Double Deletion Research Articles

Optimized psilocybin production in tryptophan catabolism-repressed fungi.

The high therapeutic potential of psilocybin, a prodrug of the psychotropic psilocin, holds great promise for the treatment of mental disorders such as therapy-refractory depression, alcohol use disorder and anorexia nervosa. Psilocybin has been designated a 'Breakthrough Therapy' by the US Food and Drug Administration, and therefore a sustainable production process must be established to meet future market demands. Here, we present the development of an invivo psilocybin production chassis based on repression of l-tryptophan catabolism. We demonstrate the proof of principle in Saccharomyces cerevisiae expressing the psilocybin biosynthetic genes. Deletion of the two aminotransferase genes ARO8/9 and the indoleamine 2,3-dioxygenase gene BNA2 yielded a fivefold increase of psilocybin titre. We transferred this knowledge to the filamentous fungus Aspergillus nidulans and identified functional ARO8/9 orthologs involved in fungal l-tryptophan catabolism by genome mining and cross-complementation. The double deletion mutant of A. nidulans resulted in a 10-fold increased psilocybin production. Process optimization based on respiratory activity measurements led to a final psilocybin titre of 267 mg/L in batch cultures with a space-time-yield of 3.7 mg/L/h. These results demonstrate the suitability of our engineered A. nidulans to serve as a production strain for psilocybin and other tryptamine-derived pharmaceuticals.

Sarbecovirus RBD indels and specific residues dictating multi-species ACE2 adaptiveness

Our comprehensive understanding of the multi-species ACE2 adaptiveness of sarbecoviruses remains elusive, particularly for those with various receptor binding motif (RBM) insertions/deletions (indels). Here, we analyzed RBM sequences from 268 sarbecoviruses categorized into four RBM indel types. We examined the ability of 20 representative sarbecovirus Spike glycoproteins (S) and derivatives in utilizing ACE2 from various bats and several other mammalian species. We reveal that sarbecoviruses with long RBMs (type-I) can achieve broad ACE2 tropism, whereas viruses with single deletions in Region 1 (type-II) or Region 2 (type-III) exhibit narrower ACE2 tropism. Sarbecoviruses with double region deletions (type-IV) completely lost ACE2 usage, which is restricted by clade-specific residues within and outside RBM. Lastly, we propose the evolution of sarbecovirus RBM indels and illustrate how loop lengths, disulfide, and residue determinants shape multi-species ACE2 adaptiveness. This study provides profound insights into the mechanisms governing ACE2 usage and spillover risks of sarbecoviruses.

NaV1.8/NaV1.9 double deletion mildly affects acute pain responses in mice.

The 2 tetrodotoxin-resistant (TTXr) voltage-gated sodium channel subtypes NaV1.8 and NaV1.9 are important for peripheral pain signaling. As determinants of sensory neuron excitability, they are essential for the initial transduction of sensory stimuli, the electrogenesis of the action potential, and the release of neurotransmitters from sensory neuron terminals. NaV1.8 and NaV1.9, which are encoded by SCN10A and SCN11A, respectively, are predominantly expressed in pain-sensitive (nociceptive) neurons localized in the dorsal root ganglia (DRG) along the spinal cord and in the trigeminal ganglia. Mutations in these genes cause various pain disorders in humans. Gain-of-function missense variants in SCN10A result in small fiber neuropathy, while distinct SCN11A mutations cause, i. a., congenital insensitivity to pain, episodic pain, painful neuropathy, and cold-induced pain. To determine the impact of loss-of-function of both channels, we generated NaV1.8/NaV1.9 double knockout (DKO) mice using clustered regularly interspaced short palindromic repeats/Cas-mediated gene editing to achieve simultaneous gene disruption. Successful knockout of both channels was verified by whole-cell recordings demonstrating the absence of NaV1.8- and NaV1.9-mediated Na+ currents in NaV1.8/NaV1.9 DKO DRG neurons. Global RNA sequencing identified significant deregulation of C-LTMR marker genes as well as of pain-modulating neuropeptides in NaV1.8/NaV1.9 DKO DRG neurons, which fits to the overall only moderately impaired acute pain behavior observed in DKO mice. Besides addressing the function of both sodium channels in pain perception, we further demonstrate that the null-background is a very valuable tool for investigations on the functional properties of individual human disease-causing variants in NaV1.8 or NaV1.9 in their native physiological environment.

