Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells.

Genome editing has become an important aspect of Chinese hamster ovary (CHO) cell line engineering for improving the production of recombinant protein therapeutics. Currently, the engineering focus is directed toward expanding product diversity while controlling and improving product quality and yields. In this chapter, we present our protocol for using the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knock out engineering target genes in CHO cells. As an example, we describe how to knock out the glutamine synthetase (GS) gene, which increases the selection efficiency of the GS-mediated gene amplification system.

CRISPR-Mediated Library Screening of Gene-Knockout Cell Lines for Investigating Antiviral Innate Immunity.

The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.

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.

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.

Renin-angiotensin blockade ameliorates the progression of glomerular injury in podocyte-specific Ctcf knockout mice.

AIM: Several studies have shown that the progression of proteinuria and renal tissue injury is associated with activation of the intrarenal renin-angiotensin system (RAS). CCCTC-binding factor (CTCF) is a DNA-binding factor that plays an essential role in the regulation of gene expression. In the present study, we aimed to investigate the phenotypic effects of CTCF deficiency in podocytes. METHODS: Angiotensin II type 1 receptor blockers (ARBs) were administered to the podocyte-specific Ctcf knockout mice, and histological and biochemical analyzes were performed. We also investigated the changes in the expression of podocin in podocyte cell cultures with or without stimulation with angiotensin II from glomeruli isolated using magnetic beads from podocyte-specific Ctcf knockout mice. RESULTS: Mice in which Ctcf was deleted from podocytes developed glomerulopathy and mice developed severe progressive proteinuria, and impaired renal function. Moreover, ARBs suppressed the development of glomerulopathy in podocyte-specific Ctcf knockout mice. Both real-time polymerase chain reaction and western blotting showed that podocin expression was decreased in cell cultures stimulated with angiotensin II. Furthermore, RAS components gene expressions in podocyte cell cultures isolated from podocyte-specific Ctcf knockout mice were significantly increased. CONCLUSION: These results suggest that RAS is involved in the development of glomerulopathy in podocyte-specific Ctcf knockout mice. Elucidation of the pathophysiology of podocyte-specific Ctcf knockout mice may provide new insights into the relationship between podocyte injury and chronic glomerulonephritis.

Investigating the effects of a cryptic splice site in the En2 splice acceptor sequence used in the IKMC knockout-first alleles.

Targeted mouse mutants are a common tool used to investigate gene function. The International Knockout Mouse Consortium undertook a large-scale screen of mouse mutants, making use of the knockout-first allele design that contains the En2 splice acceptor sequence coupled to the lacZ reporter gene. Although the knockout-first allele was designed to interfere with splicing and thus disrupt gene function, the En2 sequence has been reported to be transcribed within the host gene mRNA due to a cryptic splice site within the En2 sequence which allows splicing to the next exon of the host gene. In some circumstances, this has the potential to permit translation of a mutant protein. Here, we describe our computational analysis of all the mouse protein-coding genes with established knockout-first embryonic stem cell lines, and our predictions of their transcription outcome should the En2 sequence be included. As part of the large-scale mutagenesis program, mutant mice underwent a broad phenotyping screen, and their phenotypes are available. No wide-scale effects on mouse phenotypes reported were found as a result of the predicted En2 insertion. However, the En2 insertion was found experimentally in the transcripts of 24 of 35 mutant alleles examined, including the five already described, two with evidence of readthrough. Splicing from the cryptic splice site also has the potential to disrupt expression of the lacZ reporter gene. It is recommended that mutant transcripts be checked for this insertion as well as for leaky transcription in studies involving knockout-first alleles.

Low dose methotrexate impaired T cell transmigration through down-regulating CXCR4 expression in rheumatoid arthritis (RA).

BACKGROUND: CXC chemokine CXCL12 is involved in the pathological development of rheumatoid arthritis (RA) through abnormal migration of peripheral immune cells in the joint. Although low dose methotrexate (MTX) is clinically used to treat RA patients, CXCL12 signaling responses to MTX-mediated treatments is still not well understood. METHODS: In this study, we examined the expression of CXCR4 (cognatic receptor for CXCL12) in peripheral T cells from RA patients and arthritis mice models received from low dose MTX therapies. The effects of low dose MTX on CXCR4 were further determined via both in vitro CD3+ T cells and Cxcr4 conditional knockout (CKO) arthritis mice models. RESULTS: Our clinical data shows that low dose MTX treatment was clinically associated with down-regulated expression of chemokine receptor CXCR4 on patient peripheral T cells. In vitro, low dose MTX significantly decreased cell transmigration through down-regulated CXCR4's expression in CD3+ T cells. Consistently, CD3+ T cells treated with low dose MTX demonstrated an increased genomic hypermethylation across the promoter region of Cxcr4 gene. Furthermore, our preclinical studies showed that low dose MTX-mediated downregulation of CXCR4 significantly improved the pathological development in mouse arthritis models. Conditional disruption of the Cxcr4 gene in peripheral immune cells potentially alleviated inflammation of joints and lung tissue in the arthritis mice, though genetic modification itself overall did not change their clinical scores of arthritis, except for a significant improvement on day 45 in CXCR4 CKO arthritis mice models during the recovery phase. CONCLUSION: Our findings suggest that the effect of low dose MTX treatment could serve to eliminate inflammation in RA patients through impairment of immune cell transmigration mediated by CXCR4.

