OPA1 helical structures give perspective to mitochondrial dysfunction.

Dominant optic atrophy is one of the leading causes of childhood blindness. Around 60-80% of cases1 are caused by mutations of the gene that encodes optic atrophy protein 1 (OPA1), a protein that has a key role in inner mitochondrial membrane fusion and remodelling of cristae and is crucial for the dynamic organization and regulation of mitochondria2. Mutations in OPA1 result in the dysregulation of the GTPase-mediated fusion process of the mitochondrial inner and outer membranes3. Here we used cryo-electron microscopy methods to solve helical structures of OPA1 assembled on lipid membrane tubes, in the presence and absence of nucleotide. These helical assemblies organize into densely packed protein rungs with minimal inter-rung connectivity, and exhibit nucleotide-dependent dimerization of the GTPase domains-a hallmark of the dynamin superfamily of proteins4. OPA1 also contains several unique secondary structures in the paddle domain that strengthen its membrane association, including membrane-inserting helices. The structural features identified in this study shed light on the effects of pathogenic point mutations on protein folding, inter-protein assembly and membrane interactions. Furthermore, mutations that disrupt the assembly interfaces and membrane binding of OPA1 cause mitochondrial fragmentation in cell-based assays, providing evidence of the biological relevance of these interactions.

Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens.

Optical imaging of the dynamics of living specimens involves tradeoffs between spatial resolution, temporal resolution, and phototoxicity, made more difficult in three dimensions. Here, however, we report that rapid three-dimensional (3D) dynamics can be studied beyond the diffraction limit in thick or densely fluorescent living specimens over many time points by combining ultrathin planar illumination produced by scanned Bessel beams with super-resolution structured illumination microscopy. We demonstrate in vivo karyotyping of chromosomes during mitosis and identify different dynamics for the actin cytoskeleton at the dorsal and ventral surfaces of fibroblasts. Compared to spinning disk confocal microscopy, we demonstrate substantially reduced photodamage when imaging rapid morphological changes in D. discoideum cells, as well as improved contrast and resolution at depth within developing C. elegans embryos. Bessel beam structured plane illumination thus promises new insights into complex biological phenomena that require 4D subcellular spatiotemporal detail in either a single or multicellular context.

Multiview confocal super-resolution microscopy.

Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.

The physical and cellular mechanism of structural color change in zebrafish.

Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.

A myosin-7B-dependent endocytosis pathway mediates cellular entry of α-synuclein fibrils and polycation-bearing cargos.

Cell-to-cell transmission of misfolding-prone α-synuclein (α-Syn) has emerged as a key pathological event in Parkinson's disease. This process is initiated when α-Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFFs to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFFs. By contrast, MYO7B regulates α-Syn PFF cell entry by maintaining a plasma membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing "scars" on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is restricted to α-Syn PFFs and other polycation-bearing cargos that enter cells via HSPGs. Thus, our study not only defines regulatory factors for α-Syn PFF endocytosis, but also reveals a previously unknown endocytosis mechanism for HSPG-binding cargos in general, which requires forces generated by MYO7B and actin filaments.

Single-shot super-resolution total internal reflection fluorescence microscopy.

We combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, thereby improving the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, and enabling imaging frame rates up to 100 Hz over hundreds of time points. We applied instant TIRF-SIM to multiple live samples and achieved rapid, high-contrast super-resolution imaging close to the coverslip surface.

Evodiamine protects against airway remodelling and inflammation in asthmatic rats by modulating the HMGB1/NF-κB/TLR-4 signalling pathway.

