Golden Gate-Assisted Gene Doctoring for Streamlined and Efficient Recombineering in Bacteria.

Gene Doctoring is a genetic modification technique for E. coli and related bacteria, in which the Red-recombinase from bacteriophage λ mediates chromosomal integration of a fragment of DNA by homologous recombination (known as recombineering). In contrast to the traditional recombineering method, the integrated fragment for Gene Doctoring is supplied on a donor plasmid rather than as a linear DNA. This protects the DNA from degradation, facilitates transformation, and ensures multiple copies are present per cell, increasing the efficiency and making the technique particularly suitable for strains that are difficult to modify. Production of the donor plasmid has, until recently, relied on traditional cloning techniques that are inflexible, tedious, and inefficient. This protocol describes a procedure for Gene Doctoring combined with Golden Gate assembly of a donor plasmid, using a custom-designed plasmid backbone, for rapid and simple production of complex, multi-part assemblies. Insertion of a gene for superfolder green fluorescent protein, with selection by tetracycline resistance, into E. coli strain MG1655 is used as an example but in principle the method can be tailored for virtually any modification in a wide range of bacteria.

CRISPR-Mediated Construction of Gene-Knockout Mice for Investigating Antiviral Innate Immunity.

With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.

BnaC09.tfl1 controls determinate inflorescence trait in Brassica napus.

Determinate inflorescence is indeed a pivotal agricultural characteristic in crops, notably impacting the architecture modification of Brassica napus (AACC, 2n = 38). Previous study identified a crucial gene Bnsdt2 that encodes the transcription factor BnaC09.TFL1 (Terminal Flower 1). Here by two alleles were cloned and sequenced from indeterminate 2982 and determinate 4769, respectively, we found that BnaC09.TFL1 harbors two T/C and G/C non-synonymous mutations in exon 1, and contains sixty-six differences in a 1.9 Kb promoter sequence. Subsequently, BnaC09.TFL1 was introduced into B. napus 571 line by genetic complementation and overexpression, transgenic plants 571CTO lines and 571TClines were all restored to the indeterminate inflorescence. Interestingly, after BnaC09.TFL1 was knocked out in 'Westar', transgenic plants WestarTcr lines were mutated to determinate inflorescences. Additionally, a NIL-4769 line was constructed to evaluate the effect of BnaC09.TFL1 on agronomic traits of Brassica napus, the results demonstrated that BnaC09.tfl1 reduced the plant height and increased the branch number and branch thousand grain weight of Brassica napus. Finally, we performed RT-qPCR, GUS staining and subcellular localization experiments to analyze the expression pattern of BnaC09.TFL1, the results showed that the expression of BnaC09.TFL1 at shoot apex of NIL-4769 was higher than that of 4769, GUS activity was detected at apical of Arabidopsis thaliana and BnC09.TFL1-GFP was detected in cell membrane, nucleus and cytoplasm. Our findings provide a firm molecular foundation for the study of rapeseed's molecular mechanism of determinate inflorescence formation, as well as theoretical guidance for the application of determinate inflorescence in rapeseed breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01503-7.

Genetic deletion of the apoptosis associated speck like protein containing a card in LysM+ macrophages attenuates spinal cord injury by regulating M1/M2 polarization through ASC-dependent inflammasome signaling axis.

Apoptosis associated speck like protein containing a card (ASC), the key adaptor protein of the assembly and activation of canonical inflammasomes, has been found to play a significant role in neuroinflammation after spinal cord injury (SCI). The previous studies indicated that widely block or knockout ASC can ameliorate SCI. However, ASC is ubiquitously expressed in infiltrated macrophages and local microglia, so further exploration is needed on which type of cell playing the key role. In this study, using the LysMcre;Ascflox/flox mice with macrophage-specifc ASC conditional knockout (CKO) and contusive SCI model, we focus on evaluating the specific role of ASC in lysozyme 2 (LysM)+ myeloid cells (mainly infiltrated macrophages) in this pathology. The results revealed that macrophage-specifc Asc CKO exhibited the follow effects: (1) A significant reduction in the numbers of infiltrated macrophages in the all phases of SCI, and activated microglia in the acute and subacute phases. (2) A significant reduction in ASC, caspase-1, interleukin (IL)-1ß, and IL-18 compared to control mice. (3) In the acute and subacute phases of SCI, M1 subset differentiation was inhibited, and M2 differentiation was increased. (4) Histology and hindlimb motor recoveries were improved. In conclusion, this study elucidates that macrophage-specific ASC CKO can improve nerve function recovery after SCI by regulating M1/M2 polarization through inhibiting ASC-dependent inflammasome signaling axis. This indicates that ASC in peripheral infiltrated macrophages may play an important role in SCI pathology, at least in mice, could be a potential target for treatment.

