Comprehensive knockout analysis of the RAB family small GTPases reveals an overlapping role of RAB2 and RAB14 in autophagosome maturation.

Macroautophagy, simply referred to below as autophagy, is an intracellular degradation system that is highly conserved in eukaryotes. Since the processes involved in autophagy are accompanied by membrane dynamics, RAB small GTPases, key regulators of membrane trafficking, are generally thought to regulate the membrane dynamics of autophagy. Although more than half of the mammalian RABs have been reported to be involved in canonical and selective autophagy, no consensus has been reached in regard to the role of RABs in mammalian autophagy. Here, we comprehensively analyzed a rab-knockout (KO) library of MDCK cells to reevaluate the requirement for each RAB isoform in basal and starvation-induced autophagy. The results revealed clear alteration of the MAP1LC3/LC3-II level in only four rab-KO cells (rab1-KO, rab2-KO, rab7a-KO, and rab14-KO cells) and identified RAB14 as a new regulator of autophagy, specifically at the autophagosome maturation step. The autophagy-defective phenotype of two of these rab-KO cells, rab2-KO and rab14-KO cells, was very mild, but double KO of rab2 and rab14 caused a severer autophagy-defective phenotype (greater LC3 accumulation than in single-KO cells, indicating an overlapping role of RAB2 and RAB14 during autophagosome maturation. We also found that RAB14 is phylogenetically similar to RAB2 and that it possesses the same properties as RAB2, i.e. autophagosome localization and interaction with the HOPS subunits VPS39 and VPS41. Our findings suggest that RAB2 and RAB14 overlappingly regulate the autophagosome maturation step through recruitment of the HOPS complex to the autophagosome.Abbreviation: AID2: auxin-inducible degron 2; ATG: autophagy related; BafA1: bafilomycin A1; CKO: conditional knockout; EBSS: Earle's balanced salt solution; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; HRP: horseradish peroxidase; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; MDCK: Madin-Darby canine kidney; mAb: monoclonal antibody; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; 5-Ph-IAA: 5-phenyl-indole-3-acetic acid; pAb: polyclonal antibody; siRNA: small interfering RNA; SNARE: soluble NSF-attachment protein receptor; TF: transferrin; WT: wild-type.

Corrigendum: ACE2 knockout hinders SARS-CoV-2 propagation in iPS cell-derived airway and alveolar epithelial cells.

[This corrects the article DOI: 10.3389/fcell.2023.1290876.].

Enhancement of Green Production of Heme by Deleting Odor-Related Genes from Bacillus amyloliquefaciens Based on CRISPR/Cas9n.

Heme is a crucial component in endowing plant-based meat analogs with flavor and color. This study aimed to develop a green strategy for heme production by reducing fermentation off-odor and accelerating heme synthesis. First, an efficient CRISPR/Cas9n system was constructed in Bacillus amyloliquefaciens to construct the odor-reducing chassis cell HZC9nΔGPSU, and the odor substances including the branched-chain short fatty acids, putrescine, and ammonia were reduced by 62, 70, and 88%, respectively. Meanwhile, the hemA gene was confirmed to be the key gene for enhanced heme synthesis. Various hemA genes were compared to obtain the best gene dhemA, and the catalysis mechanism was explained by molecular docking simulation. After further expression of dhemA in HZC9nΔGPSU, the heme titer of HZC9nΔGPSU/pHY-dhemA reached 11.31 ± 0.51 mg/L, 1.70-fold higher than that of HZC9n/pHY-dhemA. The knockout of off-odor-related genes reduced the odor substances and enhanced the heme synthesis, which is promising for the green production of high-quality heme.

The ldh Gene Plays a Crucial Role in Mediating the Pathogen Control of Lactiplantibacillus plantarum AR113.

Effectively managing foodborne pathogens is imperative in food processing, where probiotics play a crucial role in pathogen control. This study focuses on the Lactiplantibacillus plantarum AR113 and its gene knockout strains, exploring their antimicrobial properties against Escherichia coli O157:H7 and Staphylococcus aureus. Antimicrobial assays revealed that the inhibitory effect of AR113 increases with its growth and the potential bacteriostatic substance is acidic. AR113Δldh, surpassed AR113Δ0273&2024, exhibited a complete absence of bacteriostatic properties, which indicates that lactic acid is more essential than acetic acid in the bacteriostatic effect of AR113. However, the exogenous acid validation test affirmed the equivalent superior bacteriostatic effect of lactic acid and acetic acid. Notably, AR113 has high lactate production and deletion of the ldh gene not only lacks lactate production but also affects acetic production. This underscores the ldh gene's pivotal role in the antimicrobial activity of AR113. In addition, among all the selected knockout strains, AR113ΔtagO and ΔccpA also had lower antimicrobial effects, suggesting the importance of tagO and ccpA genes of AR113 in pathogen control. This study contributes insights into the antimicrobial potential of AR113 and stands as the pioneering effort to use knockout strains for comprehensive bacteriostatic investigations.

