Effect Of Gene Knockout Research Articles

Sevoflurane-induced cognitive decline in aged mice: Involvement of toll-like receptors 4

Toll-like receptors 4 (TLR4) contributes to the pathogenesis of some neurodegenerative diseases. However, little is known about whether TLR4 is associated with sevoflurane-induced cognitive decline. This investigation aims to address the effect of global TLR4 gene knockout on cognitive decline following sevoflurane exposure to mice. Wild-type and TLR4−/− mice were exposed to 3% sevoflurane. Novel object recognition test and Y-maze test were used to analyze cognitive function. Tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in plasma and hippocampus were measured by ELISA. Peripheral administration of recombinant TNF-α to TLR4−/− mice was used to observed the role of TNF-α in cognitive function following sevoflurane. Our results showed that, in contrast to wild-type mice, TLR4 deficiency protected against the cognitive function impairment following sevoflurane exposure, and abrogated IL-1β, IL-6 and TNF-α response to sevoflurane in the system and the hippocampus. Subcutaneous administration of recombinant TNF-α elevated these cytokine levels in the hippocampus, and resulted in cognitive decline in TLR4−/− mice exposed to sevoflurane. Taken together, our results identify the crucial role of TLR4 in sevoflurane-induced cognitive decline, and showed that TLR4 mediated pro-inflammatory cytokine response to sevoflurane, and consequent cognitive decline in aged mice exposed to sevoflurane, and imply a novel target for improvement and therapy of sevoflurane-associated cognitive decline.

Alzheimer's disease risk modifier genes do not affect tau aggregate uptake, seeding or maintenance in cell models.

Alzheimer's disease (AD) afflicts millions of people worldwide and is caused by accumulated amyloid beta and tau pathology. Progression of tau pathology in AD may utilize prion mechanisms of propagation in which pathological tau aggregates released from one cell are taken up by neighboring or connected cells and act as templates for their own replication, a process termed 'seeding'. We have used HEK293T cells to model various aspects of pathological tau propagation, including uptake of tau aggregates, induced seeding by exogenous aggregates, seeding caused by Lipofectamine-mediated delivery to the cell interior, and stable maintenance of aggregates in dividing cells. The factors that regulate these processes are not well understood, and we hypothesized that AD risk modifier genes might play a role. We identified 22 genes strongly linked to AD via meta-analysis of genome-wide association study (GWAS). We used CRISPR/Cas9 to individually knock out each gene in HEK293T cells and verified disruption using genomic sequencing. We then tested the effect of gene knockout in tau aggregate uptake, naked and Lipofectamine-mediated seeding, and aggregate maintenance in these cultured cell lines. GWAS gene knockouts had no effect in these models of tau pathology. With obvious caveats due to the model systems used, these results imply that the 22 AD risk modifier genes are unlikely to directly modulate tau uptake, seeding, or aggregate maintenance in a cell-autonomous fashion.

Microglial activation in the dorsal striatum participates in anxiety-like behavior in Cyld knockout mice

CYLD lysine 63 deubiquitinase (CYLD), that is mainly involved in immune responses and inflammation, is expressed at high levels in the brain, especially in the dorsal striatum, but its physiological function of CYLD in the brain remains unexplored. The present study investigated the effect of Cyld gene knockout on behavior relevant to the dorsal striatum, such as motor activity and depression-like and anxiety-like behavior. Microglia and the pro-inflammatory cytokines including interleukin (IL)-1 β and tumor necrosis factor (TNF)- α were evaluated in the dorsal striatum to elucidate the underlying mechanism. Cyld knockout (Cyld-/-) mice exhibited anxiety-like behavior, but not motor deficits or depression-like behavior. Microglia were activated and the mRNA levels of IL-1 β and TNF- α were increased in the dorsal striatum of Cyld-/- mice compared to Cyld+/+ mice. The microglial modulator minocycline partially reversed the anxiety-like behavior, microglial activation and increase in IL-1 β and TNF- α mRNA and protein levels in the dorsal striatum of Cyld-/- mice. Collectively, these results suggest that Cyld knockout leading to microglial activation promotes IL-1 β and TNF- α expression and acts as a critical pathway in the pathophysiology of anxiety.