The marine environmental microbiome mediates physiological outcomes in host nematodes.

BACKGROUND: Nematodes are the most abundant metazoans in marine sediments, many of which are bacterivores; however, how habitat bacteria affect physiological outcomes in marine nematodes remains largely unknown.  RESULTS: Here, we used a Litoditis marina inbred line to assess how native bacteria modulate host nematode physiology. We characterized seasonal dynamic bacterial compositions in L. marina habitats and examined the impacts of 448 habitat bacteria isolates on L. marina development, then focused on HQbiome with 73 native bacteria, of which we generated 72 whole genomes sequences. Unexpectedly, we found that the effects of marine native bacteria on the development of L. marina and its terrestrial relative Caenorhabditis elegans were significantly positively correlated. Next, we reconstructed bacterial metabolic networks and identified several bacterial metabolic pathways positively correlated with L. marina development (e.g., ubiquinol and heme b biosynthesis), while pyridoxal 5'-phosphate biosynthesis pathway was negatively associated. Through single metabolite supplementation, we verified CoQ10, heme b, acetyl-CoA, and acetaldehyde promoted L. marina development, while vitamin B6 attenuated growth. Notably, we found that only four development correlated metabolic pathways were shared between L. marina and C. elegans. Furthermore, we identified two bacterial metabolic pathways correlated with L. marina lifespan, while a distinct one in C. elegans. Strikingly, we found that glycerol supplementation significantly extended L. marina but not C. elegans longevity. Moreover, we comparatively demonstrated the distinct gut microbiota characteristics and their effects on L. marina and C. elegans physiology. CONCLUSIONS: Given that both bacteria and marine nematodes are dominant taxa in sedimentary ecosystems, the resource presented here will provide novel insights to identify mechanisms underpinning how habitat bacteria affect nematode biology in a more natural context. Our integrative approach will provide a microbe-nematodes framework for microbiome mediated effects on host animal fitness.

A double ttrA and pduA knock-out mutant of Salmonella Typhimurium is not attenuated for mice (Mus musculus).

Despite numerous studies on Salmonella enterica subsp. enterica serovar Typhimurium, the underlying mechanisms of several aspects of its virulence are still under investigation, including the role of the pdu and ttrA genes, associated with the metabolism of 1,2-propanediol using tetrathionate as an electron acceptor respectively. Our objective was to contribute to an understanding of the role of these genes inbacterial virulence for mice (Mus musculus) using an S. Typhumirum ΔttrApduA mutant. The experiment was conducted with a group infected by the S. Typhimurium mutant and a control group infected with a wild-type strain. The mutant was not attenuated compared with the parent strain. There were no differences in the bacterial numbers recovered from the mesenteric lymph nodes and Peyer's patches but at 8-day after oral infection higher numbers were recovered from the spleen, liver, and cecum. Unlike the single pduA and ttrA mutants, the double ΔttrApduA mutation did not affect invasion and survival in mice, which highlights the need for further studies to clarify the role of these important metabolism genes under reduced redox conditions linked to Salmonella virulence.

The Effects of swnN Gene Function of Endophytic Fungus Alternaria oxytropis OW 7.8 on Its Swainsonine Biosynthesis.

