Knockout Research Articles

The impact of inducible-whole body or intestine-specific Cyp24a1 gene knockout on vitamin D metabolism in mice.

The impact of inducible-whole body or intestine-specific Cyp24a1 gene knockout on vitamin D metabolism in mice.

OsCHR728 encodes a chromatin remodeling factor involved in seed size and grain chalkiness in rice.

OsCHR728 encodes a chromatin remodeling factor involved in seed size and grain chalkiness in rice.

Do progesterone receptor membrane components (PGRMC)s play a role in the chorions refractoriness to epithelial-to-mesenchymal transition (EMT)?

Do progesterone receptor membrane components (PGRMC)s play a role in the chorions refractoriness to epithelial-to-mesenchymal transition (EMT)?

GEF-H1 drives breast cancer cells to tumor progression.

GEF-H1 drives breast cancer cells to tumor progression.

Targeting the TGFβ signaling pathway to mitigate tumor metastasis in 9p21-loss urothelial bladder cancer.

4566 Background: We previously identified that 9p21-loss (containing MTAP and CDK2NA genes) occurs in 25% of metastatic urothelial cancer (mUC) and is associated with worse survival and increased visceral metastasis. Additionally, we found significantly increased expression of the TGFβ-SMAD3 pathway in 9p21-loss UC, which is well studied to induce epithelial mesenchymal transition (EMT) and lead to metastasis. We hypothesize that the TGFβ signaling pathway mediates visceral metastasis in 9p21-loss UC. Methods: We included mUC patients at MD Anderson Cancer Center (MDACC) who had MTAP testing completed (surrogate for 9p21-loss) and received chemotherapy, immune checkpoint therapy (ICT), and/or antibody drug conjugate between 2012 and 2022. Survival and metastasis were compared between MTAP deficient patients and MTAP proficient patients. The Memorial Sloan Kettering-Metastatic Events and Tropisms (MSK-MET) cohort of mUC patients and IMVigor210 dataset were assessed for genomic alterations. Mouse models bearing CDKN2A-MTAP double knock out (DKO) MB49 tumors and wild type (WT) MB49 tumors, as well as corresponding in vitro models were used for mechanistic studies. Results: 298 mUC patients at MDACC were identified with 27% (n = 81) MTAP deficient. MTAP deficient patients experienced significantly worse overall survival (16.2 vs 21.1 months; p = 0.002; HR 1.61; 95% CI 1.2-2.2), progression-free survival (3.9 vs 5.8 months; p < 0.001; HR 1.75; 95% CI 1.3-2.4) and increased visceral metastasis (62% vs 39%; p = 0.001) with lung predominance (44% vs 26%; p = 0.003) compared to MTAP proficient patients. We also found in the MSK-MET cohort that 26% (186/714) of patients with mUC to the bladder had lung metastasis and those with lung metastasis had significantly increased frequency of CDK2NA deletion compared to patients without lung metastasis (30% vs 18%; OR 2.0; 95% CI 1.3-3.0). Our preclinical data also demonstrated that CDKN2A-MTAP DKO mice resulted in significantly larger primary bladder tumors and worse survival compared to mice bearing WT MB49 tumors. Additionally, mice bearing orthotopic CDKN2A-MTAP DKO MB49 tumors readily developed lung metastasis. Analysis of the IMvigor210 dataset (N = 298) showed that 9p21-loss UC patients had significantly increased expression of TGFβ and EMT pathway genes. These data are consistent with data from our DKO MB49 tumor models. We are currently assessing the impact of a TGFβ inhibitor on mitigating CDKN2A-MTAP DKO-mediated metastasis. Conclusions: Our clinical and pre-clinical data demonstrates that 9p21-loss mUC leads to worse survival and increased visceral metastasis, especially to the lung. We found that the TGFβ signaling pathway may play a role in the development of metastasis. Moving forward, we plan to prospectively investigate the therapeutic potential of TGFβ inhibition in patients with 9p21-loss UC.

Unraveling antipsychotic induced weight gain in schizophrenia - A proof-of-concept study exploring the impact of the cumulative historical occupancy of different receptors by antipsychotics.

Unraveling antipsychotic induced weight gain in schizophrenia - A proof-of-concept study exploring the impact of the cumulative historical occupancy of different receptors by antipsychotics.