CONTEXT: Evodiamine, which is isolated from Evodia rutaecarpa (Rutaceae), possess strong anti-inflammatory, immunomodulatory, and antibacterial properties. OBJECTIVE: The protective effects of evodiamine in asthma were evaluated. MATERIALS AND METHODS: Thirty-two Sprague-Dawley (SD) rats were used, asthma was induced by injecting intraperitoneally with a mixture of Al(OH)3 (100 mg) and ovalbumin (OA; 1 mg/kg), further exposing them to a 2% OA aerosol for 1 week. All animals were divided into four groups: control, asthma, and evodiamine 40 and 80 mg/kg p.o. treated group. Serum levels of inflammatory cytokines, interferon gamma (IFN-γ), and immunoglobulin E (IgE) and infiltrations of inflammatory cells in the bronchoalveolar lavage fluid (BALF) of the animals were determined. The thickness of the smooth muscle layer and airway wall in the intact small bronchioles of asthmatic rats was examined as well. RESULTS: Cytokine levels in the serum and BALF were lower in the evodiamine-treated group than in the asthma group. Evodiamine treatment reduced IgE and IFN-γ levels as well as the inflammatory cell infiltrate in the lung tissue of asthmatic rats. The thickness of the smooth muscle layer and airway wall of intact small bronchioles was less in the evodiamine-treated group than in the asthma group. Lower levels of TLR-4, MyD88, NF-κB, and HMGB1 mRNA in lung tissue were measured in the evodiamine-treated group than in the asthma group. DISCUSSION AND CONCLUSION: The effect of evodiamine treatment protects the asthma, as evodiamine reduces airway inflammation and remodelling in the lung tissue by downregulating the HMGB1/NF-κB/TLR-4 pathway in asthma.

Circular RNA hsa_circ_0000263 participates in cervical cancer development by regulating target gene of miR-150-5p.

Circular RNA (circRNA) is a new class of noncoding RNA, and plays an important role in many pathological processes. Cervical cancer is the most common gynecologic malignant tumor. Recently, studies have shown that there is a variety of circRNA involved in the pathogenesis of cervical cancer. We screened out the highly expressed hsa_circ_0000263 from GSE102686 by the quantitative real-time polymerase chain reaction assay in cervical cancer cell lines. In this study, we investigated whether hsa_circ_0000263 might affect cell proliferation, migration, cell cycle and apoptosis in cervical cancer in vitro and in vivo. The luciferase reporter assay and RNA immunoprecipitation assay confirmed the direct interaction between miR-150-5p and hsa_circ_0000263. By using western blot and immunohistochemistry, we confirmed that hsa_circ_0000263 can regulate the expression of murine double minute 4 (MDM4) by affecting miR-150-5p, and finally affect the expression of p53 gene. We found that hsa_circ_0000263 was significantly upregulated in cervical cancer cells. In addition, the knockdown of hsa_circ_0000263, would inhibit cell proliferation and migration ability. In conclusion, our current research reveals the important role of hsa_circ_0000263/miR-150-5p/MDM4/p53 regulatory network in cervical cancer and provides a new insight into the pathogenesis of cervical cancer.

V-1 regulates capping protein activity in vivo.

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

Cooperative interactions of LPPR family members in membrane localization and alteration of cellular morphology.

The lipid phosphate phosphatase-related proteins (LPPRs), also known as plasticity-related genes (PRGs), are classified as a new brain-enriched subclass of the lipid phosphate phosphatase (LPP) superfamily. They induce membrane protrusions, neurite outgrowth or dendritic spine formation in cell lines and primary neurons. However, the exact roles of LPPRs and the mechanisms underlying their effects are not certain. Here, we present the results of a large-scale proteome analysis to determine LPPR1-interacting proteins using co-immunoprecipitation coupled to mass spectrometry. We identified putative LPPR1-binding proteins involved in various biological processes. Most interestingly, we identified the interaction of LPPR1 with its family member LPPR3, LPPR4 and LPPR5. Their interactions were characterized by co-immunoprecipitation and colocalization analysis using confocal and super-resolution microscopy. Moreover, co-expressing two LPPR members mutually elevated their protein levels, facilitated their plasma membrane localization and resulted in an increased induction of membrane protrusions as well as the phosphorylation of S6 ribosomal protein. Taken together, we revealed a new functional cooperation between LPPR family members and discovered for the first time that LPPRs likely exert their function through forming complex with its family members.

Melanoregulin regulates a shedding mechanism that drives melanosome transfer from melanocytes to keratinocytes.