[Knockdown of motility-related genes of Komagataeibacter xylinus and its effect on bacterial cellulose synthesis].

Bacterial cellulose (BC) is a biopolymer synthesized by bacteria, which possess excellent characteristics such as high water holding capacity, high crystallinity, and high purity. It is widely used in food, medical, cosmetics, and functional films. Komagataeibacter xylinus is a model strain used in BC synthesis research. In bacteria, motility-related genes are associated with BC synthesis, whereas in Komagataeibacter xylinus CGMCC 2955, the functions of motility-related genes and their effects on BC synthesis are not known. To address this gap, we used the λ Red recombinant system to individually knock out motA, motB, and mot2A respectively, and constructed the knockout strains K. x-ΔmotA, K. x-ΔmotB, and K. x-Δmot2A. Additionally, both motA and motB were disrupted to construct the K. x-ΔmotAB mutant. The results demonstrated that knockout strain K. x-ΔmotAB exhibited the highest BC yield, reaching (5.05±0.26) g/L, which represented an increase of approximately 24% compared to wild-type strains. Furthermore, the BC synthesized by this strain exhibited the lowest porosity, 54.35%, and displayed superior mechanical properties with a Young's modulus of up to 5.21 GPa. As knocking out motA and motB genes in K. xylinus CGMCC 2955 did not reduce BC yield; instead, it promoted BC synthesis. Consequently, this research further deepened our understanding of the relationship between motility and BC synthesis in acetic acid bacteria. The knockouts of motA and motB genes resulted in reduced BC porosity and improved mechanical properties, provides a reference for BC synthesis and membrane structure regulation modification.

SntB Affects Growth to Regulate Infecting Potential in Penicillium italicum.

Penicillium italicum, a major postharvest pathogen, causes blue mold rot in citrus fruits through the deployment of various virulence factors. Recent studies highlight the role of the epigenetic reader, SntB, in modulating the pathogenicity of phytopathogenic fungi. Our research revealed that the deletion of the SntB gene in P. italicum led to significant phenotypic alterations, including delayed mycelial growth, reduced spore production, and decreased utilization of sucrose. Additionally, the mutant strain exhibited increased sensitivity to pH fluctuations and elevated iron and calcium ion stress, culminating in reduced virulence on Gannan Novel oranges. Ultrastructural analyses disclosed notable disruptions in cell membrane integrity, disorganization within the cellular matrix, and signs of autophagy. Transcriptomic data further indicated a pronounced upregulation of hydrolytic enzymes, oxidoreductases, and transport proteins, suggesting a heightened energy demand. The observed phenomena were consistent with a carbon starvation response potentially triggering apoptotic pathways, including iron-dependent cell death. These findings collectively underscored the pivotal role of SntB in maintaining the pathogenic traits of P. italicum, proposing that targeting PiSntB could offer a new avenue for controlling citrus fungal infections and subsequent fruit decay.

Mice Lacking Mrs2 Magnesium Transporter are Hypophagic and Thin When Maintained on a High-Fat Diet.

Genes regulating body fat are shared with high fidelity by mice and humans, indicating that mouse knockout (KO) phenotyping might identify valuable antiobesity drug targets. Male Mrs2 magnesium transporter (Mrs2) KO mice were recently reported as thin when fed a high-fat diet (HFD). They also exhibited increased energy expenditure (EE)/body weight and had beiged adipocytes that, along with isolated hepatocytes, demonstrated increased oxygen consumption, suggesting that increased EE drove the thin phenotype. Here we provide our data on these and additional assays in Mrs2 KO mice. We generated Mrs2 KO mice by homologous recombination. HFD-fed male and female Mrs2 KO mice had significantly less body fat, measured by quantitative magnetic resonance, than wild-type (WT) littermates. HFD-fed Mrs2 KO mice did not demonstrate increased EE by indirect calorimetry and could not maintain body temperature at 4 °C, consistent with their decreased brown adipose tissue stores but despite increased beige white adipose tissue. Instead, when provided a choice between HFD and low-fat diet (LFD), Mrs2 KO mice showed a significant 15% decrease in total energy intake resulting from significantly lower HFD intake that offset numerically increased LFD intake. Food restriction studies performed using WT mice suggested that this decrease in energy intake could explain the loss of body fat. Oral glucose tolerance test studies revealed significantly improved insulin sensitivity in Mrs2 KO mice. We conclude that HFD-fed Mrs2 KO mice are thin with improved insulin sensitivity, and that this favorable metabolic phenotype is driven by hypophagia. Further evaluation is warranted to determine the suitability of MRS2 as a drug target for antiobesity therapeutics.