Effect of urokinase gene knockout and chronic neurogenic pain on melanoma growth in the experiment.

e22099 Background: Studies have shown that the axis of the urokinase-type plasminogen activator (uPA–uPAR) has a pleiotropic effect in different stages of cancer. The purpose of the study was to reveal characteristics of the B16/F10 melanoma development in mice with the uPA gene knockout with and without chronic neurogenic pain (CNP). Methods: The study included male and female С57ВL/6 mice (n = 102) and C57BL/6-Plautm1.1BugThisPlauGFDhu/GFDhu mice with the uPA gene knockout (n = 48). B16/F10 melanoma was transplanted subcutaneously to the animals in the main groups 2 weeks after bilateral sciatic nerve ligation (CNP model). Mice with the standard melanoma transplantation were the controls. Results: The survival of uPA gene-knockout mice with melanoma was similar to that in controls and was 1.5 times (p < 0.05) longer in females than in males; the melanoma onset in these mice was observed a week earlier; the dynamics of tumor growth showed a pronounced gender dependence: in females, the tumor practically did not grow, and its volume before the death did not exceed 1.0 cm3, while in males the tumor was characterized by an active growth with two peaks of the volume increase (weeks 2 and 4); melanoma metastasized weakly - single metastases to the lungs (in females) or no metastases but with hemorrhages in the lungs and heart (in males). CNP reduced the survival of uPA gene-knockout females, similar to mice with the normal genome, but did not affect the survival of males; primary tumors in uPA gene-knockout mice emerged a few days later than in the control, but grew more intensively, with gender differences smoothed out; increased metastasis was demonstrated by the initiation of metastatic lesions of the lungs and liver in males, while hemorrhages in the lungs maintained, and by an increased number of metastases in the lungs with the appearance of lung hemorrhages in females. Conclusions: The uPA gene knockout differently changes the course of B16/F10 melanoma in male and female mice. CNP enhances the tumor growth, erasing gender differences, and activates metastasis of melanoma.

Role of Nrf2 in Regulation of Expression of CYP 2C11 and CYP 4X1 Epoxygenases and Production of Epoxyeicosatrienoic Acids (EETs) in the Rat Brain

Nuclear factor (erythroid‐derived 2)‐like‐2 (Nrf2) is the master antioxidant and cell‐protective transcription factor native in the rat brain. The CYP epoxygenases 2C11 and 4X1 are expressed in the brain and metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) that are known to induce cerebral vasodilation and increase nutritive blood flow to areas of heightened neuronal activity and also provide antioxidant and anti‐inflammatory actions. The influence of Nrf2 gene deletion on CYP 2C11 and 4X1 epoxygenase expression and activity in the rat brain is not known. In this study we used isoform specific antibodies and Western blotting to determine the effect of Nrf2 gene knockout (KO) on the expression of proteins for CYP 2C11 and CYP 4X1 epoxygenases in whole brain and isolated cerebral arteries. We also measured the production of epoxyeicosatrienoic acids (EETs) in homogenates of whole brain and isolated cerebral arteries of Nrf2 wild type (WT) and Nrf2 KO rats. CYP epoxygenase expression and EETs production were reduced in whole brain and arteries of the NRF2 KO rats compared to WT controls. These findings indicate that knockout of the Nrf2 gene leads to a marked reduction in the expression of proteins for CYP 2C11 and CYP 4X1 epoxygenases and in the production of 8, 9‐, 11,12‐ and 14,15‐EETs compared to those measured in the Nrf2 WT rat brain issues and cerebral arteries. The results of the current study suggest that CYP 2C11 and 4X1 epoxygenases may be components of the Nrf2‐induced phase 2 genes that are known to limit oxidative stress and provide cellular defense in the brain. We postulate that Nrf2 may regulate functions of the cerebral circulation and provide protection against oxidative stress, in part, via modulation of the expression level of CYP 2C11 and 4X1 epoxygenase proteins and the production of EETs.Support or Funding InformationNIH R01 #HL‐128242 and NIH #R21‐OD018309