Mammalian pigmentation is driven by the intercellular transfer of pigment-containing melanosomes from the tips of melanocyte dendrites to surrounding keratinocytes. Tip accumulation of melanosomes requires myosin Va, because melanosomes concentrate in the center of melanocytes from myosin Va-null (dilute) mice. This distribution defect results in inefficient melanosome transfer and a dilution of coat color. Dilute mice that simultaneously lack melanoregulin, the product of the dilute suppressor locus, exhibit a nearly complete restoration of coat color, but, surprisingly, melanosomes remain concentrated in the center of their melanocytes. Here we show that dilute/dsu melanocytes, but not dilute melanocytes, readily transfer the melanosomes concentrated in their center to surrounding keratinocytes in situ. Using time-lapse imaging of WT melanocyte/keratinocyte cocultures in which the plasma membranes of the two cells are marked with different colors, we define an intercellular melanosome transfer pathway that involves the shedding by the melanocyte of melanosome-rich packages, which subsequently are phagocytosed by the keratinocyte. Shedding, which occurs primarily at dendritic tips but also from more central regions, involves adhesion to the keratinocyte, thinning behind the forming package, and apparent self-abscission. Finally, we show that shedding from the cell center is sixfold more frequent in cultured dilute/dsu melanocytes than in dilute melanocytes, consistent with the in situ data. Together, these results explain how dsu restores the coat color of dilute mice without restoring intracellular melanosome distribution, indicate that melanoregulin is a negative regulator of melanosome transfer, and provide insight into the mechanism of intercellular melanosome transfer.

Mechanisms underlying Myosin 10's contribution to the maintenance of mitotic spindle bipolarity.

Myosin 10 (Myo10) couples microtubules and integrin-based adhesions to movement along actin filaments via its microtubule-binding MyTH4 domain and integrin-binding FERM domain, respectively. Here we show that Myo10-depleted HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in Myo10-depleted MEFs and in Myo10-depleted HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates y-tubulin-positive acentriolar foci that serve as extra spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both microtubules and integrins to promote PCM/pole integrity. Conversely, Myo10 only needs interact with integrins to promote supernumerary centrosome clustering. Importantly, images of metaphase Halo-Myo10 knockin cells show that the myosin localizes exclusively to the spindle and the tips of adhesive retraction fibers. We conclude that Myo10 promotes PCM/pole integrity in part by interacting with spindle microtubules, and that it promotes supernumerary centrosome clustering by supporting retraction fiber-based cell adhesion, which likely serves to anchor the microtubule-based forces driving pole focusing.

Protective effect of zerumbone on sepsis-induced acute lung injury through anti-inflammatory and antioxidative activity via NF-κB pathway inhibition and HO-1 activation.

Sepsis is a systemic illness for which there are no effective preventive or therapeutic therapies. Zerumbone, a natural molecule, has anti-oxidative and anti-inflammatory properties that may help to prevent sepsis. In the present study, we have assessed the protective effect of zerumbone against sepsis-induced acute lung injury (ALI) and its underlying mechanisms. During the experiment, mice were divided into five groups: a sham group, a sepsis-induced ALI group, and three sepsis groups that are pre-treated with zerumbone at different concentrations. We found that zerumbone greatly decreased the sepsis-induced ALI using histological investigations. Also, zerumbone treatment reduced the sepsis-induced inflammatory cytokine concentrations as well as the number of infiltrating inflammatory cells in BALF compared to non-treated sepsis animals. The zerumbone-pretreated sepsis groups had reduced pulmonary myeloperoxidase (MPO) activity than the sepsis groups. Moreover, the mechanism underlying the protective action of zerumbone on sepsis is accomplished by the activation of antioxidant genes such as nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), superoxide dismutase (SOD), and heme oxygenase 1 (HO-1). The obtained results revealed that zerumbone inhibited the sepsis-induced ALI through its anti-inflammatory and antioxidative activity via inhibition of the NF-κB pathway and activation of HO-1 pathway. Our findings demonstrate that zerumbone pretreatment suppresses sepsis-induced ALI via antioxidative activities and anti-inflammatory, implying that zerumbone could be a viable preventive agent for sepsis-induced ALI.