Reverse Genetics Applied to Immunobiology of Tumor Necrosis Factor, a Multifunctional Cytokine.

Tumor necrosis factor (TNF) is one of many cytokines - protein molecules responsible for communication between the cells of immune system. TNF was discovered and given its grand name because of its striking antitumor effects in experimental systems, but its main physiological functions in the context of whole organism turned out to be completely unrelated to protection against tumors. This short review discusses "man-made" mouse models generated by early genome-editing technologies, which enabled us to establish true functions of TNF in health and certain diseases as well as to unravel potential strategies for improving therapy of TNF-dependent diseases.

Skeletal phenotypes and molecular mechanisms in aging mice.

Aging is an inevitable physiological process, often accompanied by age-related bone loss and subsequent bone-related diseases that pose serious health risks. Research on skeletal diseases caused by aging in humans is challenging due to lengthy study durations, difficulties in sampling, regional variability, and substantial investment. Consequently, mice are preferred for such studies due to their similar motor system structure and function to humans, ease of handling and care, low cost, and short generation time. In this review, we present a comprehensive overview of the characteristics, limitations, applicability, bone phenotypes, and treatment methods in naturally aging mice and prematurely aging mouse models (including SAMP6, POLG mutant, LMNA, SIRT6, ZMPSTE24, TFAM, ERCC1, WERNER, and KL/KL-deficient mice). We also summarize the molecular mechanisms of these aging mouse models, including cellular DNA damage response, senescence-related secretory phenotype, telomere shortening, oxidative stress, bone marrow mesenchymal stem cell (BMSC) abnormalities, and mitochondrial dysfunction. Overall, this review aims to enhance our understanding of the pathogenesis of aging-related bone diseases.

Producing Human Livers From Human Stem Cells Via Blastocyst Complementation.

The need for organ transplants exceeds donor organ availability. In the quest to solve this shortage, the most remarkable area of advancement is organ production through the use of chimeric embryos, commonly known as blastocyst complementation. This technique involves the combination of different species to generate chimeras, where the extent of donor cell contribution to the desired tissue or organ can be regulated. However, ethical concerns arise with the use of brain tissue in such chimeras. Furthermore, the ratio of contributed cells to host animal cells in the chimeric system is low in the production of chimeras associated with cell apoptosis. This review discusses the latest innovations in blastocyst complementation and highlights the progress made in creating organs for transplant.

Effects of MAL gene knockout on lung tissue morphology and on E-cad and α-SMA expression in asthma mouse models.

OBJECTIVES: To investigate the effects of myelin- and lymphocyte-associated protein (MAL) gene knockout on the morphological structure of lung tissue and the expression of E-cadherin (E-cad) and alpha-smooth muscle actin (α-SMA) in an asthmatic mouse model. METHODS: Twenty-four specific pathogen-free (SPF) C57BL/6J mice were divided into four groups: the wild-type normal (WT/SAL), wild-type asthmatic (WT/OVA), gene knockout normal (MAL-/-/SAL), and gene knockout asthmatic (MAL-/-/OVA) groups. The establishment of the asthma mouse models was confirmed by evaluating behavioral symptoms and histopathological H&E and Masson staining. Western blotting and RT-qPCR were used to measure E-cad and α-SMA expression levels in lung tissues. RESULTS: H&E staining of mouse lung tissues from WT/OVA, MAL-/-/SAL, and MAL-/-/OVA groups revealed a thickened bronchial wall, irregular lumen edge, locally fallen mucosal epithelium, and inflammatory cell infiltration compared with those of the WT/SAL group. In the WT and MAL-/- groups, the proportion of Masson-stained tissues in the OVA group was greater than that in the SAL group (p < 0.05). Compared with those in the WT/SAL group, the expression levels of α-SMA mRNA and protein were increased, while those of E-cad were decreased in the WT/OVA group (p < 0.01). Similarly, compared with those in the MAL-/-/SAL group, the expression levels of E-cad mRNA and protein were increased, while those of α-SMA were decreased in the MAL-/-/OVA group (p < 0.01). All these differences were statistically significant (p < 0.01). CONCLUSIONS: The MAL gene contributes to EMT inhibition and the stability of the airway barrier under normal physiological conditions by regulating E-cad and α-SMA expression.