Sexually dimorphic renal expression of mouse Klotho is directed by a kidney-specific distal enhancer responsive to HNF1b

Transcription enhancers are genomic sequences regulating common and tissue-specific genes and their disruption can contribute to human disease development and progression. Klotho, a sexually dimorphic gene specifically expressed in kidney, is well-linked to kidney dysfunction and its deletion from the mouse genome leads to premature aging and death. However, the sexually dimorphic regulation of Klotho is not understood. Here, we characterize two candidate Klotho enhancers using H3K27ac epigenetic marks and transcription factor binding and investigate their functions, individually and combined, through CRISPR-Cas9 genome engineering. We discovered that only the distal (E1), but not the proximal (E2) candidate region constitutes a functional enhancer, with the double deletion not causing Klotho expression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism of Klotho as evidenced by qPCR and RNA-seq. Despite the sharp reduction of Klotho mRNA, unlike germline Klotho knockouts, mutant mice present normal phenotype, including weight, lifespan, and serum biochemistry. Lastly, only males lacking E1 display more prominent acute, but not chronic kidney injury responses, indicating a remarkable range of potential adaptation to isolated Klotho loss, especially in female E1 knockouts, retaining renoprotection despite over 80% Klotho reduction.

121 Copy number variation: From mapping to variance components in Holsteins

Abstract Copy number variations (CNVs) modify transcription levels mainly through changing genes and regulatory elements dosages. Hence, CNVs can contribute to the additive genetic variance of quantitative traits, which might not be fully captured by SNPs. Here, CNVs were mapped from 3,601 Holsteins and their contribution to complex traits was explored by i) quantitative trait loci (QTL) enrichment analysis, and ii) variance component analysis for metritis (1 = disease, 0 = no disease). The 4,113 CNVs derived from high-density (777k) genotypic information were compiled into 1,184 CNV regions (CNVRs) overlapping 7,832 QTLs reported in dairy cattle and available in the AnimalQTLdb. Fisher’s exact test was applied to perform the QTL enrichment analysis followed by False Discovery Rate (FDR) correction. Then, QTLs overlapping CNVRs were enriched for traits when the corrected P-value (PFDR) was lower than 0.05. For variance components estimation, genomic information from all CNVs loci was used to build the CNV-derived genomic relationship matrix (CNV_GRM). CNV loci showing double deletions, single deletions, and normal state in the population were coded as 0, 1, and 2, respectively, whereas CNV loci presenting normal state, single duplications, and double duplications were coded as 0, 1, and 2, respectively. Given that some CNVs presented both deletions and duplications at the same position in the population, these mixed-CNVs were treated as two distinct CNVs loci, one to represent deletions and the other to represent duplications. The variance components for metritis were estimated by fitting either only a SNP-derived GRM (SNP_GRM) or jointly SNP_GRM and CNV_GRM considering 3,272 cows in models that included the female category and farm-year-season as fixed effects. QTLs overlapped with CNVRs were enriched for milk (e.g., milk fat yield, milk yield, and milk protein percentage), reproduction (e.g., calving ease, non-return rate, and heat intensity), exterior (e.g., teat length, udder depth, and udder height), production (e.g., length of productive life, lifetime profit index, and net merit), and health traits (e.g., immune globulin G level, somatic cell, and bovine respiratory susceptibility). Regarding variance components, copy number variations were able to capture a fraction of the additive genetic variance not captured by SNPs alone, increasing the heritability estimate for metritis. In summary, our findings suggest that CNVs overlap QTLs associated with economically important traits more than only by chance and CNVs can capture additional additive genetic variance not fully captured by SNPs alone.