Profiling of lincRNAs and differential regulatory mechanisms in response to nanoplastic toxicity at environmentally relevant concentrations in Caenorhabditis elegans.

Profiling of lincRNAs and differential regulatory mechanisms in response to nanoplastic toxicity at environmentally relevant concentrations in Caenorhabditis elegans.

Iron overload exacerbates metabolic dysfunction-associated steatohepatitis via the microbiota-gut-liver axis through lipopolysaccharide-mediated Akr1b8 activation.

Iron overload exacerbates metabolic dysfunction-associated steatohepatitis via the microbiota-gut-liver axis through lipopolysaccharide-mediated Akr1b8 activation.

Comparative analysis of the anti-tumor activity and mechanism of action of TARA-002 and BCG in bladder cancer models.

e16613 Background: Intravesical instillations of Bacillus Calmette-Guérin (BCG) is the current standard of care for high-risk Non-Muscle Invasive Bladder Cancer (NMIBC) patients. TARA-002, a preparation of inactivated Streptococcus pyogenes, is currently in a Phase-2 (ADVANCED-2) clinical trial for the treatment of NMIBC. Interim analysis found that the rates of high-grade complete response (CR) at month 6 were 72% overall, 64% in BCG-naïve subjects and 100% in BCG-unresponsive patients with TARA-002 treatment. As TARA-002 and BCG are regarded as broad immunopotentiators, we compared the anti-tumor activity and mechanisms of action of TARA-002 and BCG using bladder cancer models. Methods: TARA-002 internalization in HEK293 Wild Type (WT) and TLR2 Knockout (KO) and Bladder Cancer (BC) cells (RT112 and T24) was evaluated by confocal microscopy. Flow Cytometry analysis of Annexin V, marker of apoptosis, was used to evaluate TARA-002 and BCG cytotoxicity in mouse and rat BC cells (MBT2, MB49 and AY27) after 24 h treatment. Cytokine analysis was conducted on 5637 BC cells co-cultured with peripheral blood mononuclear cells (PBMCs) following treatment with either BCG or TARA-002 for 24 h. Lastly, the MB49 orthotopic bladder cancer mouse model evaluated tumor growth inhibition and survival upon TARA-002 intravesical treatment. Results: Unlike BCG, which relies on fibronectin receptors for internalization, TARA-002 internalization was unaffected by fibronectin receptor blockade and was instead shown to be TLR2-dependent as the HEK293 TLR2-KO cells consistently displayed lower levels of intracellular TARA-002 compared to the WT counterpart. Furthermore, TARA-002 exhibited significant dose-dependent cytotoxicity against BC cell lines, while BCG showed only modest tumor-killing effects. Co-culture of 5637 BC cells and PBMCs showed that TARA-002 treatment increased pro-inflammatory Th1 cytokines (TNF-a, IFN-g, IL-12p70) and reduced IL-8 levels, a marker linked to poor BC prognosis, compared to BCG. Finally, intravesical instillations of TARA-002 in the BCG-unresponsive MB49 orthotopic model showed a strong anti-tumor effect and significantly enhanced survival outcomes. Since BCG exposure induces trained immunity, it is plausible that BCG-induced trained immunity may enhance the response to TARA-002, potentially explaining its improved efficacy in BCG-treated patients compared to the BCG-naïve population. To evaluate this hypothesis, preclinical tests are being conducted at the time of submission. Conclusions: Our results suggest that the mechanism of action of TARA-002 differs from that of BCG, leading to variations in their biological activity. This leads to more potent anti-tumor activity of TARA-002 in both in vitro and in vivo models compared to BCG, accompanied by an enhanced immune-mediated pro-inflammatory response.

CNPY2 drives DSS-induced colitis via the macrophage-ROS axis.

CNPY2 drives DSS-induced colitis via the macrophage-ROS axis.

Mitochondrial Ca2+ uniporter b (MCUb) regulates neuronal Ca2+ dynamics and resistance to ischemic stroke.

Mitochondrial Ca2+ uniporter b (MCUb) regulates neuronal Ca2+ dynamics and resistance to ischemic stroke.