Aquaporin 4 knockout increases complete freund's adjuvant-induced spinal central sensitization

Growing evidence suggests a critical role of astrocytes for pain regulation. The water channel protein aquaporin 4 (AQP4), a functional regulator of astrocytes, is involved in various neurological disorders. However, the pathophysiological roles of AQP4 in pain conditions remain unclear. In the present study, we investigated the effect of AQP4 gene knockout in central sensitization induced by complete Freund's adjuvant (CFA). The behavioral analysis revealed that mechanical allodynia and thermal hyperalgesia were more severe in AQP4 null mice than those of wild-type controls over the course of 11 days following CFA intraplantar injection. CFA caused activation of astrocytes with upregulated expression levels of AQP4 and glutamate transporter 1 (GLT1) in the dorsal horn of the spinal cord. AQP4 deficiency reduced GLT1 up-regulation, causing persistent expression of the neuronal activation marker Fos within superficial dorsal horn neurons, including glutamatergic neurons. However, AQP4 deletion did not affect CFA-evoked proinflammatory cytokine expression in the spinal cord. Together, these results have shown that AQP4 absence intensifies CFA-induced spinal central sensitization, which is associated with reduced compensatory up-regulation of GLT1, subsequently increasing glutamatergic overexcitation. Therefore, targeting spinal cord AQP4 may serve as a potential strategy for treatment of peripheral inflammation-evoked hyperalgesia.

Whole-transcriptome Analysis of Fully Viable Energy Efficient Glycolytic-null Cancer Cells Established by Double Genetic Knockout of Lactate Dehydrogenase A/B or Glucose-6-Phosphate Isomerase.

Nearly all mammalian tumors of diverse tissues are believed to be dependent on fermentative glycolysis, marked by elevated production of lactic acid and expression of glycolytic enzymes, most notably lactic acid dehydrogenase (LDH). Therefore, there has been significant interest in developing chemotherapy drugs that selectively target various isoforms of the LDH enzyme. However, considerable questions remain as to the consequences of biological ablation of LDH or upstream targeting of the glycolytic pathway. In this study, we explore the biochemical and whole transcriptomic effects of CRISPR-Cas9 gene knockout (KO) of lactate dehydrogenases A and B [LDHA/B double KO (DKO)] and glucose-6-phosphate isomerase (GPI KO) in the human colon cancer cell line LS174T, using Affymetrix 2.1 ST arrays. The metabolic biochemical profiles corroborate that relative to wild type (WT), LDHA/B DKO produced no lactic acid, (GPI KO) produced minimal lactic acid and both KOs displayed higher mitochondrial respiration, and minimal use of glucose with no loss of cell viability. These findings show a high biochemical energy efficiency as measured by ATP in glycolysis-null cells. Next, transcriptomic analysis conducted on 48,226 mRNA transcripts reflect 273 differentially expressed genes (DEGS) in the GPI KO clone set, 193 DEGS in the LDHA/B DKO clone set with 47 DEGs common to both KO clones. Glycolytic-null cells reflect up-regulation in gene transcripts typically associated with nutrient deprivation / fasting and possible use of fats for energy: thioredoxin interacting protein (TXNIP), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), PPARγ coactivator 1α (PGC-1α), and acetyl-CoA acyltransferase 2 (ACAA2). Other changes in non-ergometric transcripts in both KOs show losses in "stemness", WNT signaling pathway, chemo/radiation resistance, retinoic acid synthesis, drug detoxification, androgen/estrogen activation, and extracellular matrix reprogramming genes. These findings demonstrate that: 1) The "Warburg effect" is dispensable, 2) loss of the LDHAB gene is not only inconsequential to viability but fosters greater mitochondrial energy, and 3) drugs that target LDHA/B are likely to be ineffective without a plausible combination second drug target.