The Myosin-V Myo51 and Alpha-Actinin Ain1p Cooperate during Contractile Ring Assembly and Disassembly in Fission Yeast Cytokinesis.

Cytokinesis is driven in part by the constriction of a ring of actin filaments, myosin motors and other proteins. In fission yeast, three myosins contribute to cytokinesis including a Myosin-V Myo51. As Myosin-Vs typically carry cargo along actin filaments, the role of Myo51 in cytokinesis remains unclear. The previous work suggests that Myo51 may crosslink actin filaments. We hypothesized that if Myo51 crosslinks actin filaments, cells carrying double deletions of ain1, which encodes the crosslinker alpha-actinin, and myo51 (∆ain1 ∆myo51 cells) will exhibit more severe cytokinesis phenotypes than cells with the single ∆ain1 mutation. Contrary to our expectations, we found that the loss of Myo51 in ∆ain1 cells partially rescued the severity of the node clumping phenotype measured in ∆ain1 cells. Furthermore, we describe a normal process of contractile ring "shedding", the appearance of fragments of ring material extending away from the contractile ring along the ingressing septum that occurs in the second half of constriction. We measured that ∆ain1 ∆myo51 cells exhibit premature and exaggerated shedding. Our work suggests that Myo51 is not a simple actin filament crosslinker. Instead, a role in effective node motion better recapitulates its function during ring assembly and disassembly.

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.

Novel Pannexin 1 isoform is increased in cancer.

Pannexin 1 (PANX1) is upregulated in many cancers, where its activity and signalling promote tumorigenic properties. Here, we report a novel ∼25 kDa isoform of human PANX1 (hPANX1-25K) which lacks the N-terminus and was detected in several human cancer cell lines including melanoma, osteosarcoma, breast cancer and glioblastoma multiforme. This isoform was increased upon hPANX1 CRISPR/Cas9 deletion targeting the first exon near M1, suggesting a potential alternative translation initiation (ATI) site. hPANX1-25K was confirmed to be a hPANX1 isoform via mass spectrometry, can be N-linked glycosylated at N254, and can interact with both β-catenin and full length hPANX1. A double deletion of hPANX1 and hPANX1-25K reduces cell growth and viability in cancer cells. hPANX1-25K is prevalent throughout melanoma progression, and its levels are increased in squamous cell carcinoma cells and patient-derived tumours, compared to keratinocytes and normal skin, indicating that it may be differentially regulated in normal and cancer cells.

Evaluating cellular roles and phenotypes associated with trehalose degradation genes in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, 2 types of trehalase activities have been described. Neutral trehalases (Nth1 and Nth2) are considered to be the main proteins that catalyze intracellular trehalose mobilization. In addition to Nth1 and Nth2, studies have shown that acid trehalase Ath1 is required for extracellular trehalose degradation. Although both neutral and acid-type trehalases have been predominantly investigated in laboratory strains of S. cerevisiae, we sought to examine the phenotypic consequences of disrupting these genes in wild strains. In this study, we constructed mutants of the trehalose degradation pathway (NTH1, NTH2, and ATH1) in 5 diverse S. cerevisiae strains to examine whether published lab strain phenotypes are also exhibited by wild strains. For each mutant, we assessed a number of phenotypes for comparison to trehalose biosynthesis mutants, including trehalose production, glycogen production, cell size, acute thermotolerance, high-temperature growth, sporulation efficiency, and growth on a variety of carbon sources in rich and minimal medium. We found that all trehalase mutants including single deletion nth1Δ, nth2Δ, and ath1Δ, as well as double deletion nth1nth2Δ, accumulated higher intracellular trehalose levels compared to their isogenic wild-type cells. Also, nth1Δ and nth1Δnth2Δ mutants exhibited mild thermal sensitivity, suggesting a potential minor role for trehalose mobilization when cells recover from stress. In addition, we evaluated phenotypes more directly relevant to trehalose degradation, including both extracellular and intracellular trehalose utilization. We discovered that intracellular trehalose hydrolysis is critical for typical spore germination progression, highlighting a role for trehalose in cell cycle regulation, likely as a storage carbohydrate providing glycolytic fuel. Additionally, our work provides further evidence suggesting Ath1 is indispensable for extracellular trehalose utilization as a carbon source, even in the presence of AGT1.