SMARCD1 is a dual regulator of PD-L1 expression and cell proliferation facilitating tumor evasion.

SMARCD1 is a dual regulator of PD-L1 expression and cell proliferation facilitating tumor evasion.

Piezo1 promotes double-directional differentiation from human periodontal ligament progenitor cells.

Piezo1 promotes double-directional differentiation from human periodontal ligament progenitor cells.

Insights from selenoprotein I mouse models for understanding biological roles of this enzyme.

Insights from selenoprotein I mouse models for understanding biological roles of this enzyme.

Plasminogen and plasmin induce specialized proresolving mediators and promote efferocytosis via 5-lipoxygenase.

Plasminogen and plasmin induce specialized proresolving mediators and promote efferocytosis via 5-lipoxygenase.

Interrogating mediators of single-cell transcriptional changes in the acute damaged cerebral cortex: Insights into endothelial-astrocyte interactions.

Interrogating mediators of single-cell transcriptional changes in the acute damaged cerebral cortex: Insights into endothelial-astrocyte interactions.

Purinergic receptors play a key role in shock wave-induced proliferation

Shock wave treatment (SWT) is a non-invasive therapy applied in musculoskeletal and urological disorders, as well as in chronic wound regeneration. As the use of medical SWT broadens, it is important to better understand the molecular mechanisms underlying its success. Here, we identified P2X4 and P2Y2 purinergic receptors to be primarily expressed in C3H/10T1/2 mouse mesenchymal stromal cells and investigated their role in the initiation of the signaling events following SWT using single- and double-receptor knock-out (KO) cell lines. We show that SWT induced the expression of c-Jun and c-Fos within 30 min after stimulation and that the SWT-induced Erk1/2 pathway activation and immediate early gene expression were decreased in P2Y2-, P2X4- and P2Y2/P2X4-deficient cells. Importantly, SWT did not promote proliferation in P2Y2/P2X4-deficient cells, while loss of either one of the receptors significantly reduced the proliferative effect, indicating a cumulative effect of their loss. Finally, our data suggests a more prominent role of the P2Y2 receptor in SWT-induced cellular effects, since primarily its loss contributed to the observed changes. With these findings, we further the understanding of the molecular mechanisms of SWT and propose that the varying expression of purinergic receptors in tissues should be considered when establishing treatment protocols.

Pharmacological Enhancement of Small Conductance Ca2+-Activated K+ Channels Suppresses Cardiac Arrhythmias in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia.

Sarcolemmal small conductance Ca2+-activated K+ channels have the unique capacity to translate intracellular Ca2+ signal into repolarization, while mitochondrial SK channels can link Ca2+ cycling to mitochondrial function. We hypothesize that pharmacological enhancement of SK channels can be protective against malignant cardiac arrhythmias associated with disturbances in Ca2+ handling machinery. A mouse CASQ2 KO (calsequestrin type 2 knockout) model of catecholaminergic polymorphic ventricular tachycardia (CPVT) was used for in vivo ECG recordings and for cell electrophysiology, Ca2+, and reactive oxygen species imaging in isolated ventricular myocytes (VMs). Bidirectional and polymorphic ventricular tachycardias in CASQ2 KO mice induced by stress challenge (epinephrine+caffeine cocktail) were attenuated by injection of NS309, a specific SK channel enhancer. Voltage-clamp experiments in isolated VMs treated with β-adrenergic agonist isoproterenol showed a reduction of sarcolemmal SK channel current (ISK) density in CPVT VMs. Application of NS309 to CPVT VMs increased ISK. Current-clamp experiments demonstrated a significant reduction of arrhythmogenic delayed afterdepolarizations and spontaneous Ca2+ waves in isoproterenol-challenged CPVT VMs pretreated with NS309. Importantly, subsequent application of membrane-impermeable SK channel inhibitor apamin did not reverse the protective effects of NS309, suggesting important roles of mitochondrial SK channels in intracellular Ca2+ handling rescue. SK channel enhancement reversed the increased rate of reactive oxygen species production by mitochondria in CPVT VMs. It also reversed increased cardiac RyR2 (ryanodine receptor 2) oxidation measured in samples from CPVT hearts of the animals after the stress challenge. Electron microscopy studies showed a significant widening of mitochondria cristae in the ventricular tissue from CPVT mice, which led to a decrease in quaternary supercomplexes of electron transport chain, resulting in impairment of ATP production in VMs under β-adrenergic stimulation. Application of NS309 facilitated cristae flattening in CPVT ventricular tissue and restored supercomplexes and ATP production in VMs from diseased animals. Sarcolemmal SK channel enhancement reduces arrhythmic potential by restoring repolarization force in CPVT VMs. Activation of mitochondrial SK channels attenuates mitochondria structural changes in CPVT, restoring more efficient electron transport chain assembly into supercomplexes and reducing mito-reactive oxygen species production. This decreases RyR2 oxidation and thus channel activity, reducing spontaneous Ca2+ waves underlying arrhythmogenic delayed afterdepolarizations.

