Genetic Ablation of GIGYF1, Associated With Autism, Causes Behavioral and Neurodevelopmental Defects in Zebrafish and Mice.

BACKGROUND: Autism spectrum disorder is characterized by deficits in social communication and restricted or repetitive behaviors. Due to the extremely high genetic and phenotypic heterogeneity, it is critical to pinpoint the genetic factors for understanding the pathology of these disorders. METHODS: We analyzed the exomes generated by the SPARK (Simons Powering Autism Research) project and performed a meta-analysis with previous data. We then generated 1 zebrafish knockout model and 3 mouse knockout models to examine the function of GIGYF1 in neurodevelopment and behavior. Finally, we performed whole tissue and single-nucleus transcriptome analysis to explore the molecular and cellular function of GIGYF1. RESULTS: GIGYF1 variants are significantly associated with various neurodevelopmental disorder phenotypes, including autism, global developmental delay, intellectual disability, and sleep disturbance. Loss of GIGYF1 causes similar behavioral effects in zebrafish and mice, including elevated levels of anxiety and reduced social engagement, which is reminiscent of the behavioral deficits in human patients carrying GIGYF1 variants. Moreover, excitatory neuron-specific Gigyf1 knockout mice recapitulate the increased repetitive behaviors and impaired social memory, suggesting a crucial role of Gigyf1 in excitatory neurons, which correlates with the observations in single-nucleus RNA sequencing. We also identified a series of downstream target genes of GIGYF1 that affect many aspects of the nervous system, especially synaptic transmission. CONCLUSIONS: De novo variants of GIGYF1 are associated with neurodevelopmental disorders, including autism spectrum disorder. GIGYF1 is involved in neurodevelopment and animal behavior, potentially through regulating hippocampal CA2 neuronal numbers and disturbing synaptic transmission.

Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies.

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma-/- mouse studies having informed our understanding of GZMA's physiological function. We show herein that Gzma-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J GzmaS211A mouse provided the first insights into GZMA's bioactivity free of background issues, with circulating proteolytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

Matrix remodeling-associated protein 8 is a marker of a subset of cancer-associated fibroblasts in pancreatic cancer.

Cancer-associated fibroblasts (CAFs), a compartment of the tumor microenvironment, were previously thought to be a uniform cell population that promotes cancer progression. However, recent studies have shown that CAFs are heterogeneous and that there are at least two types of CAFs, that is, cancer-promoting and -restraining CAFs. We previously identified Meflin as a candidate marker of cancer-restraining CAFs (rCAFs) in pancreatic ductal adenocarcinoma (PDAC). The precise nature of rCAFs, however, has remained elusive owing to a lack of understanding of their comprehensive gene signatures. Here, we screened genes whose expression correlated with Meflin in single-cell transcriptomic analyses of human cancers. Among the identified genes, we identified matrix remodeling-associated protein 8 (MXRA8), which encodes a type I transmembrane protein with unknown molecular function. Analysis of MXRA8 expression in human PDAC samples showed that MXRA8 was differentially co-expressed with other CAF markers. Moreover, in patients with PDAC or syngeneic tumors developed in MXRA8-knockout mice, MXRA8 expression did not affect the roles of CAFs in cancer progression, and the biological importance of MXRA8+ CAFs is still unclear. Overall, we identified MXRA8 as a new CAF marker; further studies are needed to determine the relevance of this marker.

Inducible Slc4a11 Knockout Triggers Corneal Edema Through Perturbation of Corneal Endothelial Pump.