The swnN gene in the endophytic fungus Alternaria oxytropis OW 7.8 isolated from Oxytropis glabra was identified, and the gene knockout mutant ΔswnN was first constructed in this study. Compared with A. oxytropis OW 7.8, the ΔswnN mutant exhibited altered colony and mycelia morphology, slower growth rate, and no swainsonine (SW) in mycelia. SW was detected in the gene function complementation strain ΔswnN/swnN, indicating that the function of the swnN gene promoted SW biosynthesis. Six differentially expressed genes (DEGs) closely associated with SW synthesis were identified by transcriptomic analysis of A. oxytropis OW 7.8 and ΔswnN, with P5CR, swnR, swnK, swnH2, and swnH1 down-regulating, and sac up-regulating. The expression levels of the six genes were consistent with the transcriptomic analysis results. Five differential metabolites (DEMs) closely associated with SW synthesis were identified by metabolomic analysis, with L-glutamate, α-ketoglutaric acid, and L-proline up-regulating, and phosphatidic acid (PA) and 2-aminoadipic acid down-regulating. The SW biosynthetic pathways in A. oxytropis OW 7.8 were predicted and refined. The results lay the foundation for in-depth elucidation of molecular mechanisms and the SW synthesis pathway in fungi. They are also of importance for the prevention of locoism in livestock, the control and utilization of locoweeds, and the protection and sustainable development of grassland ecosystems.

The Car1 Knockout Mice Exhibit Antidepressant-like Behaviors Accompanied with Gut Microbiota Disturbance.

Major depressive disorder (MDD) is a severe mental disorder with largely unknown mechanisms. Carbonic anhydrases convert CO2 to carbonates and protons, playing roles in various brain functions. Carbonic anhydrase 1 (Car1) is particularly abundant and may be linked to microbiota at interstitial sites. We developed Car1-deficient mice to explore the relationship between depression-like behaviors and gut microbiota. Behavioral tests confirmed depression-like behavior in Car1-/- mice. Fecal samples from Car1-/- and WT mice were collected, and 16S rRNA gene sequencing identified distinct microbiota components between the groups. Car1-/- mice exhibited significantly increased immobility in the tail suspension test (TST) compared to WT mice. The gut microbiota composition differed at the phylum level in p_Bacteroidetes, p_Verrucomicrobia, p_Firmicutes, and p_Tenericutes. At the family level, Car1-/- mice had significantly different abundances in eight microbiota groups compared to WT mice. Car1 deficiency is associated with depressive-like behavior and gut microbiota dysbiosis, potentially linked to depressive-like phenotypes.

Plasmodium berghei HMGB1 controls the host immune responses and splenic clearance by regulating the expression of pir genes.

High mobility group box (HMGB) proteins belong to high mobility group (HMG) superfamily of non-histone nuclear proteins that are involved in chromatin remodeling, regulation of gene expression and DNA repair. When extracellular, HMGBs serve as alarmins inducing inflammation and this is attributed to the proinflammatory activity of box B. Here, we show that Plasmodium HMGB1 has key amino acid changes in box B resulting in the loss of TNF-α stimulatory activity. Site-directed mutagenesis of the critical amino acids in box B with respect to mouse HMGB1 renders recombinant Plasmodium berghei (Pb) HMGB1 capable of inducing TNF-α release. Targeted deletion of PbHMGB1 and a detailed in vivo phenotyping show that PbHMGB1 knockout (KO) parasites can undergo asexual stage development. Interestingly, Balb/c mice-infected with PbHMGB1KO parasites display a protective phenotype with subsequent clearance of blood parasitemia, and develop long-lasting protective immunity against the challenges performed with Pb wildtype parasites. The characterization of splenic responses show prominent germinal centres leading to effective humoral responses and enhanced T follicular helper cells. There is also a complete protection from experimental cerebral malaria in CBA/CaJ mice susceptible for cerebral pathogenesis with subsequent parasite clearance. Transcriptomic studies suggest the involvement of PbHMGB1 in pir expression. Our findings highlight the gene regulatory function of parasite HMGB1 and its in vivo significance in modulating the host immune responses. Further, clearance of asexual stages in PbHMGB1KO-infected mice underscores the important role of parasite HMGB1 in host immune evasion. These findings have implications in developing attenuated blood-stage vaccine for malaria.

Construction of an Efficient Agrobacterium tumefaciens-Based Transformation System in the Entomopathogenic fungus Metahizium rileyi.