Sex-Specific Roles of Hypocretin Receptor Signaling in CRF Neurons on Alcohol Drinking, Anxiety, and BNST Neuronal Excitability.

Alcohol use disorder (AUD) is characterized by compulsive alcohol consumption and negative emotional states during withdrawal, often perpetuating a cycle of addiction through arousal dysfunction. The hypocretin/orexin (Hcrt) neuropeptide system, a key regulator of arousal, has been implicated in these processes, particularly in its interactions with corticotropin-releasing factor (CRF) neurons within the bed nucleus of the stria terminalis (BNST). We investigated the role of Hcrt receptor signaling in CRF neurons in modulating alcohol intake, anxiety behaviors, and BNST excitability, with a focus on sex-specific differences. Using CRF-specific genetic deletion of HcrtR1 and/or HcrtR2 receptors in mice, we found that deletion of HcrtR1 significantly reduced alcohol intake, with sex-specific effects on BNST excitability. CRF-specific HcrtR2 deletion, while not affecting alcohol consumption, decreased baseline anxiety-like behaviors in males relative to females. Moreover, the double deletion of both Hcrt receptors from CRF neurons led to reduced alcohol drinking in males and dampened anxiety behaviors and BNST excitability in both sexes during protracted withdrawal. These findings suggest that Hcrt signaling in CRF neurons plays a critical role in the persistence of excessive alcohol consumption and the development of negative affective states, with distinct contributions from HcrtR1 and HcrtR2. The observed sex-specific differences underscore the need for tailored therapeutic approaches targeting the Hcrt system in the treatment of AUD.

Construction of the Aspergillus nidulans pmtC∆hogA∆yA∆ triple deletion strain by mating and benlate inducing

In Aspergillus nidulans, construction of double or triple deletion strain is widely carried out to investigate genetic coordination, relation, or interaction. In this study, a fungal mutant, in which pmtC, hogA, and yA are deleted, is constructed for a recent experimental purpose. The mutation strain is screened and isolated by auxotrophic mutations from progenies of a sexual mating between the two A. nidulans fungi, pmtC∆yA∆ deletion strain and hogA∆ deletion strain. The genotype of the strain obtained from the matting is confirmed by PCR indicating that the obtained strain contains deleted hogA, both wild-type pmtC and deleted pmtC deletion. Moreover, the obtained strain forms morphology with irregular edges, abnormal growth, and derived colony, which is like general morphology of diploid strains, suggesting that the mutant is a diploid strain. As benlate is a haploidization-inducer for the diploid strain, the spores of the diploid strain are spread on media with 0.05ppm of benlate. Forming colonies performing pmtC-like characteristics, such as poor development, impaired vegetation and limited conidiation, are considered as target haploid strains and confirmed by PCR. The PCR results show that, among four isolated strains, there is only one strain showing PCR band which is approximate to pmtC knock-out gene, indicating the strain carry pmtC deletion gene. As a result, the fungal strains, namely AnTLE36.HC, is a pmtC∆hogA∆yA∆ deletion strain.