The urokinase gene knockout effects on growth factor dynamics in mice with melanoma, developing on the background of chronic neurogenic pain

Purpose of the study . Studying the dynamics of growth factors (GF) in urokinase (uPA)-deficient mice with chronic neurogenic pain (CNP) and B16/F10 melanoma. Materials and methods . Levels of VEGFA, VEGFC, sVEGFRl, sVEGFR3, IGF1, IGF2, TGFp1 and FGF21 were determined by ELISA in tumors, perifocal tissues (PT) and the skin of male and female С 57 BL/6 mice (with a normal genome, n = 75) and C57BL/6-Plautm1.1BugThisPlauGFDhu/GFDhu mice (uPA-deficient animals, n = 46) at 3nd week of the carcinogenesis and CNP. Results . The skin of intact uPA-deficient mice demonstrated higher GF levels than in C57BL/6 mice, but VEGF- А and TGF-p1 in males (unlike females) were 4.4 and 5 times lower than in C57BL/6 males. This changes were generally similar in the skin of C57BL/6 mice with CNP. uPA-deficient females showed elevated GF in PT, especially VEGF- А and IGF1 — by 21.5 and 8.1 times, respectively in simultaneously CNP and growth of the melanoma. uPA gene-knockout males had similar changes in GF, although less marked. The levels of all studied GF in tumor tissue were lower than levels in PT in both males and females, except for VEGFA in males — 5.6 times higher in tumor tissue. Changes in PT of C57BL/6 mice were similar: maximally increased levels of all GF, especially VEGF, IGF and TGF-p1 — in females on average by 6.2, 15.9 and 5.5 times, respectively, in males by 9.4, 5.9 and 6.7 times, respectively, compared to the skin levels. While the absolute values of GF concentrations and the intensity of changes were higher than in uPA-deficient mice. Conclusion . In general, skin levels of GF in intact uPA-deficient mice were similar to the levels in mice without uPA-deficient with CNP. The GF dynamics was analogous in mice of both lines at simultaneously CNP and growth of the melanoma, but the intensity of changes in mice without uPA-deficient was significantly higher implying a synergic effect of CNP and paracrine influence of melanoma on the GF levels.

Global and site-specific analysis of bone in a rat model of spinal cord injury-induced osteoporosis

Micro-Computed Tomography bone analysis is the gold standard method for assessing trabecular and cortical bone microarchitecture in small animal bones. This technique reports morphometric parameters as averages over selected volumes of interest (VOIs). This study proposes the introduction of an additional global 2D morphometric step into the analysis process, that provides a survey of the underlying morphometric variation present throughout both trabecular and cortical bone. The visualisation of these morphometric distributions provides a systematic approach to VOI selection that provides rationale and adds confidence to subsequent 3D morphometric analysis. To test the applicability and value of this methodological addition it was applied to the distal femur of a rat model of spinal cord injury (SCI)-induced osteoporosis. The 2D morphometric variation of both trabecular and cortical bone was quantified as a function of bone length. SCI-induced osteoporosis was localised in i) trabecular bone, where metaphyseal bone was more severely affected than epiphyseal bone, and there was a significant reduction in Distal Femoral Trabecular Extent, a new parameter defined here that quantifies how far trabecular bone penetrates in to the marrow cavity, ii) cortical bone, where diaphyseal bone underwent significant lowering of both cortical area and thickness, while distal-metaphyseal bone did not. Theses site-specific changes were validated, further elucidated and compared with follow-up conventional 3D analysis. The techniques applied here are equally applicable to other long bones (tibia, humerus, radius, ulna), other types of imaging modality and other types of experimental design including the effects of rehabilitation, aging, loading, gene knockout and pharmacological intervention.