Metarhizium rileyi is a filamentous entomopathogenic fungus that is highly pathogenic to lepidopteran insects. In our study, we constructed an Agrobacterium tumefaciens-mediated transgene system using the hygromycin resistance gene (Hyg R) as a selection marker in M. rileyi through homologous recombination. Binary knockout vectors for two genes (NOR_03501, longevity assurance gene, and NOR_03153, ATP-binding domain protein domain gene) in the M. rileyi strain SZCY201010 were successfully developed. We compared the genetic transformation efficiency using five kinds of asexual spores. The initial genetic transformation rates using a competent blastospore for NOR_03501 and NOR_03153 were 54.35 and 47.19%, respectively. Subsequently, both genes were successfully knocked out, and the transformed fungi were verified by PCR, RT-qPCR, and green fluorescent protein labeling. The biological phenotypes of the two genes were analyzed. The NOR_03501 gene plays a crucial role in carbon source utilization, stress resistance, and cuticle infection of fungal mycelium growth, while the NOR_03153 gene is significant for conidial production, stress resistance, and body wall infection. This study provides a promising tool for gene manipulation in M. rileyi, enhancing research in functional genomics and the exploration of fungal gene resources.

RNA lipid nanoparticles as efficient in vivo CRISPR-Cas9 gene editing tool for therapeutic target validation in glioblastoma cancer stem cells.

In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ∼80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research.

Deletion of protein kinase C θ attenuates hepatic ischemia/reperfusion injury and further elucidates its mechanism in pathophysiology.

Objectives: Hepatic ischemia-reperfusion (HIR) is a severe process in pathophysiology that occurs clinically in hepatectomy, and hepatic transplantations. The present study aimed to investigate the effect of PKC θ deletion against HIR injury and elucidate its mechanism in pathophysiology. Materials and Methods: HIR injury was induced in wild-type and PKC θ deletion mice treated with or without heme. The ALT and AST levels were determined to evaluate liver function. HIR injury was observed via histological examination. Oxidative stress and inflammatory response markers, and their signaling pathways were detected. Results: The study found that PKC θ knockout decreased serum AST and ALT levels when compared to the WT mice. Furthermore, heme treatment significantly reduced the ALT and AST levels of the PKC θ deletion mice compared with the untreated PKC θ deletion mice. PKC θ deletion markedly elevated superoxide dismutase activity in the liver tissue, reduced malondialdehyde content in the tissue, and the serum TNF-α and IL-6 levels compared with the WT mice. Heme treatment was observed to elevate the activity of SOD and reduced MDA content and serum of TNF-α and IL 6 in the PKC θ deletion animals. Meanwhile, heme treatment increased HO-1 and Nrf 2 protein expression, and reduced the levels of TLR4, phosphorylated NF-κB, and IKB-α. Conclusion: These findings suggested that PKC θ deletion ameliorates HIR, and heme treatment further improves HIR, which is related to regulation of PKC θ deletion on Nrf 2/HO-1 and TLR4/NF-κB/IKB α pathway.

An Additional L451G452N453 in the RpoB Protein Suppressed the Synthetic Lethality in Escherichia coli at 37 Degrees Caused by Depletion of DnaK/J and Trigger Factor.

Escherichia coli depletion of chaperone trigger factor and DnaK/J were not viable at 37°C, but viable below 30°C. Among the engineered E. coli depleted of trigger factor and DnaK/J, one strain Z625, exhibited survival at 37°C, while another strain Z629 only survived below 30°C. Comparative analysis of fatty acid profiles of Z625 and Z629 revealed absence of numerous saturated fatty acids in Z625 as compared to the wild-type E. coli BW25113. In addition, increased unsaturated fatty acids were present in Z625, whereas the fatty acids profile of Z629 closely resembled that of BW25113. Whole genome sequencing revealed a 9-bp insertion in rpoB of Z625. Combined structural analysis of simulated RpoB protein bearing the amino acid sequence L451G452N453 insertion and susceptibility analysis to rifampicin suggested that the insertion did not disturb the individual RpoB structure as beta subunit of RNA polymerase. Comparative transcriptomic analysis of Z625 and Z629 suggested that this insertion impacted transcription of the overall RNA polymerase in Z625, leading to potential repression of some genes whose overexpression was toxic to E. coli. Additionally, Z625 exhibited distinctive metabolic adaptations, likely contributing to its survival at 37°C. In summary, our study elucidated one LGN insertion in rpoB that impacts transcriptional regulation in E. coli, thereby explaining the survival of E. coli depletion of trigger factor and DnaK/J at 37°C, and these founding suggested that some simple mutations in critical genes like rpoB might play an important role in driving adaptive evolution.

Molecular mechanism of a coastal cyanobacterium Synechococcus sp. PCC 7002 adapting to changing phosphate concentrations.

