya disease resistance mechanisms and will facilitate the genetic improvement of papaya through molecular breeding.