Purpose: The conventional Slc4a11 knockout (KO) shows significant corneal edema at eye opening, a fact that complicates the study of the initial events leading to edema. An inducible KO would provide opportunities to examine early events following loss of Slc4a11 activity. Methods: Slc4a11 Flox (SF) mice were crossed with mice expressing the estrogen receptor Cre Recombinase fusion protein and fed tamoxifen (Tm) for two weeks. Corneal thickness (CT) was measured by OCT. At eight weeks endpoint, oxidative damage, tight junction integrity, stromal lactate concentration, endothelial permeability, differentially expressed transporters, and junction proteins were determined. Separately, a keratocyte only inducible Slc4a11 KO was also examined. Results: At four weeks post-Tm induction Slc4a11 transcript levels were 2% of control. Corneal thickness increased gradually and was 50% greater than Wild Type (WT) after eight weeks with significantly altered endothelial morphology, increased nitrotyrosine staining, significantly higher stromal lactate, decreased expression of lactate transporters and Na-K ATPase activity, higher ATP, altered expression of tight and adherens junctions, and increased fluorescein permeability. No significant differences in CT were found between WT and keratocyte only Slc4a11 KO. Conclusions: The Slc4a11 inducible KO shows development of a similar phenotype as the conventional KO, thereby validating the model and providing a tool for further use in examining the sequence of cellular events by use of noninvasive in vivo physiological probes.

Efficiency comparison of B6(Cg)-Tyrc-2j /J and C57BL/6NTac embryos as hosts for the generation of knockout mice.

Careful selection of the host embryo is critical to the efficient production of knockout (KO) mice when injecting mouse embryonic stem (mES) cells into blastocysts. B6(Cg)-Tyrc-2j/J (B6 albino) and C57BL/6NTac (B6NTac) strains of mice are widely used to produce host blastocysts for such procedures. Here, we tested these two strains to identify an appropriate match for modified agouti C57BL/6N (JM8A3.N1) mES cells. When comparing blastocyst yield, super-ovulated B6NTac mice produced more injectable blastocysts per female than B6 albino mice (8.2 vs. 5.4). There was no significant difference in birth rate when injected embryos were transferred to the same pseudopregnant recipient strain. However, the live birth rate was significantly higher for B6NTac blastocysts than B6 albino blastocysts (62.7% vs. 50.2%). In addition, the proportion of pups exhibiting high-level and complete chimerism, as identified by coat color, was also significantly higher in the B6NTac strain. There was no obvious difference in the efficiency of germline transmission (GLT) when compared between B6NTac and B6 albino host embryos (61.5% vs. 63.3% for mES clones; 64.5% vs. 67.9% for genes, respectively), thus suggesting that an equivalent GLT rate could be obtained with only a few blastocyst injections for B6NTac embryos. In conclusion, our data indicate that B6NTac blastocysts are a better choice for the microinjection of JM8A3.N1 mES cells than B6 albino blastocysts.

The Scope of Astrocyte Elevated Gene-1/Metadherin (AEG-1/MTDH) in Cancer Clinicopathology: A Review.

Since its initial cloning in 2002, a plethora of studies in a vast number of cancer indications, has strongly established AEG-1 as a bona fide oncogene. In all types of cancer cells, overexpression and knockdown studies have demonstrated that AEG-1 performs a seminal role in regulating proliferation, invasion, angiogenesis, metastasis and chemoresistance, the defining cancer hallmarks, by a variety of mechanisms, including protein-protein interactions activating diverse oncogenic pathways, RNA-binding promoting translation and regulation of inflammation, lipid metabolism and tumor microenvironment. These findings have been strongly buttressed by demonstration of increased tumorigenesis in tissue-specific AEG-1 transgenic mouse models, and profound resistance of multiple types of cancer development and progression in total and conditional AEG-1 knockout mouse models. Additionally, clinicopathologic correlations of AEG-1 expression in a diverse array of cancers establishing AEG-1 as an independent biomarker for highly aggressive, chemoresistance metastatic disease with poor prognosis have provided a solid foundation to the mechanistic and mouse model studies. In this review a comprehensive analysis of the current and up-to-date literature is provided to delineate the clinical significance of AEG-1 in cancer highlighting the commonality of the findings and the discrepancies and discussing the implications of these observations.

Mitochondrial arginase-2 is essential for IL-10 metabolic reprogramming of inflammatory macrophages.

