Significance and Possible Biological Mechanism for CLDN8 Downregulation in Kidney Renal Clear Cell Carcinoma Tissues.

Background: The clinical role of claudin 8 (CLDN8) in kidney renal clear cell carcinoma (KIRC) remains unclarified. Herein, the expression level and potential molecular mechanisms of CLDN8 underlying KIRC were determined. Methods: High-throughput datasets of KIRC were collected from GEO, ArrayExpress, SRA, and TCGA databases to determine the mRNA expression level of the CLDN8. In-house tissue microarrays and immunochemistry were performed to examine CLDN8 protein expression. A summary receiver operating characteristic curve (SROC) and standardized mean difference (SMD) forest plot were generated using Stata v16.0. Single-cell analysis was conducted to further prove the expression level of CLDN8. A clustered regularly interspaced short palindromic repeats knockout screen analysis was executed to assess the growth impact of CLDN8. Functional enrichment analysis was conducted using the Metascape database. Additionally, single-sample gene set enrichment analysis was implied to explore immune cell infiltration in KIRC. Results: A total of 17 mRNA datasets comprising 1,060 KIRC samples and 452 non-cancerous control samples were included in this study. Additionally, 105 KIRC and 16 non-KIRC tissues were analyzed using in-house immunohistochemistry. The combined SMD was -5.25 (95% confidence interval (CI): -6.13 to -4.37), and CLDN8 downregulation yielded an SROC area under the curve (AUC) close to 1.00 (95% CI: 0.99 - 1.00). CLDN8 downregulation was also confirmed at the single-cell level. Knocking out CLDN8 stimulated KIRC cell proliferation. Lower CLDN8 expression was correlated with worse overall survival of KIRC patients (hazard ratio of CLDN8 downregulation = 1.69, 95% CI: 1.2 - 2.4). Functional pathways associated with CLDN8 co-expressed genes were centered on carbon metabolism obstruction, with key hub genes ACADM, ACO2, NDUFS1, PDHB, SDHD, SUCLA2, SUCLG1, and SUCLG2. Conclusions: CLDN8 is downregulated in KIRC and is considered a potential tumor suppressor. CLDN8 deficiency may promote the initiation and progression of KIRC, potentially in conjunction with metabolic dysfunction.

Distinct roles of the major binding residues in the cation-binding pocket of the melibiose transporter MelB.

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with Na+, Li+, or H+ but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt and the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport remain poorly understood. In this study, we solved two x-ray crystal structures of MelBSt, the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na+-binding residues for the selection of Na+ and H+ by free energy simulations. Transport assays showed that the D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelBSt.

Case report: Multiple organ failure caused by Japanese spotted fever.

A 71-year-old male had disseminated multiple organ dysfunction syndrome (MODS). Following treatment with cefotaxime and piperacillin-tazobactam, his symptoms have worsened instead. Multiple organ failure caused by Japanese Spotted Fever (JSF) was diagnosed based on metagenomic next-generation sequencing (mNGS), we rapidly treated the patient with doxycycline. Thereafter, his symptoms gradually improved. In this report, we emphasized the importance of rapid microbial diagnostic tools and the early use of tetracyclines for the treatment of JSF.

[Examples of Professor MA Kun's treatment of infertility caused by uterine fibroids using method of tonifying kidney and activating blood].

Uterine fibroids are a prevalent factor that impacts fertility in women of reproductive age. This study discusses the theoretical foundation and formula principles of Professor MA Kun's clinical treatment for infertility caused by uterine fibroids. The kidney stores essence and is responsible for reproduction, while blood serves as a vital material basis for women's physiological functions. Kidney deficiency is the fundamental pathogenesis of infertility, and imbalances in kidney Qi and essence or deficiencies in kidney Yin and Yang can result in blood stasis. Blood stasis plays a significant role throughout this condition by impeding the flow of blood, which is crucial for nourishing Qi. Therefore, both kidney deficiency and blood stasis are key factors contributing to infertility caused by uterine fibroids. Professor MA Kun treats infertility caused by uterine fibroids using an approach that involves tonifying the kidneys and activating blood circulation based on changes in Qi and blood during the menstrual cycle as well as follicular growth processes. By identifying stage-specific evidence, appropriate treatments can be applied accordingly. During menstruation when the uterus opens and menstrual blood flows out, promoting follicular development through nourishing kidney Yin and activating blood circulation becomes essential. In later stages of menstruation, additional measures are taken to remove blood stasis, alleviate symptoms, disperse knots, attack pathogens while simultaneously replenishing vital energy. During intermenstrual periods when Yin holds greater importance than Yang, tonifying the kidneys and activating blood circulation helps facilitate smooth discharge of eggs by promoting transformation between Yin and Yang energies. Premenstrual period to warm kidney Yang to promote pregnant egg implantation, and at the same time to dredge the liver and regulate Qi, Qi elimination stagnation, complementary in the line, with the symptoms of additional subtractions. Clinical effect is remarkable, for the reference of colleagues.

Distinct roles of the major binding residues in the cation-binding pocket of MelB.

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with either H+, Li+, or Na+, but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt, as well as the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport still remain poorly understood. We have solved two x-ray crystal structures of MelBSt cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published previously. We determined the energetic contributions of three major Na+-binding residues in cation selectivity for Na+ and H+ by the free energy simulations. The D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport with poor activities at higher ΔpH and better activities at reversal ΔpH was observed, supporting that the membrane potential is the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket.

Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter.