Phosphorus concentration on the surface of seawater varies greatly with different environments, especially in coastal. The molecular mechanism by which cyanobacteria adapt to fluctuating phosphorus bioavailability is still unclear. In this study, transcriptomes and gene knockouts were used to investigate the adaptive molecular mechanism of a model coastal cyanobacterium Synechococcus sp. PCC 7002 during periods of phosphorus starvation and phosphorus recovery (adding sufficient phosphorus after phosphorus starvation). The findings indicated that phosphorus deficiency affected the photosynthesis, ribosome synthesis, and bacterial motility pathways, which recommenced after phosphorus was resupplied. Even more, most of the metabolic pathways of cyanobacteria were enhanced after phosphorus recovery compared to the control which was kept in continuous phosphorus replete conditions. Based on transcriptome, 54 genes potentially related to phosphorus-deficiency adaptation were selected and knocked out individually or in combination. It was found that five mutants showed weak growth phenotype under phosphorus deficiency, indicating the importance of the genes (A0076, A0549-50, A1094, A1320, A1895) in the adaptation of phosphorus deficiency. Three mutants were found to grow better than the wild type under phosphorus deficiency, suggesting that the products of these genes (A0079, A0340, A2284-86) might influence the adaptation to phosphorus deficiency. Bioinformatics analysis revealed that cyanobacteria exposed to highly fluctuating phosphorus concentrations have more sophisticated phosphorus acquisition strategies. These results elucidated that Synechococcus sp. PCC 7002 have variable phosphorus response mechanisms to adapt to fluctuating phosphorus concentration, providing a novel perspective of how cyanobacteria may respond to the complex and dynamic environments. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00244-y.

FDX2, an iron-sulfur cluster assembly factor, is essential to prevent cellular senescence, apoptosis or ferroptosis of ovarian cancer cells.

Recent studies reveal that biosynthesis of iron-sulfur clusters (Fe-Ss) is essential for cell proliferation, including that of cancer cells. Nonetheless, it remains unclear how Fe-S biosynthesis functions in cell proliferation/survival. Here, we report that proper Fe-S biosynthesis is essential to prevent cellular senescence, apoptosis, or ferroptosis, depending on cell context. To assess these outcomes in cancer, we developed an ovarian cancer line with conditional KO of FDX2, a component of the core Fe-S assembly complex. FDX2 loss induced global downregulation of Fe-S-containing proteins and Fe2+ overload, resulting in DNA damage and p53 pathway activation, and driving the senescence program. p53 deficiency augmented DNA damage responses upon FDX2 loss, resulting in apoptosis rather than senescence. FDX2 loss also sensitized cells to ferroptosis, as evidenced by compromised redox homeostasis of membrane phospholipids. Our results suggest that p53 status and phospholipid homeostatic activity are critical determinants of diverse biological outcomes of Fe-S deficiency in cancer cells.

Proteomic Analysis of Rap1A GTPase Signaling-Deficient C57BL/6 Mouse Pancreas and Functional Studies Identify an Essential Role of Rap1A in Pancreas Physiology.

Ras-related Rap1A GTPase is implicated in pancreas ß-cell insulin secretion and is stimulated by the cAMP sensor Epac2, a guanine exchange factor and activator of Rap1 GTPase. In this study, we examined the differential proteomic profiles of pancreata from C57BL/6 Rap1A-deficient (Null) and control wild-type (WT) mice with nanoLC-ESI-MS/MS to assess targets of Rap1A potentially involved in insulin regulation. We identified 77 overlapping identifier proteins in both groups, with 8 distinct identifier proteins in Null versus 56 distinct identifier proteins in WT mice pancreata. Functional enrichment analysis showed four of the eight Null unique proteins, ERO1-like protein ß (Ero1lß), triosephosphate isomerase (TP1), 14-3-3 protein γ, and kallikrein-1, were exclusively involved in insulin biogenesis, with roles in insulin metabolism. Specifically, the mRNA expression of Ero1lß and TP1 was significantly (p < 0.05) increased in Null versus WT pancreata. Rap1A deficiency significantly affected glucose tolerance during the first 15-30 min of glucose challenge but showed no impact on insulin sensitivity. Ex vivo glucose-stimulated insulin secretion (GSIS) studies on isolated Null islets showed significantly impaired GSIS. Furthermore, in GSIS-impaired islets, the cAMP-Epac2-Rap1A pathway was significantly compromised compared to the WT. Altogether, these studies underscore an essential role of Rap1A GTPase in pancreas physiological function.