A metabolic reconstruction of Lactobacillus reuteri JCM 1112 and analysis of its potential as a cell factory

BackgroundLactobacillus reuteri is a heterofermentative Lactic Acid Bacterium (LAB) that is commonly used for food fermentations and probiotic purposes. Due to its robust properties, it is also increasingly considered for use as a cell factory. It produces several industrially important compounds such as 1,3-propanediol and reuterin natively, but for cell factory purposes, developing improved strategies for engineering and fermentation optimization is crucial. Genome-scale metabolic models can be highly beneficial in guiding rational metabolic engineering. Reconstructing a reliable and a quantitatively accurate metabolic model requires extensive manual curation and incorporation of experimental data.ResultsA genome-scale metabolic model of L. reuteri JCM 1112T was reconstructed and the resulting model, Lreuteri_530, was validated and tested with experimental data. Several knowledge gaps in the metabolism were identified and resolved during this process, including presence/absence of glycolytic genes. Flux distribution between the two glycolytic pathways, the phosphoketolase and Embden–Meyerhof–Parnas pathways, varies considerably between LAB species and strains. As these pathways result in different energy yields, it is important to include strain-specific utilization of these pathways in the model. We determined experimentally that the Embden–Meyerhof–Parnas pathway carried at most 7% of the total glycolytic flux. Predicted growth rates from Lreuteri_530 were in good agreement with experimentally determined values. To further validate the prediction accuracy of Lreuteri_530, the predicted effects of glycerol addition and adhE gene knock-out, which results in impaired ethanol production, were compared to in vivo data. Examination of both growth rates and uptake- and secretion rates of the main metabolites in central metabolism demonstrated that the model was able to accurately predict the experimentally observed effects. Lastly, the potential of L. reuteri as a cell factory was investigated, resulting in a number of general metabolic engineering strategies.ConclusionWe have constructed a manually curated genome-scale metabolic model of L. reuteri JCM 1112T that has been experimentally parameterized and validated and can accurately predict metabolic behavior of this important platform cell factory.

The testis-specific expressed gene Spata34 is not required for fertility in mice.

It is estimated that more than two thousand genes exhibit testis-predominant expression pattern. The functions of hundreds of these genes have been explored during mouse spermatogenesis. However, there are still many genes whose relevance to reproduction in vivo remains unexplored. Our previous studies, as well as the other documented study, have indicated that Spata34, an evolutionarily conserved gene in metazoan species, was exclusively expressed in mouse testes and involved in spermatogenesis by regulating cell cycle progression. The present study aims to determine the effect of Spata34 gene knockout on mouse reproduction in vivo by generating a Spata34 gene knockout model using CRISPR/Cas9-mediated genome editing technology. We found that the Spata34 gene KO mice had normal fertility compared with wild type mice, and no overt detectable difference was found in testis/body weight ratios, testicular histology, sperm counts and spermatozoa motility parameters between WT and Spata34 KO mice. Our report indicated that the testis-specific-expressed gene Spata34 was not required for male mouse fertility, which will help to avoid unnecessary expenditures and effort by other researchers.

Repression of mitochondrial metabolism for cytosolic pyruvate-derived chemical production in Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae.ResultsThe growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. 13C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production.ConclusionThese results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.

Whole Genome CRISPR-Cas9 Screen Identifies STING as a Determinant of Intrinsic Radiosensitivity in Head and Neck Squamous Cell Carcinoma