Abstract MP33: A20 in the Kidney Epithelium Attenuates Angiotensin II-induced Hypertension by Constraining Renal Tubular NHE3 Expression

Whereas the ubiquitin editor A20 limits NF-κB-mediated signaling in myeloid leukocytes, the functions of A20 in renal epithelial cells during hypertension have not been described. We recently found that A20 in the kidney tubule ameliorates hypertension and cardiac hypertrophy induced by chronic angiotensin (Ang) II infusion (500ng/kg/min) in mice. Here we report that following 3 weeks of hypertension, the kidneys of mice with tubular deletion of A20 in the kidney epithelium (“A20 iKKO” = A20 flox/flox Pax8-rtTA TetO-Cre + ) have augmented protein expression of the proximal tubular sodium transporter NHE3 (1.5±0.1 vs. 1.0±0.1; p <0.01) but similar levels of ENaC subunit expression in the collecting tubule ( p =NS for all 3 subunits) vs wild-type (WT) controls. Moreover, kidneys from A20 iKKO mice have upregulated mRNA levels for the T cell chemokine CCL5 (2.4±0.3 vs. 1.0±0.3; p <0.01) and higher proportions of effector memory CD8 + T cells (51.2±2.3 vs. 39.3±2.9 % of CD8 + ; p =0.013) compared to WTs despite similar numbers of effector memory T cells in the spleen ( p =NS). To examine whether A20 in the kidney attenuates hypertension by constraining tubular NHE3 expression, we bred A20 iKKO mice with an NHE3 flox/flox line, thereby generating mice with double A20 and NHE3 deletion in the kidney tubule (“Dual KKO”) mice. At baseline, kidneys from Dual KKO mice showed robust reductions in mRNA for A20 (0.5±0.1 vs. 1.0±0.1; p =0.022) and NHE3 (0.2±0.05 vs. 1.0±0.15; p =0.002) compared to WT littermates. During the first ten days of Ang II infusion, mean arterial pressures (MAPs) in the Dual KKO mice were lower than the MAPs previously recorded in our A20 iKKO cohort (129±3 vs. 152±4 mmHg; p <0.01). Moreover, during 3 weeks of Ang II infusion, Dual KKO and WT mice maintained similar MAPs (145±7 vs. 149±13 mmHg; p =0.78), and heart weight to body weight ratios (5.9±0.3 vs. 5.5±0.2 mg/g; p =0.37) in concomitant measurements. Thus, A20 in the kidney tubule blunts the chronic hypertensive response by curtailing NHE3 expression. Our data further suggest that A20 in the nephron can blunt renal inflammation without altering systemic T cell immunity. Stimulating the A20 signaling pathway in the kidney may thus attenuate NHE3-dependent hypertension without conferring attendant risks of global immunosuppression.

The role of aroA and ppk1 in Aeromonas veronii pathogenicity and the efficacy evaluation of mutant strain AV-ΔaroA/ppk1 as a live attenuated vaccine

Aeromonas veronii is an opportunistic pathogen that poses great threat to aquaculture and human health, so there is an urgent need for green and efficient methods to deal with its infection. In this study, single and double gene deletion strains (AV-ΔaroA, AV-Δppk1 and AV-ΔaroA/ppk1) that can be stably inherited were constructed. Pathogenicity test showed that the toxicity of AV-ΔaroA and AV-ΔaroA/ppk1 was significantly lower compared to wild-type A. veronii. Biological characterization analysis revealed that the decrease in pathogenicity might be due to the declined growth, motility, biofilm formation abilities and the expression of virulence-related genes in mutants. Subsequently, we evaluated the efficacy of AV-ΔaroA/ppk1 as a live attenuated vaccine (LAV). Safety assessment experiments showed that AV-ΔaroA/ppk1 injected at a concentration of 3 × 107 CFU/mL was safe for C. carassius. The relative percentage survival of AV-ΔaroA/ppk1 was 67.85 %, significantly higher than that of the inactivated A. veronii, which had an RPS of 54.84 %. This improved protective effect was mainly attributed to the increased levels of A. veronii specific IgM antibody, enhanced alkaline phosphatase, lysozyme and superoxide dismutase activities, as well as higher expression levels of several immune related genes. Together, these findings deepen our understanding of the functional roles of aroA and ppk1 in A. veronii pathogenicity, provide a good candidate of LAV for A. veronii.