Mitochondria are important regulators of macrophage polarisation. Here, we show that arginase-2 (Arg2) is a microRNA-155 (miR-155) and interleukin-10 (IL-10) regulated protein localized at the mitochondria in inflammatory macrophages, and is critical for IL-10-induced modulation of mitochondrial dynamics and oxidative respiration. Mechanistically, the catalytic activity and presence of Arg2 at the mitochondria is crucial for oxidative phosphorylation. We further show that Arg2 mediates this process by increasing the activity of complex II (succinate dehydrogenase). Moreover, Arg2 is essential for IL-10-mediated downregulation of the inflammatory mediators succinate, hypoxia inducible factor 1α (HIF-1α) and IL-1ß in vitro. Accordingly, HIF-1α and IL-1ß are highly expressed in an LPS-induced in vivo model of acute inflammation using Arg2-/- mice. These findings shed light on a new arm of IL-10-mediated metabolic regulation, working to resolve the inflammatory status of the cell.

Dopamine and the phosphorylated dopamine transporter are increased in the diacylglycerol kinase η-knockout mouse brain.

The molecular mechanisms generating the mania-like abnormal behaviors caused by diacylglycerol (DG) kinase (DGK) η deficiency remain unclear. Here, we found that DGKη knockout markedly increased dopamine (DA) levels in the midbrain (DA-producing region, 2.8-fold) and cerebral cortex (DA projection region, 1.2-fold). Moreover, DGKη deficiency significantly augmented phosphorylated DA transporter (DAT) levels (1.4-fold increase), which induce DA efflux to the synaptic cleft, in the cerebral cortex. Moreover, phosphorylation levels of protein kinase C-ß, which is activated by DG and involved in DAT phosphorylation, were also increased. DAT expressed in Neuro-2a cells recruited DGKη to the plasma membrane and colocalized with it. These results strongly suggest that dopaminergic hyperfunction caused by DGKη deficiency in the brain leads to mania-like behaviors.

Estrogens and development of the rete testis, efferent ductules, epididymis and vas deferens.

Estrogen has always been considered the female hormone and testosterone the male hormone. However, estrogen's presence in the testis and deleterious effects of estrogen treatment during development have been known for nearly 90 years, long before estrogen receptors (ESRs) were discovered. Eventually it was learned that testes actually synthesize high levels of estradiol (E2) and sequester high concentrations in the reproductive tract lumen, which seems contradictory to the overwhelming number of studies showing reproductive pathology following exogenous estrogen exposures. For too long, the developmental pathology of estrogen has dominated our thinking, even resulting in the "estrogen hypothesis" as related to the testicular dysgenesis syndrome. However, these early studies and the development of an Esr1 knockout mouse led to a deluge of research into estrogen's potential role in and disruption of development and function of the male reproductive system. What is new is that estrogen action in the male cannot be divorced from that of androgen. This paper presents what is known about components of the estrogen pathway, including its synthesis and target receptors, and the need to achieve a balance between androgen- and estrogen-action in male reproductive tract differentiation and adult functions. The review focuses on what is known regarding development of the male reproductive tract, from the rete testis to the vas deferens, and examines the expression of estrogen receptors and presence of aromatase in the male reproductive system, traces the evidence provided by estrogen-associated knockout and transgenic animal models and discusses the effects of fetal and postnatal exposures to estrogens. Hopefully, there will be enough here to stimulate discussions and new investigations of the androgen:estrogen balance that seems to be essential for development of the male reproductive tract.

Ablation of Zfhx4 results in early postnatal lethality by disrupting the respiratory center in mice.

Breathing is an integrated motor behavior that is driven and controlled by a network of brainstem neurons. Zfhx4 is a zinc finger transcription factor and our results showed that it was specifically expressed in several regions of the mouse brainstem. Mice lacking Zfhx4 died shortly after birth from an apparent inability to initiate respiration. We also found that the electrical rhythm of brainstem‒spinal cord preparations was significantly depressed in Zfhx4-null mice compared to wild-type mice. Immunofluorescence staining revealed that Zfhx4 was coexpressed with Phox2b and Math1 in the brainstem and that Zfhx4 ablation greatly decreased the expression of these proteins, especially in the retrotrapezoid nucleus. Combined ChIP‒seq and mRNA expression microarray analysis identified Phox2b as the direct downstream target gene of Zfhx4, and this finding was validated by ChIP‒qPCR. Previous studies have reported that both Phox2b and Math1 play key roles in the development of the respiratory center, and Phox2b and Math1 knockout mice are neonatal lethal due to severe central apnea. On top of this, our study revealed that Zfhx4 is a critical regulator of Phox2b expression and essential for perinatal breathing.