Radiation therapy is a critical component of curative therapy for HNSCC. However, the mechanisms of radioresistance in HPV-Negative HNSCC, a molecularly distinct disease with poor prognosis relative to HPV-Positive HNSCC, are incompletely understood. Although RNA interference based screening methods have been used to understand resistance to radiotherapy, they are hampered by off-target effects and partial target suppression. The recent introduction of CRISPR technology has provided a more efficient method for gene targeting and suppression. As such we hypothesized that a whole genome CRISPR-Cas9 screen in HPV-Negative HNSCC would identify novel components of the cellular response to ionizing radiation. A whole genome CRISPR-Cas9 library containing 123,411 guide RNAs (gRNAs) targeting 19,050 genes was amplified, sequenced to ensure diversity, and packaged into lentivirus. HPV-Negative HNSCC cells (160 million) were transduced with lentivirus in a pooled format and irradiated. Following irradiation, 50 million cells per condition were collected (400x coverage) and gRNA enrichment or depletion determined via MAGeCK analysis of next-generation sequencing (NGS) results. Clonogenic assay (CA) was used to determine the effects of gene knockout (KO) on radiation and cisplatin sensitivity. Immunoblotting was performed to determine effects of knockout on protein expression. Tumor growth delay assays were performed using tumor cells implanted subcutaneously in athymic nude mice. Cas9 expression alone showed no effect on radiosensitivity by CA indicating feasibility to proceed with genetic screening. Analysis of NGS results show the top-ranked gRNAs enriched after radiation target STING (stimulator of interferon genes; p < 0.001; FDR <0.01), suggesting that STING KO imparts a radioresistant phenotype. Isogenic STING KO cell lines were established by CRISPR-Cas9 KO. CA shows that STING KO significantly enhanced radiation survival in multiple KO lines by a factor of 5-10. STING KO also conferred resistance to cisplatin treatment (3-7 fold) in CAs suggesting that STING regulates an intrinsic response to DNA damage. To determine the effects of STING KO on in vivo radiosensitivity, tumor growth delay assays were performed in immunodeficient mice. STING KO tumors were significantly more radioresistant when compared to isogenic STING proficient tumors (p< 0.05) with a dose modifying factor of 0.35. Whereas STING has previously been shown to regulate the extrinsic (immune-based) response of tumors to radiation, our data suggest that STING, as identified by a whole genome CRISPR-Cas9 screen, regulates the intrinsic response to radiation in HNSCC. This supports future investigation of strategies activating the STING pathway in combination with radiotherapy to enhance therapeutic response.

Abstract P2045: Genetic Disruption of Npr1 Depletes T Regulatory Cells and Provokes High Levels of Proinflammatory Cytokines and Fibrosis in the Kidneys of Female Mutant Mice

The aim of the present study was to determine the effects of gene-knockout of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) on immunogenic responses affecting kidney function and blood pressure (BP) in Npr1 (coding for GC-A/NPRA) null mutant mice. We used female Npr1 gene-disrupted ( Npr1 -/- , 0-copy), heterozygous ( Npr1 +/- , 1-copy), wild-type ( Npr1 +/+ , 2-copy) and gene-duplicated ( Npr1 ++/++ , 4-copy) mice. Expression levels of Toll-like receptor 2/4 (TLR2/TLR4) mRNA were increased 4- to 5-fold in 1-copy mice and 6- to 10-fold in 0-copy mice; protein levels were increased 2.5- to 3-fold in 1-copy mice and 4- to 5-fold in 0-copy mice. Expression of proinflammatory cytokines and BP was significantly elevated in 1-copy and 0-copy mice compared with 2-copy and 4-copy mice. In addition, 0-copy and 1-copy mice exhibited drastic reductions in T regulatory cells (Tregs). After rapamycin treatment, Tregs were increased by 17% ( P &lt; 0.001) in 0-copy mice and 8% ( P &lt; 0.001) in 1-copy mice. Renal mRNA and protein levels of TLR2 and TLR4 were decreased by 70% in 0-copy mice and 50% in 1-copy mice. There were significantly higher levels of Tregs and very low levels of TLR2/TLR4 expression in 4-copy mice (p &lt; 0.001). These findings indicate that the disruption of Npr1 in female mice triggers renal immunogenic pathways, which transactivate the expression of proinflammatory cytokines and renal fibrosis with elevated BP in mutant animals. The data suggest that rapamycin treatment attenuates proinflammatory cytokine expression, dramatically increases anti-inflammatory cytokines, and substantially reduces BP and renal fibrosis in mutant animals.

Effects of Stathmin 1 Gene Knockout on Behaviors and Dopaminergic Markers in Mice Exposed to Social Defeat Stress

Stathmin (STMN), a microtubule-destabilizing factor, can regulate fear, anxiety, and learning. Social defeat stress (SDS) has detrimental effects on mental health and increases the risk of various psychiatric diseases. This study investigated the effects of STMN1 gene knockout (KO) on behavioral parameters and dopaminergic markers using an SDS mouse model. The STMN1 KO mice showed anxious hyperactivity, impaired object recognition, and decreased levels of neutral and social investigating behaviors at baseline compared to wild-type (WT) mice. The impact of SDS on neutral, social investigating and dominant behaviors differed markedly between the STMN1 WT and KO mice. In addition, different levels of total DARPP-32 and pDARPP-32 Thr75 expression were observed among the control, unsusceptible, and susceptible groups of STMN1 KO mice. Our results show that STMN1 has specific roles in locomotion, object recognition, and social interactions. Moreover, SDS had differential impacts on social interactions and dopaminergic markers between STMN1 WT and KO mice.