Altered interactions between cis-regulatory elements partially resolve BLADE-ON-PETIOLE genetic redundancy in Capsella rubella.

Duplicated genes are thought to follow one of three evolutionary trajectories that resolve their redundancy: neofunctionalization, subfunctionalization, or pseudogenization. Differences in expression patterns have been documented for many duplicated gene pairs and interpreted as evidence of subfunctionalization and a loss of redundancy. However, little is known about the functional impact of such differences and about their molecular basis. Here, we investigate the genetic and molecular basis for the partial loss of redundancy between the two BLADE-ON-PETIOLE genes BOP1 and BOP2 in red shepherd's purse (Capsella rubella) compared to Arabidopsis (Arabidopsis thaliana). While both genes remain almost fully redundant in A. thaliana, BOP1 in C. rubella can no longer ensure wild-type floral organ numbers and suppress bract formation, due to an altered expression pattern in the region of the cryptic bract primordium. We use two complementary approaches, transgenic rescue of A. thaliana atbop1 atbop2 double mutants and deletions in the endogenous AtBOP1 promoter, to demonstrate that several BOP1 promoter regions containing conserved noncoding sequences interact in a nonadditive manner to control BOP1 expression in the bract primordium and that changes in these interactions underlie the evolutionary divergence between C. rubella and A. thaliana BOP1 expression and activity. Similarly, altered interactions between cis-regulatory regions underlie the divergence in functional promoter architecture related to the control of floral organ abscission by BOP1. These findings highlight the complexity of promoter architecture in plants and suggest that changes in the interactions between cis-regulatory elements are key drivers for evolutionary divergence in gene expression and the loss of redundancy.

Malaria diagnosis challenges and pfhrp2 and pfhrp3 gene deletions using pregnant women as sentinel population in Nanoro region, Burkina Faso

ABSTRACT Malaria in pregnancy causes adverse consequences and prompt and accurate diagnosis is essential for case management. In malaria endemic countries, diagnosis is mainly based on rapid diagnostic tests (RDT) and microscopy. However, increasing reports of false negatives caused by low parasitemia and pfhrp2/3 deletions raise concerns about HRP2-based RDT usefulness. This study aimed to assess RDT and microscopy performance and to describe pfhrp2/3 deletions in a cohort of 418 pregnant women in Burkina Faso. Malaria was diagnosed using RDT and microscopy and blood samples were collected during antenatal care visits. Diagnostic results were compared to PCR as gold standard. Pfhrp2 and pfhrp3 deletions were characterized for patients with confirmed P. falciparum infection. RDT had better sensitivity (76%) but lower specificity (83%) than microscopy (sensitivity = 57%; specificity = 98%). Low parasitemia (<150 parasites/µL), especially in multigravidae, was the principal factor causing false negatives by both methods. Moreover, pfhrp2 deletion frequency among overall false negatives by RDT was 21.43%. Higher frequency of deletions was found among all samples, independently of RDT result, for example around 2% of samples had double deletions meaning that the majority of deletions had no effect on RDT testing. Finally, it was found higher pfhrp2 deletion in women with lower uterine height during the first trimester. Wider and National surveillance study of deletions is recommended among pregnant women and in Burkina Faso.