Deletion of Mgat2 in spermatogonia blocks spermatogenesis.

Identifying factors required for spermatogenesis is important for understanding mechanisms of male fertility. Inactivation of either the Mgat1 or Man2a2 gene leads to a block in spermatogenesis causing infertility in male mice. MGAT1 GlcNAc-transferase initiates complex N-glycan synthesis and MAN2A2 mannosidase generates the substrate for MGAT2 GlcNAc-transferase to form a biantennary complex N-glycan. In this paper, we show that conditional deletion of Mgat2 in spermatogonia via Stra8-iCre caused a novel block in spermatogenesis, largely prior to the formation of round spermatids. Mgat2[-/-] germ cells did not bind the lectins Phaseolus vulgaris leucoagglutinin (L-PHA) or Griffonia simplicifolia II (GSA-II), similar to germ cells lacking MGAT1 and complex N-glycans. However, overall spermatogenic defects were distinct in germ cells with deleted Mgat2 versus Mgat1. In addition, RNA-seq analysis at 15 days after birth revealed a unique transcriptomic landscape in Mgat2[-/-] germ cells with genes required for sperm formation and functions being most downregulated. Bioinformatic analyses using the ingenuity pathway analysis (IPA) algorithm identified ERK and AKT as central activities. Western blot analyses of 15-day germ cell lysates confirmed that both AKT and ERK1/2 signaling were increased by loss of MGAT2 in germ cells. By contrast, Mgat1[-/-] germ cells were previously shown to have reduced ERK signaling and unchanged AKT activity. Therefore, since the loss of all complex N-glycans is common to each mutant model, the different immature N-glycans that accumulate in Mgat2[-/-] versus Mgat1[-/-] germ cells are proposed to be the basis of their unique spermatogenic phenotypes.

ASGR1 Deficiency Inhibits Atherosclerosis in Western Diet-Fed ApoE-/- Mice by Regulating Lipoprotein Metabolism and Promoting Cholesterol Efflux.

BACKGROUND: Atherosclerosis is the most common cause of cardiovascular diseases. Clinical studies indicate that loss-of-function ASGR1 (asialoglycoprotein receptor 1) is significantly associated with lower plasma cholesterol levels and reduces cardiovascular disease risk. However, the effect of ASGR1 on atherosclerosis remains incompletely understood; whether inhibition of ASGR1 causes liver injury remains controversial. Here, we comprehensively investigated the effects and the underlying molecular mechanisms of ASGR1 deficiency and overexpression on atherosclerosis and liver injury in mice. METHODS: We engineered Asgr1 knockout mice (Asgr1-/-), Asgr1 and ApoE double-knockout mice (Asgr1-/-ApoE-/-), and ASGR1-overexpressing mice on an ApoE-/- background and then fed them different diets to assess the role of ASGR1 in atherosclerosis and liver injury. RESULTS: After being fed a Western diet for 12 weeks, Asgr1-/-ApoE-/- mice exhibited significantly decreased atherosclerotic lesion areas in the aorta and aortic root sections, reduced plasma VLDL (very-low-density lipoprotein) cholesterol and LDL (low-density lipoprotein) cholesterol levels, decreased VLDL production, and increased fecal cholesterol contents. Conversely, ASGR1 overexpression in ApoE-/- mice increased atherosclerotic lesions in the aorta and aortic root sections, augmented plasma VLDL cholesterol and LDL cholesterol levels and VLDL production, and decreased fecal cholesterol contents. Mechanistically, ASGR1 deficiency reduced VLDL production by inhibiting the expression of MTTP (microsomal triglyceride transfer protein) and ANGPTL3 (angiopoietin-like protein 3)/ANGPTL8 (angiopoietin-like protein 8) but increasing LPL (lipoprotein lipase) activity, increased LDL uptake by increasing LDLR (LDL receptor) expression, and promoted cholesterol efflux through increasing expression of LXRα (liver X receptor-α), ABCA1 (ATP-binding cassette subfamily A member 1), ABCG5 (ATP-binding cassette subfamily G member 5), and CYP7A1 (cytochrome P450 family 7 subfamily A member 1). These underlying alterations were confirmed in ASGR1-overexpressing ApoE-/- mice. In addition, ASGR1 deficiency exacerbated liver injury in Western diet-induced Asgr1-/-ApoE-/- mice and high-fat diet-induced but not normal laboratory diet-induced and high-fat and high-cholesterol diet-induced Asgr1-/- mice, while its overexpression mitigated liver injury in Western diet-induced ASGR1-overexpressing ApoE-/- mice. CONCLUSIONS: Inhibition of ASGR1 inhibits atherosclerosis in Western diet-fed ApoE-/- mice, suggesting that inhibiting ASGR1 may serve as a novel therapeutic strategy to treat atherosclerosis and cardiovascular diseases.