Detection of DNA Methylation in Gene Loci ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671 for Diagnosis of Cervical Cancer.

Objective: To evaluate the diagnostic value of DNA methylation detection of multiple gene loci in cervical cancer. Methods: A total of 61 cases requiring cervical biopsy were selected from the outpatient clinic of Maternal and Child Health Hospital of Hubei Province between January 2018 and December 2019. The patients were divided into four groups based on histopathologic diagnosis: cervical cancer (CC) group, high-grade squamous intraepithelial lesion (HSIL) group, low-grade squamous intraepithelial lesion (LSIL) group, and control group. HPV examination, liquid-based cytology examination, and DNA methylation detection at multiple gene sites were performed. The positive rate of DNA methylation, sensitivity, specificity, area under the curve (AUC), and other efficacy indexes were calculated to evaluate the diagnostic value of DNA methylation detection at multiple gene loci in cervical cancer. Results: The positive rates of DNA methylation in CC, HSIL, LSIL, and control groups were 100%, 88%, 83% and 17%, respectively. The ZNF671 gene had the highest positive rate among the cervical lesion group, with rates of 57%, 76%, and 100% in LSIL, HSIL, and CC groups respectively. The combination of DNA methylation detection at multiple gene loci showed the highest diagnostic efficacy for HSIL and cervical cancer, with AUC value of 0.850 (95% CI:0.746-0.954), a Youden index of 0.654, and a sensitivity and specificity of 85% and 85.4%, respectively. The diagnostic efficacy of the combined detection was significantly higher than that of HPV examination and liquid-based cytology examination (P < 0.05). Conclusion: DNA methylation detection at multiple gene loci is highly effective and diagnostic tool for cervical cancer, and has potential application value in clinical practice.

Exploring Cantharidin and its Analogues as Anticancer Agents: A Review.

BACKGROUND: Cantharidin (CTD) is a highly toxic substance which can be used to treat a variety of cancers. However, the clinical application of CTD is restricted due to the serious side effects. In recent years, screening its analogues, exploring the mechanism of action and using combinatory therapy with certain substances are considered to be feasible methods which can reduce side effects and improve the therapeutic activity of CTD. This review aims to describe SAR (structure-activity relationship) of CTD analogues, CTD induction mechanisms, and combinatory therapy exploration. METHODS: We searched for research about CTD by entering the database. Important information was screened and extracted purposefully, including SAR, mechanisms, methods, etc. Finally, these contents were unified into a framework to form a review. RESULTS: Some CTD analogues with imidazolium salt or double bonds at C-5 and C-6 positions demonstrate good anticancer activity. Through introducing methyl and acetoxy groups at the C-1 or C-4 position, the inhibitory effect of PP was weakened or even inactivated. Removing the two methyl groups of C-2 and C-3 can reduce side effects and improve efficacy. Replacing methyl with fluorine can also improve the activity and reduce toxicity. Water solubility and bioavailability could be improved by opening the five fivemembered anhydride ring to form carboxylic acid, salt, amide, and ester derivatives. The anticancer mechanism can be divided into the following aspects, including inhibiting cell invasion and metastasis, inducing apoptosis, regulating cell cycle and enhancing immunity. The proper formulation of CTD and its analogues (liposomes, nanoparticles and micelles) can improve the targeting of liver cancer and reduce toxic and side effects. CTD combined with anti-angiogenic therapeutics (Ginsenoside Rg3, Bevacizumab, Apatinib and Endostar) showed additive anti-pancreatic cancer effects. CONCLUSION: It was found that the potential mechanism was closely related to multi-channel and multi-target interactions, which provided a guiding direction for the later exploration of new clinical therapeutic applications. However, some detailed mechanisms are still unclear, and more evidence is required to verify. In addition, the new methods to improve the therapeutic potential of CTD and its analogues still need more clinical trials to be tested in the future. This prospect is very broad and worthy of further study.