Knockout of Sphingosine Kinase 1 Attenuates Renal Fibrosis in Unilateral Ureteral Obstruction Model

Background: Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in various diseases. S1P also plays significant roles in the differentiation of fibroblasts into myofibroblasts, being implicated in fibrotic diseases. S1P is produced by the phosphorylation of sphingosine catalyzed by sphingosine kinases (SphK1 and SphK2). It remains unclear if the activation of endogenous SphK1 contributes to fibrogenesis in kidneys. The present study determined the effect of SphK1 gene knockout (KO) on fibrotic markers in kidneys. Methods: The renal fibrosis was produced using the unilateral ureteral obstruction (UUO) model in wild-type (WT) and SphK1 gene KO mice. Renal mRNA levels of SphK1 and S1P receptors (S1PR) were measured by real-time RT-PCR. Fibrotic and immune cell markers in kidneys were measured by Western blot analysis and immunostaining, respectively. Renal morphological damage was examined by Periodic-Acid Schiff staining. Results: The mRNA levels of SphK1 and S1PRs were dramatically increased in renal tissues of WT-UUO mice, whereas the increase in renal SphK1 mRNA was blocked in KO-UUO mice. Interestingly, the increased levels of fibrotic markers, collagen and α-smooth muscle actin, in kidneys were significantly attenuated in KO-UUO versus WT-UUO mice. Meanwhile, kidney damage indices were remarkably attenuated in KO-UUO mice compared with WT-UUO mice. However, increased numbers of CD43+ and CD48+ cells, markers for T cell and macrophage, respectively, showed no significant difference between ­WT-UUO and KO-UUO kidneys. Conclusion: The activation of the SphK1-S1P pathway may contribute to tubulointerstitial fibrosis in UUO kidneys by affecting fibrotic signaling within renal cells independent of immune modulation.

Flux balance analysis with or without molecular crowding fails to predict two thirds of experimentally observed epistasis in yeast

Computational predictions of double gene knockout effects by flux balance analysis (FBA) have been used to characterized genome-wide patterns of epistasis in microorganisms. However, it is unclear how in silico predictions are related to in vivo epistasis, as FBA predicted only a minority of experimentally observed genetic interactions between non-essential metabolic genes in yeast. Here, we perform a detailed comparison of yeast experimental epistasis data to predictions generated with different constraint-based metabolic modeling algorithms. The tested methods comprise standard FBA; a variant of MOMA, which was specifically designed to predict fitness effects of non-essential gene knockouts; and two alternative implementations of FBA with macro-molecular crowding, which account approximately for enzyme kinetics. The number of interactions uniquely predicted by one method is typically larger than its overlap with any alternative method. Only 20% of negative and 10% of positive interactions jointly predicted by all methods are confirmed by the experimental data; almost all unique predictions appear to be false. More than two thirds of epistatic interactions are undetectable by any of the tested methods. The low prediction accuracies indicate that the physiology of yeast double metabolic gene knockouts is dominated by processes not captured by current constraint-based analysis methods.