An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation.

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

Genome-Wide Identification and Characterization of Homeobox Transcription Factors in Phoma sorghina var. saccharum Causing Sugarcane Twisted Leaf Disease.

A homeobox transcription factor is a conserved transcription factor, ubiquitous in eukaryotes, that regulates the tissue formation of structure, cell differentiation, proliferation, and cancer. This study identified the homeobox transcription factor family and its distribution in Phoma sorghina var. saccharum at the whole genome level. It elucidated the gene structures and evolutionary characteristics of this family. Additionally, knockout experiments were carried out and the preliminary function of these transcription factors was studied. Through bioinformatics approaches, nine homeobox transcription factors (PsHOX1-PsHOX9) were identified in P. sorghina var. saccharum, and these contained HOX-conserved domains and helix-turn-helix secondary structures. Nine homeobox gene deletion mutants were obtained using the homologous recombinant gene knockout technique. Protoplast transformation was mediated by polyethylene glycol (PEG) and the transformants were identified using PCR. The knockouts of PsHOX1, PsHOX2, PsHOX3, PsHOX4, PsHOX6, PsHOX8, and PsHOX9 genes resulted in a smaller growth diameter in P. sorghina var. saccharum. In contrast, the knockouts of the PsHOX3, PsHOX6, and PsHOX9 genes inhibited the formation of conidia and led to a significant decrease in the pathogenicity. This study's results will provide insights for understanding the growth and development of P. sorghina var. saccharum. The pathogenic mechanism of the affected sugarcane will provide an essential theoretical basis for preventing and controlling sugarcane twisted leaf disease.

Conditional knockout mouse model reveals a critical role of peroxiredoxin 1 in oral leukoplakia carcinogenesis.

Peroxiredoxin 1 (Prx1) is an antioxidant protein that may promote the carcinogenesis in oral leukoplakia (OLK). To investigate the effect of Prx1 on the oral mucosal epithelium of OLK, we generated a Prx1 conditional knockout (cKO) mouse model. The mRNA and gRNA were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technique. An infusion cloning method was used to construct a homologous recombination vector. To obtain the F0 generation mice, fertilized eggs of C57BL/6J mice were microinjected with Cas9 mRNA, gRNA, and a donor vector. Polymerase chain reaction (PCR) amplification and sequencing were used to identify F1 generation mice. Using the cyclization recombination-enzyme-locus of the X-overP1 (Cre-loxP) system, we created a Prx1 cKO mouse model, and the effectiveness of the knockout was confirmed through immunohistochemistry. We examined the influence of Prx1 knockout on the occurrence of OLK in mice by constructing a model of tongue mucosa carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO). Prx1 modification was present in the F1 generation, as evidenced by PCR amplification and sequencing. Prx1flox/flox: Cre + mice exhibited normal growth and fertility. Immunohistochemical analysis revealed that tongue epithelial cells in Prx1flox/flox: Cre + mice displayed a distinct deletion of Prx1. An examination of the heart, liver, spleen, lung, and kidney tissues revealed no visible histological changes. Histological analysis showed a reduction in the occurrence of the malignant transformation of OLK in the tongue tissues of Prx1flox/flox: Cre + mice. Ki67 immunostaining showed that Prx1 knockout significantly inhibited cell proliferation in the tongue epithelial. Our research developed a conditional knockout mouse model for Prx1. The obtained results provide insights into the function of Prx1 in the development of oral cancer and emphasize its potential as a therapeutic target for precancerous oral lesions.

Molecular mechanism of reduced biological fitness of fludioxonil-resistant strains of Botrytis cinerea based on transcriptome analysis.