Functions of Muscarinic Receptor Subtypes in Gastrointestinal Smooth Muscle: A Review of Studies with Receptor-Knockout Mice.

Parasympathetic signalling via muscarinic acetylcholine receptors (mAChRs) regulates gastrointestinal smooth muscle function. In most instances, the mAChR population in smooth muscle consists mainly of M2 and M3 subtypes in a roughly 80% to 20% mixture. Stimulation of these mAChRs triggers a complex array of biochemical and electrical events in the cell via associated G proteins, leading to smooth muscle contraction and facilitating gastrointestinal motility. Major signalling events induced by mAChRs include adenylyl cyclase inhibition, phosphoinositide hydrolysis, intracellular Ca2+ mobilisation, myofilament Ca2+ sensitisation, generation of non-selective cationic and chloride currents, K+ current modulation, inhibition or potentiation of voltage-dependent Ca2+ currents and membrane depolarisation. A lack of ligands with a high degree of receptor subtype selectivity and the frequent contribution of multiple receptor subtypes to responses in the same cell type have hampered studies on the signal transduction mechanisms and functions of individual mAChR subtypes. Therefore, novel strategies such as genetic manipulation are required to elucidate both the contributions of specific AChR subtypes to smooth muscle function and the underlying molecular mechanisms. In this article, we review recent studies on muscarinic function in gastrointestinal smooth muscle using mAChR subtype-knockout mice.

Dietary Erythrodiol Modifies Hepatic Transcriptome in Mice in a Sex and Dose-Dependent Way.

Erythrodiol is a terpenic compound found in a large number of plants. To test the hypotheses that its long-term administration may influence hepatic transcriptome and this could be influenced by the presence of APOA1-containing high-density lipoproteins (HDL), Western diets containing 0.01% of erythrodiol (10 mg/kg dose) were provided to Apoe- and Apoa1-deficient mice. Hepatic RNA-sequencing was carried out in male Apoe-deficient mice fed purified Western diets differing in the erythrodiol content. The administration of this compound significantly up- regulated 68 and down-regulated 124 genes at the level of 2-fold change. These genes belonged to detoxification processes, protein metabolism and nucleic acid related metabolites. Gene expression changes of 21 selected transcripts were verified by RT-qPCR. Ccl19-ps2, Cyp2b10, Rbm14-rbm4, Sec61g, Tmem81, Prtn3, Amy2a5, Cyp2b9 and Mup1 showed significant changes by erythrodiol administration. When Cyp2b10, Dmbt1, Cyp2b13, Prtn3 and Cyp2b9 were analyzed in female Apoe-deficient mice, no change was observed. Likewise, no significant variation was observed in Apoa1- or in Apoe-deficient mice receiving doses ranging from 0.5 to 5 mg/kg erythrodiol. Our results give evidence that erythrodiol exerts a hepatic transcriptional role, but this is selective in terms of sex and requires a threshold dose. Furthermore, it requires an APOA1-containing HDL.

Role of collagen XII in skin homeostasis and repair.