Effect of gene knockout of L-tyrosine transport system on L-tyrosine production in Escherichia coli

L-tyrosine is one of three aromatic amino acids that are widely used in food, pharmaceutical and chemical industries. The transport system engineering provides an important research strategy for the metabolic engineering of Escherichia coli to breed L-tyrosine producing strain. The intracellular transport of L-tyrosine in E. coli is mainly regulated by two distinct permeases encoded by aroP and tyrP genes. The aroP and tyrP gene knockout mutants were constructed by CRISPR-Cas technique on the basis of L-tyrosine producing strain HGXP, and the effects of regulating transport system on L-tyrosine production were investigated by fermentation experiments. The fermentation results showed that the aroP and tyrP knockout mutants produced 3.74 and 3.45 g/L L-tyrosine, respectively, which were 19% and 10% higher than that of the original strain. The optimum induction temperature was determined to be 38 °C. Fed-batch fermentation was carried out on a 3-L fermentor. The L-tyrosine yields of aroP and tyrP knockout mutants were further increased to 44.5 and 35.1 g/L, respectively, which were 57% and 24% higher than that of the original strain. The research results are of great reference value for metabolic engineering of E. coli to produce L-tyrosine.

Basal Autophagy Deficiency Causes Thyroid Follicular Epithelial Cell Death in Mice.

Autophagy is a catabolic process that involves the degradation of cellular components through the lysosomal machinery, relocating nutrients from unnecessary processes to more pivotal processes required for survival. It has been reported that systemic disruption of the Atg5 or Atg7 gene, a component of autophagy, is lethal and that its tissue-specific disruption causes tissue degeneration in several organs. However, the functional significance of autophagy in the thyroid glands remains unknown. Our preliminary data imply the possible involvement of dysfunctional autophagy in radiation-induced thyroid carcinogenesis. Therefore, we evaluated the effect of Atg5 gene knockout (KO) on thyroid morphology and function. To this end, Atg5flox/flox mice were crossed with TPO-Cre mice, yielding the thyroid follicular epithelial cell (thyrocyte)‒specific ATG5-deficient mice (Atg5thyr-KO/KO). Atg5 gene KO was confirmed by a lack of ATG5 expression, and disruption of autophagy was demonstrated by a decrease in microtubule-associated protein 1 light chain 3-II puncta and an increase in p62. Atg5thyr-KO/KO mice were born normally, and thyroid morphology, thyroid weights, and serum T4 and TSH levels were almost normal at 4 months. However, at 8 and 12 months, a decrease in the number of thyrocytes and an increase in TUNEL+-thyrocytes were observed in Atg5thyr-KO/KO mice even though thyroid function was still normal. The number of irregularly shaped (gourd-shaped) follicles was also increased. Excess oxidative stress was indicated by increased 8-hydroxy-2'-deoxyguanosine and 53BP1 foci in Atg5thyr-KO/KO mice. These data demonstrate that thyrocytes gradually undergo degradation/cell death in the absence of basal levels of autophagy, indicating that autophagy is critical for the quality control of thyrocytes.

Effects of FABP7 on functional recovery after spinal cord injury in adult mice.

Elucidating the mechanisms of neuronal injury is crucial for the development of spinal cord injury (SCI) treatments. Brain-type fatty acid-binding protein 7 (FABP7) is expressed in the adult rodent brain, especially in astrocytes, and has been reported to play a role in astrocyte function in various types of brain damage; however, its role after SCI has not been well studied. In this study, the authors evaluated the expression change of FABP7 after SCI using a mouse spinal cord compression model and observed the effect of FABP7 gene knockout on neuronal damage and functional recovery after SCI. Female FABP7 knockout (KO) mice with a C57BL/6 background and their respective wild-type littermates were subjected to SCI with a vascular clip. The expression of FABP7, neuronal injury, and functional recovery after SCI were analyzed in both groups of mice. Western blot analysis revealed upregulation of FABP7 in the wild-type mice, which reached its peak 14 days after SCI, with a significant difference in comparison to the control mice. Immunohistochemistry also showed upregulation of FABP7 at the same time points, mainly in proliferative astrocytes. The number of surviving ventral neurons in the FABP7-KO mice at 28 days after SCI was significantly lower than that observed in the wild-type mice. In addition, motor functional recovery in the FABP7-KO mice was significantly worse than that of the wild-type mice. The findings of this study indicate that FABP7 could have a neuroprotective role that might be associated with modulation of astrocytes after SCI. FABP7 could potentially be a therapeutic target in the treatment of SCI.