Disruption of Retriever Function Impacts Retrograde Trafficking From Endosomes.

Within endosomes cargo proteins are sorted and packaged into endosomal-transport carriers (ETCs) enabling their delivery to other intracellular compartments. Retromer, a conserved multimeric protein complex, has defined functions in sorting cargo mediating formation of ETCs for both retrograde trafficking back to the trans-Golgi network (TGN) and recycling of cargo to the cell surface. Recent studies have identified the retriever complex, which is structurally like retromer, that also can function in the recycling of cargo from endosomes. However, retriever's function in retrograde trafficking from endosomes has not been investigated. CrispR mediated knock-out cell models for retromer and retriever in A549 lung carcinoma epithelial cells were generated. Retriever's role in recycling of established cargo Integrin β1 in A549 cells was confirmed. Cation-independent mannose 6-phosphate receptor and TGN46, two well-established retrograde cargos showed a redistribution from the TGN to early endosomes in retromer knockout (KO) and retriever KO cells which is consistent with decreased retrograde trafficking. Application of a ETC redirection assay in A549 cells identified that ETCs dependent on either retromer or retriever can be tethered by Golgin97 and Golgin245, but not GCC88. Overall, the presence of retriever is required for efficient endosomal retrograde trafficking. Within A549 cells, the requirement of retromer for Wiskott-Aldrich Syndrome Protein and SCAR Homolog (WASH) endosomal recruitment was confirmed while recruitment of WASH in the absence of retriever was not reduced. Furthermore, the efficient recruitment of retriever to endosomes was dependent on retromer. The underlying mechanism by which these complexes initiate the formation of retrograde ETCs therefore seems distinct.

Prebiotics Rescue Gut Microbiome Dysregulation and Enhance Cognitive and Gastrointestinal Function in a Mouse Model of Schizophrenia.

Schizophrenia is a devastating psychiatric disorder characterized by positive (eg, hallucinations) and negative (eg, reduced motivation) symptoms, and cognitive deficits. Chronic gastrointestinal tract issues exist as comorbid symptoms of schizophrenia. Recent findings indicate the involvement of the microorganisms that inhabit the gut, the microbiota (and the broader microbiome which also includes microbial genomes, etc.) in schizophrenia pathogenesis. In the present study, we hypothesized that chronic administration with prebiotics fructooligosaccharide and galactooligosaccharide (FOS and GOS; a combination used clinically for other disorders) would restore gut microbiome composition of the metabotropic glutamate receptor 5 (mGlu5) knockout (KO) mouse model of schizophrenia, which we previously demonstrated to exhibit gut dysbiosis. We assessed the impact of prebiotics on gut microbiome composition and function, as well as the gastrointestinal function and schizophrenia-like phenotype of mGlu5 KO mice and wild-type littermates. We administered a combination of the prebiotics FOS and GOS, vs vehicle control administration, in both the mouse model of schizophrenia and wild-type littermates. The present study firstly corroborated the altered gut microbiome composition in the mGlu5 KO mouse model of schizophrenia. Importantly, we have revealed an altered microbial metabolic profile. We have also shown that the prebiotics we administered were not only able to rescue these gut microbiome changes but also had additional beneficial effects including cognitive enhancement and improved gastrointestinal function. These preclinical findings indicate that prebiotics, such as the combination of FOS and GOS used in the present study, may have therapeutic potential in schizophrenia as an add-on intervention with an exceptional safety profile.