Skin integrity and function depends to a large extent on the composition of the extracellular matrix, which regulates tissue organization. Collagen XII is a homotrimer with short collagenous domains that confer binding to the surface of collagen I-containing fibrils and extended flexible arms, which bind to non-collagenous matrix components. Thereby, collagen XII helps to maintain collagen suprastructure and to absorb stress. Mutant or absent collagen XII leads to reduced muscle and bone strength and lax skin, whereas increased collagen XII amounts are observed in tumor stroma, scarring and fibrosis. This study aimed at uncovering in vivo mechanisms by which collagen XII may achieve these contrasting outcomes. We analyzed skin as a model tissue that contains abundant fibrils, composed of collagen I, III and V with collagen XII decorating their surface, and which is subject to mechanical stress. The impact of different collagen XII levels was investigated in collagen XII-deficient (Col12-KO) mice and in mice with collagen XII overexpression in the dermis (Col12-OE). Unchallenged skin of these mice was histologically inconspicuous, but at the ultrastructural level revealed distinct aberrations in collagen network suprastructure. Repair of excisional wounds deviated from controls in both models by delayed healing kinetics, which was, however, caused by completely different mechanisms in the two mouse lines. The disorganized matrix in Col12-KO wounds failed to properly sequester TGFß, resulting in elevated numbers of myofibroblasts. These are, however, unable to contract and remodel the collagen XII-deficient matrix. Excess of collagen XII, in contrast, promotes persistence of M1-like macrophages in the wound bed, thereby stalling the wounds in an early inflammatory stage of the repair process and delaying healing. Taken together, we demonstrate that collagen XII is a key component that assists in orchestrating proper skin matrix structure, controls growth factor availability and regulates cellular composition and function. Together, these functions are pivotal for re-establishing homeostasis after injury.

Backcrossing to an appropriate genetic background improves the birth rate of carbohydrate sulfotransferase 14 gene-deleted mice.

Ehlers-Danlos syndromes (EDSs) are heterogeneous group of heritable connective tissue disorders characterized by joint and skin hyperextensibility as well as fragility of various organs. Recently, we described a new type of EDS, musculocontractual EDS (mcEDS-CHST14), caused by pathogenic variants of the carbohydrate sulfotransferase 14 (CHST14) gene mutation. B6;129S5-Chst14tm1Lex/Mmucd (B6;129-Chst14 KO) mice are expected to be an animal model of mcEDS-CHST14. However, >90% of B6;129-Chst14 KO homozygous (B6;129-Chst14-/-) mice show perinatal lethality. Therefore, improvement of the birth rate of Chst14-/- mice is needed to clarify the pathophysiology of mcEDS-CHST14 using this animal model. Some B6;129-Chst14-/- embryos had survived at embryonic day 18.5 in utero, suggesting that problems with delivery and/or childcare may cause perinatal lethality. However, in vitro fertilization and egg transfer did not improve the birth rate of the mice. A recent report showed that backcrossing to C57BL/6 strain induces perinatal death of all Chst14-/- mice, suggesting that genetic background influences the birthrate of these mice. In the present study, we performed backcrossing of B6;129-Chst14 KO mice to a BALB/c strain, an inbred strain that shows lower risks of litter loss than C57BL/6 strain. Upon backcrossing 1 to 12 times, the birth rate of Chst14-/- mice was improved with a birth rate of 6.12-18.64%. These results suggest that the genetic background influences the birth rate of Chst14-/- mice. BALB/c congenic Chst14-/- (BALB.Chst14-/-) mice may facilitate investigation of mcEDS-CHST14. Furthermore, backcrossing to an appropriate strain may contribute to optimizing animal experiments.

Gender difference in development of steatohepatitis in p62/Sqstm1 and Nrf2 double-knockout mice.

Gender and menopause influence the severity and development manner of nonalcoholic steatohepatitis (NASH). Male p62/Sqstm1 and nuclear factor E2-related factor-2 (p62 and Nrf2) double-knockout (DKO) mice exhibit severe steatohepatitis caused by hyperphagia-induced obesity, overload of lipopolysaccharide (LPS) into the liver, and potentiation of the inflammatory response in Kupffer cells. However, the pathogenetic phenotype of steatohepatitis in female DKO mice remains unknown. Phenotypic changes of steatohepatitis in DKO mice were compared in terms of gender differences. Compared with DKO male mice, DKO female mice exhibited later onset of steatohepatitis with obesity after 30 weeks of age, as well as milder severity of hepatic inflammation and fibrosis. Serum estradiol was higher in female than male mice, with levels increasing up to 30 weeks of age before decreasing until 50 weeks of age (corresponding to the post-menopausal period). Fecal and serum LPS were lower in female mice than male mice, and inflammatory signaling in the liver was attenuated in female compared with male mice. Correlating with LPS levels, the composition of intestinal microbiota in female mice was different from male mice. Gender differences were observed for the development of steatohepatitis in DKO mice. Low-grade inflammatory hit in the liver under in vivo conditions of high estradiol may be attributable to the milder pathological features of steatohepatitis in female mice.