Abcb4-defect cholangitis mouse model with hydrophobic bile acid composition by in vivo liver-specific gene deletion

Progressive familial intrahepatic cholestasis (PFIC) is a liver disease that occurs during childhood and requires liver transplantation. ABCB4 is localized along the canalicular membranes of hepatocytes, transports phosphatidylcholine into bile, and its mutation causes PFIC3. Abcb4 gene-deficient mice established as animal models of PFIC3 exhibit cholestasis-induced liver injury. However, their phenotypes are often milder than those of human PFIC3, partly because of the existence of large amounts of less toxic hydrophilic bile acids synthesized by the rodent-specific enzymes Cyp2c70 and Cyp2a12. Mice with double deletions of Cyp2c70/Cyp2a12 (CYPDKO mice) have a human-like hydrophobic bile acid composition. PFIC-related gene mutations were induced in CYPDKO mice to determine whether these triple-gene-deficient mice are a better model for PFIC. To establish a PFIC3 mouse model using CYPDKO mice, we induced abcb4 gene deletion in vivo using adeno-associated viruses expressing SaCas9 under the control of a liver-specific promoter and abcb4-target gRNAs. Compared to Abcb4-deficient wild-type mice, Abcb4-deficient CYPDKO mice showed more pronounced liver injury along with an elevation of inflammatory and fibrotic markers. The proliferation of intrahepatic bile ductal cells and hematopoietic cell infiltration were also observed. CYPDKO/abcb4-deficient mice show a predominance of taurine-conjugated chenodeoxycholic acid and lithocholic acid in the liver. In addition, phospholipid levels in the gallbladder bile were barely detectable. Mice with both human-like bile acid composition and Abcb4-defect exhibit severe cholestatic liver injury and are useful for studying human cholestatic diseases and developing new treatments.

High Frequency of Deletions in the pfhrp2 and pfhrp3 Genes of Plasmodium falciparum in the Middle Rio Negro Region of the Brazilian Amazon.

Several countries are reporting natural populations of P. falciparum with deletions in the pfhrp2/3 genes that can lead to false-negative results in rapid diagnostic tests. To investigate the prevalence of deletion in the pfhrp2/3 genes in the Rio Negro basin in the Brazilian Amazon and identify whether there is clinical differentiation between individuals infected by these parasites, clinical samples collected from 2003 to 2016 were analyzed from symptomatic and asymptomatic P. falciparum-infected individuals. The molecular deletion of pfhrp2 and pfhrp3 genes was evaluated using the protocols recommended by the WHO. From 82 samples used, 28 (34.2%) had a single deletion in pfhrp2, 19 (23.2%) had a single deletion in pfhrp3, 15 (18.3%) had a double deletion (pfhrp2/3), and 20 (24.4%) did not have a deletion in either gene. In total, 29.3% of individuals had an asymptomatic plasmodial infection and were 3.64 times more likely to have parasites with a double deletion (pfhrp2/3) than patients with clinical malaria (p = 0.02). The high prevalence of parasites with pfhrp2/3 deletions shows the need to implement a surveillance program in this area. Deletions in parasites may be associated with the clinical pattern of the disease in this area. More studies must be carried out to elucidate these findings.

The role of cytolethal distending toxin in Glaesserella parasuis JS0135 strain infection: Cytotoxicity, phagocytic resistance and pathogenicity

Glaesserella parasuis is an important porcine pathogen that commonly colonizes the upper respiratory tract of pigs and is prone to causing Glässer’s disease under complex conditions. As yet, the disease has led to serious economic losses to the swine industry worldwide. Studies so far have found that several virulence factors are associated with the pathogenicity of G. parasuis, but the pathogenic mechanism is still not fully understood. Cytolethal distending toxin (CDT), a potential virulence factor in G. parasuis, is involved in cytotoxicity, serum resistance, adherence to and invasion of host cells in vitro. Here, to further investigate the pathogenic role of CDT during G. parasuis infection in vitro and in vivo, a double cdt1 and cdt2 deletion mutant (Δcdt1Δcdt2) without selectable marker was first generated in G. parasuis JS0135 strain by continuous natural transformations and replica plating. Morphological observation and lactate dehydrogenase assay showed that the Δcdt1Δcdt2 mutant was defective in cytotoxicity. Additionally, the Δcdt1Δcdt2 mutant was more susceptible to phagocytosis caused by 3D4/2 macrophages compared to the wild-type JS0135 strain. Moreover, by focusing on clinical signs, necropsy, bacterial recovery and pathological observation, we found that the deletion of cdt1 and cdt2 genes led to a significant attenuation of virulence in G. parasuis. Taken together, these findings suggest that as an important virulence factor, CDT can significantly affect the pathogenicity of G. parasuis.