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.

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.

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.

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.

The role of cerebellar FOXP1 in the development of motor and communicative behaviors in mice.

The gene FOXP2 is well established for a role in human speech and language; far less is known about FOXP1. However, this related gene has also been implicated in human language development as well as disorders associated with features of autism spectrum disorder (ASD). FOXP1 protein expression has also recently been identified in the cerebellum-a neural structure previously shown to express FOXP2 protein. The current study sought to elucidate the behavioral implications of a conditional knock-out of Foxp1 using an En1-Cre driver, which is active in the entirety of the cerebellum and a subset of neurons in the midbrain and spinal cord, in mice using a test battery including motor tasks associated with cerebellar dysfunction, as well as communicative and autistic-relevant behaviors. Male and female mice with a conditional knock-out (cKO, n = 31) and wildtype littermate controls (WT, n = 34) were assessed for gross and orofacial motor control, motor-coordination learning, locomotion, social behavior, anxiety, auditory processing and expressive vocalizations. Overall results suggest Foxp1 plays a specific role in the development of communicative systems, and phenotypic expression of disruptions may interact with sex. Robust motor deficits associated with Foxp1 protein loss may particularly affect vocalizations based on significant orofacial motor deficits in cKO subjects could also contribute to vocalization anomalies. In summary, the current study provides key insights into the role of Foxp1 in cerebellar function and associated behaviors in mice, with implications for an improved understanding of communicative and motor-based neurodevelopmental disabilities in humans.

A Multiple Targeting Genome Editing System for Remodulation of Circulating Malignant Cells to Eliminate Cancer Immunosuppression and Restore Immune Responses.

Cancer immunotherapy, which aims to eliminate cancer immunosuppression and reactivate anticancer immunity, holds great promise in oncology treatments. However, it is challenging to accurately study the efficacy of immunotherapy based on human-derived cells through animal experiments due to xenogeneic immune rejection. Herein, a personalized and precise strategy to evaluate the effectiveness of immunotherapy using the blood samples of cancer patients is presented. Through the utilization of multiple cancer-targeting delivery system decorated with the epidermal growth factor receptor (EGFR)-specific aptamer CL4 and the AXL-specific aptamer GL21.T to achieve superior efficiency in delivering the genome editing plasmid for MUC1 knockout, effective modulation on the behavior of circulating malignant cells (CMCs) is realized. After genome editing, both mucin 1 (MUC1) and programmed death-ligand 1 (PD-L1) are significantly downregulated in CMCs. The elimination of immunosuppression results in markedly enhanced secretion of pro-inflammatory anticancer cytokines encompassing interleukins 2, 12, and 15 and interferon-γ by immune cells. The study not only provides a strategy to overcome immunosuppression but also yields critical insights for personalized immunotherapy approaches.

The Application of Duck Embryonic Fibroblasts CCL-141 as a Cell Model for Adipogenesis.

The duck embryo fibroblast cell line CCL-141, which is currently the only commercialized duck cell line, has been underexplored in adipogenesis research. (1) Background: This study establishes an experimental protocol to induce adipogenesis in CCL-141 cells, addressing the importance of understanding gene functions in this process. (2) Methods: Chicken serum, fatty acids, insulin, and all-trans retinoic acid were used to treat CCL-141 cells, with adipogenesis confirmed by Oil Red O staining and gene expression quantification. CRISPR/Cas9 technology was applied to knockout PPARγ, and the resulting adipogenic phenotype was assessed. (3) Results: The treatments promoted adipogenesis, and the knockout of PPARγ validated the cell line's utility for gene function studies. (4) Conclusions: CCL-141 cells are a suitable model for investigating duck adipogenesis, contributing to the understanding of regulatory factors in this biological process.

The Effects of swnH1 Gene Function of Endophytic Fungus Alternaria oxytropis OW 7.8 on Its Swainsonine Biosynthesis.