Epididymal proteins Binder of SPerm Homologs 1 and 2 (BSPH1/2) are dispensable for male fertility and sperm motility in mice.

The binder of sperm family of proteins has been reported to be indispensable for sperm maturation and capacitation. However, their physiological functions in fertility have only been studied in vitro. CRISPR/Cas9 genome editing was utilized to generate double knockout (DKO) mice by simultaneously targeting the two murine binder of sperm genes, Bsph1 and Bsph2. To confirm that the homologous genes and proteins were completely eliminated in the DKO mice, different methods such as reverse transcription polymerase chain reaction, digital droplet-polymerase chain reaction and liquid chromatography tandem mass spectrometry were applied. Bsph1/2 DKO male mice were bred by intercrossing. Compared to wild type counterparts, male Bsph1/2 null mice, lacking BSPH1/2 proteins, were fertile with no differences in sperm motility and sperm count. However, the weights of male pups were significantly increased in Bsph1/2 double knockout mice in a time dependent manner spanning days 6 and 21, as well as 6 weeks of age. No change was detected in the weights of female pups during the same period. Taken together, these data indicate that BSPH1/2 proteins are dispensable for male fertility in mice but may influence growth.

Aldehyde dehydrogenase 2 deficiency significantly exacerbates tert-butyl alcohol-induced toxicity in mice.

Owing to the use of ethyl tert-butyl ether (ETBE) as a fuel additive, the possible adverse effects of ETBE exposure have become a public concern. Our previous study showed that ETBE-induced toxicity in aldehyde dehydrogenase 2 (Aldh2) gene knockout (KO) mice was caused by its primary metabolite acetaldehyde, which was toxic. However, it is unclear whether tert-butyl alcohol (TBA), another main metabolite of ETBE, plays a role in ETBE-induced toxicity. To investigate this relationship, we analyzed the changes of TBA concentrations in tissues after ETBE exposure, and then evaluated the toxicity after direct TBA treatment in both KO and wild-type (WT) mice. An exposure to 500 ppm ETBE via inhalation resulted in the formation of its three metabolites, TBA, 2-methyl-1,2-propanediol and ethanol, whose concentrations in the liver, brain, fat and testis of male KO mice were significantly higher than the corresponding concentrations observed in male WT mice. Direct treatment to TBA (20 mg/mL of drinking water) caused significant changes in relative organ weights and histopathology, and increased levels of genetic damages in both types of mice. These toxic effects were also seen in KO mice exposed to a lower concentration of TBA (5 mg/mL), which was associated with increased oxidative stress in serum (reduced glutathione and reduced glutathione/oxidized glutathione ratio decreased). Our findings indicate that ALDH2 is involved in the metabolism of ETBE and TBA, and ALDH2 deficiency could greatly increase the sensitivity to TBA-induced toxicity.

Mouse Genetic Background Affects Transfer of an Antibiotic Resistance Plasmid in the Gastrointestinal Tract.