BACKGROUND: Fludioxonil is a fungicide used to control gray mold. However, the frequency of resistance in the field is low, and highly resistant strains are rarely isolated. The biological fitness of the resistant strain is lower than that of the wild strain. Therefore, the molecular mechanism underlying the decrease in the fitness of the fludioxonil-resistant strain of Botrytis cinerea was explored to provide a theoretical basis for resistance monitoring and management. RESULTS: Transcriptome analysis was performed on five different-point mutant resistant strains of fludioxonil, focusing on mining and screening candidate genes that lead to reduced fitness of the resistant strains and the functional verification of these genes. The differentially expressed genes (DEGs) of the five point-mutation resistant strains intersected with 1869 DEGs. Enrichment analysis showed that three downregulated genes (Bcin05g07030, Bcgad1, and Bcin03g05840) were enriched in multiple metabolic pathways and were downregulated in both domesticated strains. Bcin05g07030 and Bcin03g05840 were involved in mycelial growth and development, pathogenicity, and conidial yield, and negatively regulated oxidative stress and cell wall synthesis. Bcgad1 was involved in mycelial growth and development, conidial yield, oxidative stress, and cell wall synthesis. Furthermore, Bcin05g07030 was involved in osmotic stress and spore germination, whereas Bcin03g05840 and Bcgad1 negatively regulated osmotic stress and cell wall integrity. CONCLUSION: These results enable us to further understand the molecular mechanism underlying the decrease in the biological fitness of B. cinerea fludioxonil-resistant strains. © 2024 Society of Chemical Industry.

Splice variants of CK1α and CK1α-like: Comparative analysis of subcellular localization, kinase activity, and function in the Wnt signaling pathway.

Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/ß-catenin pathway, which promotes the degradation of ß-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/ß-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of ß-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/ß-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate ß-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/ß KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-ß-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/ß-catenin pathway at the level of ß-catenin and Axin.

Live Attenuated Vaccines against Tuberculosis: Targeting the Disruption of Genes Encoding the Secretory Proteins of Mycobacteria.

Tuberculosis (TB), a chronic infectious disease affecting humans, causes over 1.3 million deaths per year throughout the world. The current preventive vaccine BCG provides protection against childhood TB, but it fails to protect against pulmonary TB. Multiple candidates have been evaluated to either replace or boost the efficacy of the BCG vaccine, including subunit protein, DNA, virus vector-based vaccines, etc., most of which provide only short-term immunity. Several live attenuated vaccines derived from Mycobacterium tuberculosis (Mtb) and BCG have also been developed to induce long-term immunity. Since Mtb mediates its virulence through multiple secreted proteins, these proteins have been targeted to produce attenuated but immunogenic vaccines. In this review, we discuss the characteristics and prospects of live attenuated vaccines generated by targeting the disruption of the genes encoding secretory mycobacterial proteins.

Effect of knocking out mouse Slc44a4 on colonic uptake of the microbiota-generated thiamine pyrophosphate and colon physiology.

Humans and mammals obtain vitamin B1 from dietary and gut microbiota sources. A considerable amount of the microbiota-generated vitamin exists in the form of thiamine pyrophosphate (TPP), and colonocytes are capable of absorbing TPP via a specific carrier-mediated process that involves the colonic TPP transporter (cTPPT encoded by SLC44A4). Little is known about the relative contribution of the SLC44A4 transporter toward total colonic carrier-mediated TPP uptake and its role in colon physiology. To address these issues, we generated an Slc44a4 knockout (KO) mouse model (by Cre-Lox recombination) and found a near-complete inhibition in colonic carrier-mediated [3H]TPP uptake in the Slc44a4 KO compared with wild-type (WT) littermates. We also observed a significant reduction in KO mice's body weight and a shortening of their colon compared with WT. Using RNAseq and Ingenuity pathway analysis (IPA) approaches, we found that knocking out the colonic Slc44a4 led to changes in the level of expression of many genes, including upregulation in those associated with intestinal inflammation and colitis. Finally, we found that the Slc44a4 KO mice were more susceptible to the effect of the colitogenic dextran sodium sulfate (DSS) compared with WT animals, a finding that lends support to the recent prediction by multiple genome-wide association studies (GWAS) that SLC44A4 is a possible colitis susceptibility gene. In summary, the results of these investigations show that Slc44a4 is the predominant or only transporter involved in the colonic uptake of TPP, that the transporter is important for colon physiology, and that its deletion increases susceptibility to inflammation.NEW & NOTEWORTHY This study shows that Slc44a4 is the predominant or only transport system involved in the uptake of the gut microbiota-generated thiamine pyrophosphate (TPP) in the colon and that its deletion affects colon physiology and increases its susceptibility to inflammation.