The swnH1 gene in the endophytic fungus Alternaria oxytropis OW 7.8 isolated from Oxytropis glabra was identified, and the gene knockout mutant ΔswnH1 was first constructed in this study. Compared with A. oxytropis OW 7.8, the ΔswnH1 mutant exhibited altered colony and mycelium morphology, slower growth rate, and no swainsonine (SW) in mycelia, indicating that the function of the swnH1 gene promoted SW biosynthesis. Five differential expressed genes (DEGs) closely associated with SW synthesis were identified by transcriptomic analysis of A. oxytropis OW 7.8 and ΔswnH1, with sac, swnR, swnK, swnN, and swnH2 down-regulating. Six differential metabolites (DEMs) closely associated with SW synthesis were identified by metabolomic analysis, with P450, PKS-NRPS, saccharopine, lipopolysaccharide kinase, L-PA, α-aminoadipic, and L-stachydrine down-regulated, while L-proline was up-regulated. The SW biosynthetic pathways in A. oxytropis OW 7.8 were predicted and refined. The results lay the foundation for in-depth exploration of the molecular mechanisms and metabolic pathways of SW synthesis in fungi and provide reference for future control of SW in locoweeds, which would benefit the development of animal husbandry and the sustainable use of grassland ecosystems.

Impact of the adenosine receptor A2BR expressed on myeloid cells on immune regulation during pregnancy.

During pregnancy, the maternal immune system must carefully balance protection against pathogens with tolerance toward the semiallogeneic fetus. Dysfunctions of the immune system can lead to severe complications such as preeclampsia, fetal growth restriction, or pregnancy loss. Adenosine plays a role in physiological processes and plasma-level increase during pregnancy. The adenosine receptor A2B (A2BR), which is expressed on both, immune and nonimmune cells, is activated by high adenosine concentrations, achieved during pregnancy. We investigated the impact of A2BR expressed on myeloid cells on immune regulation during pregnancy using a mouse model with myeloid deficiency for A2BR. We demonstrate systemic changes in myeloid and lymphoid cell populations during pregnancy in A2BR-KO (Adora2B923f/f-LysMCre) mice with increased monocytes, neutrophils, and T cells but decreased B cells as well as altered T-cell subpopulations with decreased Th1 cells and Tregs and increased Th17 cells. Lack of A2BR on myeloid cells caused an increased systemic expression of IL-6 but decreased systemic accumulation and function of MDSC and reduced numbers of uterine natural killer cells. The pregnancy outcome was only marginally affected. Our results demonstrate that A2BR on myeloid cells plays a role in immune regulation during pregnancy, but the clinical impact on pregnancy remains unclear.

Roles of Three FgPel Genes in the Development and Pathogenicity Regulation of Fusarium graminearum.

Fusarium head blight (FHB) is a devastating fungal disease caused by Fusarium graminearum. Pectin lyase, a pectinase, acts on the α-1,4-glycosidic linkage of galacturonic acid primarily by ß-elimination. In this study, three pectin lyase genes (FgPel1, 2, 3) in F. graminearum were selected, and deletion mutants (ΔFgPel1, 2, 3) were constructed by homologous recombination for functional characterization. The gene deletions affected the morphology and growth rate of F. graminearum on pectin medium at various concentrations, with the growth rate of ΔFgPel1 being more significant. The growth of ΔFgPel1 colonies slowed at pH 4, with optimal growth at pH 6.5, whereas ΔFgPel2 and ΔFgPel3 exhibited greater inhibition at pH 8. Colony morphology and diameter of the deletion mutants showed no significant differences compared to the wild-type strain PH-1, and there was no effect on conidial production or germination rate. Pathogenicity assays demonstrated that gene deletion significantly reduced the ability of F. graminearum to infest corn silks and wheat ears, and that ΔFgPel2 showed a more pronounced reduction in pathogenicity on wheat spikes. In summary, the pectin lyase genes (FgPel1, 2, 3) are involved in pectin utilization and are influenced by external pH conditions, which attenuate the pathogenicity of F. graminearum without affecting its vegetative growth or asexual spore formation. These findings elucidate the roles of these genes and provide a basis for controlling FHB.

The Ubiquitous Wilt-Inducing Pathogen Fusarium oxysporum-A Review of Genes Studied with Mutant Analysis.

Fusarium oxysporum is one of the most economically important plant fungal pathogens, causing devastating Fusarium wilt diseases on a diverse range of hosts, including many key crop plants. Consequently, F. oxysporum has been the subject of extensive research to help develop and improve crop protection strategies. The sequencing of the F. oxysporum genome 14 years ago has greatly accelerated the discovery and characterization of key genes contributing to F. oxysporum biology and virulence. In this review, we summarize important findings on the molecular mechanisms of F. oxysporum growth, reproduction, and virulence. In particular, we focus on genes studied through mutant analysis, covering genes involved in diverse processes such as metabolism, stress tolerance, sporulation, and pathogenicity, as well as the signaling pathways that regulate them. In doing so, we hope to present a comprehensive review of the molecular understanding of F. oxysporum that will aid the future study of this and related species.