Myosin 10 uses its MyTH4 and FERM domains differentially to support two aspects of spindle pole biology required for mitotic spindle bipolarity.

Myosin 10 (Myo10) has the ability to link actin filaments to integrin-based adhesions and to microtubules by virtue of its integrin-binding FERM domain and microtubule-binding MyTH4 domain, respectively. Here we used Myo10 knockout cells to define Myo10's contribution to the maintenance of spindle bipolarity, and complementation to quantitate the relative contributions of its MyTH4 and FERM domains. Myo10 knockout HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in knockout MEFs and knockout HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates γ-tubulin-positive acentriolar foci that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both integrins and microtubules to promote PCM/pole integrity. Conversely, Myo10's ability to promote the clustering of supernumerary centrosomes only requires that it interact with integrins. Importantly, images of Halo-Myo10 knock-in cells show that the myosin localizes exclusively within adhesive retraction fibers during mitosis. Based on these and other results, we conclude that Myo10 promotes PCM/pole integrity at a distance, and that it facilitates supernumerary centrosome clustering by promoting retraction fiber-based cell adhesion, which likely provides an anchor for the microtubule-based forces driving pole focusing.

Characteristics of HPV integration in cervical adenocarcinoma and squamous carcinoma.

PURPOSE: HPV integration usually occurs in HPV-related cancer, and is the main cause of cancer. But the carcinogenic mechanism of HPV integration is unclear. The study aims to provide a theoretical basis for understanding the pathogenesis of cervical adenocarcinoma (AC) and cervical squamous carcinoma (SCC). METHODS: We used HPV capture sequencing to obtain HPV integration sites in AC and SCC, and analyzed cytobands, distribution of genetic and genomic elements, identified integration hotspot genes, clinicopathological parameters, breakpoints of HPV16 and performed pathway analysis. Then we conducted immunohistochemical (IHC) assay to preliminarily verify the expression of most frequently integrated genes in AC, STARD3 and ERBB2. RESULTS: The results revealed that the most frequently observed integrated cytoband was 17q12 in AC and 21p11.2 in SCC, respectively. The breakpoints in both AC and SCC were more tended to occur within gene regions, compared to intergenetic regions. Compared to SCC samples, AC samples had a higher prevalence of genomic elements. In AC, HPV integration has no significantly difference with clinicopathological parameters, but in SCC integration correlated with differentiation (P < 0.05). Breakpoints of HPV in SCC located in LCR more frequently compared to AC, which destroyed the activation of promoter p97. Hotspot genes of HPV integration were STARD3 and ERBB2 in AC, and RNA45S rDNA and MIR3648-1 in SCC, respectively. Meanwhile, we preliminarily proved that the expression of STARD3 and ERBB2, the most frequently integrated genes, would increase after integration. CONCLUSION: These results suggested that HPV may utilize the powerful hosts' promoters to express viral oncogenes and overexpression of viral oncogenes plays a significant role in the carcinogenesis of SCC. In AC, HPV integration may affect hosts' oncogenes, and the dysregulation of oncogenes may primarily contribute to progression of AC.

The delta subunit of the GABAA receptor is necessary for the GPT2-promoted breast cancer metastasis.

Objectives: Glutamic pyruvate transaminase (GPT2) catalyzes the reversible transamination between alanine and α-ketoglutarate (α-KG) to generate pyruvate and glutamate during cellular glutamine catabolism. The glutamate could be further converted to γ-aminobutyric acid (GABA). However, the role of GPT2 in tumor metastasis remains unclear. Methods: The wound healing and transwell assays were carried out to analyze breast cancer cell migration and invasion in vitro. Gene ontology analysis was utilized following RNA-sequencing to discover the associated molecule function. The mass spectrometry analysis following phosphoprotein enrichment was performed to discover the associated transcription factors. Most importantly, both the tail vein model and Mammary gland conditional Gpt2-/- spontaneous tumor mouse models were used to evaluate the effect of GPT2 on breast cancer metastasis in vivo. Results: GPT2 overexpression increases the content of GABA and promotes breast cancer metastasis by activating GABAA receptors. The delta subunit GABRD is necessary for the GPT2/GABA-induced breast cancer metastasis in xenograft and transgenic mouse models. Gpt2 knockout reduces the lung metastasis of the genetic Gpt2-/- breast cancer in mice and prolongs the overall survival of tumor burden mice. Mechanistically, GPT2-induced GABAA receptor activation increases Ca2+ influx by turning on its associated calcium channel, and the surged intracellular calcium triggers the PKC-CREB pathway activation. The activated transcription factor CREB accelerates breast cancer metastasis by upregulating metastasis-related gene expressions, such as PODXL, MMP3, and MMP9. Conclusion: In summary, this study demonstrates that GPT2 promotes breast cancer metastasis through up-regulated GABA activation of GABAAR-PKC-CREB signaling, suggesting it is a potential target for breast cancer therapy.