Eighteen genes primarily expressed in the testis are not required for male fertility in mice.

There are approximately 20,000 protein-coding genes in humans and mice. More than 1000 of these genes are predominantly expressed in the testis or are testis-specific and thought to play an important role in male reproduction. Through the production of gene knockout mouse models and phenotypic evaluations, many genes essential for spermatogenesis, sperm maturation, and fertilization have been discovered, greatly contributing to the elucidation of their molecular mechanisms. On the other hand, there are many cases in which single-gene knockout models do not affect fertility, indicating that tissue-specific genes are not always critical. Here, we selected 18 genes whose mRNA expression is restricted to the testis or higher than in other tissues, but whose function in male reproduction is unknown. We then created single-gene KO mouse models using the CRISPR/Cas9 system. The established KO males were subjected to mating tests and screened for effects on fecundity, revealing that these genes were not essential for spermatogenesis and male fertility. This knowledge will contribute to understanding the functions of genes characteristic of the testis and identify the cause of male infertility.

Optimization of a DiCre recombinase system with reduced leakage for conditional genome editing of Cryptosporidium.

BACKGROUND: The dimerizable Cre recombinase system (DiCre) exhibits increased leaky activity in Cryptosporidium, leading to unintended gene editing in the absence of induction. Therefore, optimization of the current DiCre technique is necessary for functional studies of essential Cryptosporidium genes. METHODS: Based on the results of transcriptomic analysis of Cryptosporidium parvum stages, seven promoters with different transcriptional capabilities were screened to drive the expression of Cre fragments (FKBP-Cre59 and FRB-Cre60). Transient transfection was performed to assess the effect of promoter strength on leakage activity. In vitro and in vivo experiments were performed to evaluate the leaky activity and cleavage efficiency of the optimized DiCre system by polymerase chain reaction (PCR), nanoluciferase, and fluorescence analyses. RESULTS: The use of promoters with lower transcriptional activity, such as pcgd6_4110 and pcgd3_260, as opposed to strong promoters such as pActin, pα-Tubulin, and pEnolase, reduced the leakage rate of the system from 35-75% to nearly undetectable levels, as verified by transient transfection. Subsequent in vitro and in vivo experiments using stable lines further demonstrated that the optimized DiCre system had no detectable leaky activity. The system achieved 71% cleavage efficiency in vitro. In mice, a single dose of the inducer resulted in a 10% conditional gene knockout and fluorescent protein expression in oocysts. These fluorescently tagged transgenic oocysts could be enriched by flow sorting for further infection studies. CONCLUSIONS: A DiCre conditional gene knockout system for Cryptosporidium with good cleavage efficiency and reduced leaky activity has been successfully established.

RAB37-mediated autophagy guards ovarian homeostasis and function.

Loss of ovarian homeostasis is associated with ovary dysfunction and female diseases; however, the underlying mechanisms responsible for the establishment of homeostasis and its function in the ovary have not been fully elucidated. Here, we showed that conditional knockout of Rab37 in oocytes impaired macroautophagy/autophagy proficiency in the ovary and interfered with follicular homeostasis and ovary development in mice. Flunarizine treatment upregulated autophagy, thus rescuing the impairment of follicular homeostasis and ovarian dysfunction in rab37 knockout mice by reprogramming of homeostasis. Notably, both the E2F1 and EGR2 transcription factors synergistically activated Rab37 transcription and promoted autophagy. Thus, RAB37-mediated autophagy ensures ovary function by maintaining ovarian homeostasis.Abbreviations: AMH: anti-Mullerian hormone; ATG: autophagy related; BECN1: beclin 1; cKO: conditional knockout; Cre: cyclization recombination enzyme; dpp: days postpartum; E2: estradiol; E2F1: E2F transcription factor 1; EBF1: EBF transcription factor 1; EGR2: early growth response 2; FSH: follicle stimulating hormone; LH: luteinizing hormone; mpp: months postpartum; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; RAB37: RAB37, member RAS oncogene family; SQSTM1: sequestosome 1; TFEB: transcription factor EB; Zp3: zona pellucida glycoprotein 3.

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.