Safety and DIVA Capability of Novel Live Attenuated Classical Swine Fever Marker Vaccine Candidates in Pregnant Sows.

Classical Swine Fever (CSF), a highly contagious viral disease affecting pigs and wild boar, results in significant economic losses in the swine industry. In endemic regions, prophylactic vaccination and stamping-out strategies are used to control CSF outbreaks. However, sporadic outbreaks and persistent infections continue to be reported. Although the conventional attenuated CSF vaccines protect pigs against the disease, they do not allow for the differentiation of infected from vaccinated animals (DIVA), limiting their use as an eradication tool. In this study, three targeted attenuation strategies were employed to generate vaccine candidates based on the current prevalent CSFV group 2 strains GD18 and QZ07: a single deletion of H79 in Erns (QZ07-sdErnsH-KARD), double deletion of H79 and C171 in Erns (GD18-ddErnsHC-KARD and QZ07-ddErnsHC-KARD), and deletion of H79 in Erns combined with a 5-168 amino acids deletion of Npro (GD18-ddNpro-ErnsH-KARD). Additionally, a negative serological marker with four substitutions in a highly conserved epitope in E2 recognized by the monoclonal antibody 6B8 was introduced in each candidate for DIVA purposes. The safety of these four resulting vaccine candidates was evaluated in pregnant sows. Two candidates, GD18-ddErnsHC-KARD and QZ07-sdErnsH-KARD were found to be safe for pregnant sows and unlikely to cause vertical transmission. Both candidates also demonstrated potential to be used as DIVA vaccines, as was shown using a proprietary blocking ELISA based on the 6B8 monoclonal antibody. These results, together with our previous work, constitute a proof-of-concept for the rational design of CSF antigenically marked modified live virus vaccine candidates.

GRA47 is important for the morphology and permeability of the parasitophorous vacuole in Toxoplasma gondii

Establishing an intact intracellular parasitophorous vacuole (PV) that enables efficient nutrient uptake and protein trafficking is essential for the survival and proliferation of Toxoplasma gondii. Although the PV membrane (PVM)-localized dense granule protein 17 (GRA17) and GRA23 mediate the permeability of the PVM to small molecules, including nutrient uptake and excretion of metabolic by-products, the molecular mechanism by which T. gondii acquires nutrients remains unclear. In this study, we showed that the secreted protein GRA47 contributed to normal PV morphology, PVM permeability to small molecules, growth, and virulence in T. gondii. Co-immunoprecipitation analysis demonstrated potential interaction of GRA47 with GRA72, and the loss of GRA72 affected PV morphology, parasite growth and infectivity. To investigate the biological relationship among GRA47, GRA72, GRA17 and GRA23, attempts were made to construct strains with double gene deletion and overexpressing strains. Only Δgra23Δgra72 was successfully constructed. This strain exhibited a significant increase in the proportion of aberrant PVs compared with the Δgra23 strain. Overexpressing one of the three related GRAs partially rescued PVs with aberrant morphology in Δgra47, Δgra72 and Δgra17, while the expression of the Plasmodium falciparum PVM protein PfExp2, an ortholog of GRA17 and GRA23, fully rescued the PV morphological defect in all three Δgra strains. These results suggest that these GRA proteins may not be functionally redundant but rather work in different ways to regulate nutrient acquisition. These findings highlight the versatility of the nutrient uptake mechanisms in T. gondii, which may contribute to the parasite’s remarkable ability to grow in different cellular niches in a very broad range of hosts.