Dissemination of antibiotic resistance (AR) genes, often on plasmids, leads to antibiotic-resistant bacterial infections, which is a major problem for animal and public health. Bacterial conjugation is the primary route of AR gene transfer in the mammalian gastrointestinal tract. Significant gaps in knowledge about which gastrointestinal communities and host factors promote plasmid transfer remain. Here, we used Salmonella enterica serovar Kentucky strain CVM29188 carrying plasmid pCVM29188_146 (harboring streptomycin and tetracycline resistance genes) to assess plasmid transfer to Escherichia coli under in vitro conditions and in various mouse strains with a conventional or defined microbiota. As an initial test, the transfer of pCVM29188_146 to the E. coli strains was confirmed in vitro Colonization resistance and, therefore, a lack of plasmid transfer were found in wild-type mice harboring a conventional microbiota. Thus, mice harboring the altered Schaedler flora (ASF), or ASF mice, were used to probe for host factors in the context of a defined microbiota. To assess the influence of inflammation on plasmid transfer, we compared interleukin-10 gene-deficient 129S6/SvEv ASF mice (proinflammatory environment) to wild-type 129S6/SvEv ASF mice and found no difference in transconjugant yields. In contrast, the mouse strain influenced plasmid transfer, as C3H/HeN ASF mice had significantly lower levels of transconjugants than 129S6/SvEv ASF mice. Although gastrointestinal members were identical between the ASF mouse strains, a few differences from C3H/HeN ASF mice were detected, with C3H/HeN ASF mice having significantly lower abundances of ASF members 356 (Clostridium sp.), 492 (Eubacterium plexicaudatum), and 502 (Clostridium sp.) than 129S6/SvEv ASF mice. Overall, we demonstrate that microbiota complexity and mouse genetic background influence in vivo plasmid transfer.IMPORTANCE Antibiotic resistance is a threat to public health. Many clinically relevant antibiotic resistance genes are carried on plasmids that can be transferred to other bacterial members in the gastrointestinal tract. The current study used a murine model to study the transfer of a large antibiotic resistance plasmid from a foodborne Salmonella strain to a gut commensal E. coli strain in the gastrointestinal tract. We found that different mouse genetic backgrounds and a different diversity of microbial communities influenced the level of Escherichia coli that acquired the plasmid in the gastrointestinal tract. This study suggests that the complexity of the microbial community and host genetics influence plasmid transfer from donor to recipient bacteria.

Deacetylation of XBP1s by sirtuin 6 confers resistance to ER stress-induced hepatic steatosis.

The active spliced form of X-box-binding protein 1 (XBP1s) is a key modulator of ER stress, but the functional role of its post-translational modification remains unclear. Here, we demonstrate that XBP1s is a deacetylation target of Sirt6 and that its deacetylation protects against ER stress-induced hepatic steatosis. Specifically, the abundance of acetylated XBP1s and concordant hepatic steatosis were increased in hepatocyte-specific Sirt6 knockout and obese mice but were decreased by genetic overexpression and pharmacological activation of Sirt6. Mechanistically, we identified that Sirt6 deacetylated a transactivation domain of XBP1s at Lys257 and Lys297 and promoted XBP1s protein degradation through the ubiquitin-proteasome system. Overexpression of XBP1s, but not its deacetylation mutant 2KR (K257/297R), in mice increased lipid accumulation in the liver. Importantly, in liver tissues obtained from patients with nonalcoholic fatty liver disease (NAFLD), the extent of XBP1s acetylation correlated positively with the NAFLD activity score but negatively with the Sirt6 level. Collectively, we present direct evidence supporting the importance of XBP1 acetylation in ER stress-induced hepatic steatosis.

Mitoferrin-1 is required for brain energy metabolism and hippocampus-dependent memory.

Disturbed iron (Fe) ion homeostasis and mitochondrial dysfunction have been implicated in neurodegeneration. Both processes are related, because central Fe ion consuming biogenetic pathways take place in mitochondria and affect their oxidative energy metabolism. Iron is imported into mitochondria by the two homologous Fe ion importers mitoferrin-1 and mitoferrin-2. To elucidate more specifically the role of mitochondrial Fe ions for brain energy metabolism and for proper neuronal function, we generated mice with a neuron-specific knockout of mitoferrin-1 (Slc25a37-/- or mfrn-1-/-) and compared them with corresponding control littermates (mfrn-1flox/flox). Mice lacking neuronal mfrn-1 exhibited no obvious anatomical or behavioral abnormalities as neonates, young or adult animals. However, they exhibited a moderate decrease in brain mitochondrial O2-consumption with complex-I substrates of the electron transport chain (p < 0.05), indicating a moderate suppression of electron transport. While these mice did not exhibit altered basal fear levels, inquisitiveness or motor skills in specific neurobiological test batteries, they clearly exhibited decreased spatial learning skills and missing establishment of stable spatial memory in Morris water maze, as compared to floxed controls (p < 0.05). We thus conclude that mitochondrial Fe ion supply is an important player in neuronal energy metabolism and proper brain function and that the carrier mitoferrin-1 cannot be completely replaced by mitoferrin-2 or other as yet unknown Fe ion carriers.