Establishment and Validation of Models for the Risk of Multi-Drug Resistant Bacteria Infection and Prognosis in Elderly Patients with Pulmonary Infection: A Multicenter Retrospective Study.

Purpose: The aim of this study was to establish risk prediction and prognosis models for multidrug-resistant bacterial infections (MDRB) in elderly patients with pulmonary infections in a multicenter setting. Patients and Methods: This study is a retrospective cohort analysis in Anhui province of China. Data dimension reduction and feature selection were performed using the lasso regression model. Multifactorial regression analysis to identify risk factors associated with MDRB infection and prognosis. The relevant risks of each patient in the prognostic training cohort were scored based on prognostic independent risk factors. Subsequently, patients were classified into high-risk and low-risk groups, and survival differences were compared between them. Finally, models were established based on independent risk factors for infection, risk groups, and independent prognostic factors, and were presented on nomograms. The predictive accuracy of the model was assessed using corresponding external validation set data. Results: The study cohort comprised 994 elderly patients with pulmonary infection. Multivariate analysis revealed that endotracheal intubation, previous antibiotic use beyond 2 weeks, and concurrent respiratory failure or cerebrovascular disease were independent risk factors associated with the incidence of MDRB infection. Cox regression analysis identified respiratory failure, malnutrition, an APACHE II score of at least 20, and higher blood creatinine levels as independent prognostic risk factors. The models were validated using an external validation dataset from multiple centers, which demonstrated good diagnostic ability and a good fit with a fair benefit. Conclusion: In conclusion, our study provides an appropriate and generalisable assessment of risk factors affecting infection and prognosis in patients with MDRB, contributing to improved early identification of patients at higher risk of infection and death, and appropriately guiding clinical management.

Melanoregulin is stably targeted to the melanosome membrane by palmitoylation.

In mammals, pigments are made by melanocytes within a specialized organelle, the melanosome. Mature, pigment-laden melanosomes are then transferred to keratinocytes to drive the visible pigmentation of the animal's hair and skin. The dilute suppressor (dsu) locus encodes an extragenic suppressor of the pigmentation defect exhibited by mice lacking myosin Va (i.e. dilute mice). We recently showed that melanoregulin, the product of the dsu locus, functions as a negative regulator of a shedding mechanism that drives the intercellular transfer of melanosomes from the melanocyte to the keratinocyte. Here we address melanoregulin's localization within the melanocyte, as well as the molecular basis for its localization. First, we confirm and extend recently published results using exogenous, GFP-tagged melanoregulin by showing that endogenous melanoregulin also targets extensively to melanosomes. Second, using site-directed mutagenesis, metabolic labeling with H(3)-palmitate, and an inhibitor of palmitoylation in vivo, we show that the targeting of melanoregulin to the limiting membranes of melanosomes in melanocytes and lysosomes in CV1 cells depends critically on the palmitoylation of one or more of six closely-spaced cysteine residues located near melanoregulin's N-terminus. Finally, using Fluorescence Recovery after Photobleaching (FRAP), we show that melanoregulin-GFP exhibits little if any tendency to cycle in and out of the melanosome membrane. We conclude that multiple palmitoylation serves to stably anchor melanoregulin in the melanosome membrane.