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of major facilitator superfamily transporters. With a conformation-selective nanobody, we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. The available outward-facing sugar-bound structures showed that the N- and C-terminal residues of the inner barrier contribute to the sugar selectivity. The inward-open conformation shows that the sugar selectivity pocket is also broken when the inner barrier is broken. Isothermal titration calorimetry measurements revealed that this inward-facing conformation trapped by this nanobody exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is supported by molecular dynamics simulations. Furthermore, the hydron/deuterium exchange mass spectrometry analyses allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

Rational Approach to Improve Detergent Efficacy for Membrane Protein Stabilization.

Membrane protein structures are essential for the molecular understanding of diverse cellular processes and drug discovery. Detergents are not only widely used to extract membrane proteins from membranes but also utilized to preserve native protein structures in aqueous solution. However, micelles formed by conventional detergents are suboptimal for membrane protein stabilization, necessitating the development of novel amphiphilic molecules with enhanced protein stabilization efficacy. In this study, we prepared two sets of tandem malonate-derived glucoside (TMG) variants, both of which were designed to increase the alkyl chain density in micelle interiors. The alkyl chain density was modulated either by reducing the spacer length (TMG-Ms) or by introducing an additional alkyl chain between the two alkyl chains of the original TMGs (TMG-Ps). When evaluated with a few membrane proteins including a G protein-coupled receptor, TMG-P10,8 was found to be substantially more efficient at extracting membrane proteins and also effective at preserving protein integrity in the long term compared to the previously described TMG-A13. This result reveals that inserting an additional alkyl chain between the two existing alkyl chains is an effective way to optimize detergent properties for membrane protein study. This new biochemical tool and the design principle described have the potential to facilitate membrane protein structure determination.

Melamine-cored glucosides for membrane protein solubilization and stabilization: importance of water-mediated intermolecular hydrogen bonding in detergent performance.

Membrane proteins play essential roles in a number of biological processes, and their structures are important in elucidating such processes at the molecular level and also for rational drug design and development. Membrane protein structure determination is notoriously challenging compared to that of soluble proteins, due largely to the inherent instability of their structures in non-lipid environments. Micelles formed by conventional detergents have been widely used for membrane protein manipulation, but they are suboptimal for long-term stability of membrane proteins, making downstream characterization difficult. Hence, there is an unmet need for the development of new amphipathic agents with enhanced efficacy for membrane protein stabilization. In this study, we designed and synthesized a set of glucoside amphiphiles with a melamine core, denoted melamine-cored glucosides (MGs). When evaluated with four membrane proteins (two transporters and two G protein-coupled receptors), MG-C11 conferred notably enhanced stability compared to the commonly used detergents, DDM and LMNG. These promising findings are mainly attributed to a unique feature of the MGs, i.e., the ability to form dynamic water-mediated hydrogen-bond networks between detergent molecules, as supported by molecular dynamics simulations. Thus, MG-C11 is the first example of a non-peptide amphiphile capable of forming intermolecular hydrogen bonds within a protein-detergent complex environment. Detergent micelles formed via a hydrogen-bond network could represent the next generation of highly effective membrane-mimetic systems useful for membrane protein structural studies.

Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter.

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of the Na+-coupled major facilitator superfamily transporters. With a conformational nanobody (Nb), we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. Collectively with the available outward-facing sugar-bound structures, both the outer and inner barriers were localized. The N- and C-terminal residues of the inner barrier contribute to the sugar selectivity pocket. When the inner barrier is broken as shown in the inward-open conformation, the sugar selectivity pocket is also broken. The binding assays by isothermal titration calorimetry revealed that this inward-facing conformation trapped by the conformation-selective Nb exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for the substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is also supported by molecular dynamics simulations. Furthermore, the use of this Nb in combination with the hydron/deuterium exchange mass spectrometry allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

Role of PI3K/Akt/mTOR pathway-mediated macrophage autophagy in affecting the phenotype transformation of lung fibroblasts induced by silica dust exposure. / PI3K/Akt/mTOR通路介导巨噬细胞自噬影响矽尘致肺成纤维细胞表型转化.

OBJECTIVES: The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy. Autophagy plays a key role in the formation of silicosis fibrosis. The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis. This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy. METHODS: The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h. Macrophages were exposed to different concentrations (0, 25, 50, 100, 200, 400 µg/mL) and different times (0, 6, 12, 24, 48 h) of SiO2 dust suspension. The survival rate of macrophages was measured by cell counting kit-8 (CCK-8) method. Enzyme linked immunosorbent assay (ELISA) was used to measure the contents of transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) in the cell supernatant. The co-culture system of macrophages and HFL-1 cells was established by transwell. A blank control group, a SiO2 group, a LY294002 group, a SC79 group, a LY294002+SiO2 group, and a SC79+SiO2 group were set up in this experiment. Macrophages in the LY294002+SiO2 group were pretreated with LY294002 (PI3K inhibitor) for 18 hours, and macrophages in the SC79+SiO2 group were pretreated with SC79 (Akt activator) for 24 hours, and then exposed to SiO2 (100 µg/mL) dust suspension for 12 hours. The expression of microtubule-associated protein 1 light chain 3 (LC3) protein in macrophages was detected by the immunofluorescence method. The protein expressions of PI3K, Akt, mTOR, Beclin-1, LC3 in macrophages, and collagen III (Col III), α-smooth muscle actin (α-SMA), fibronectin (FN), matrix metalloproteinase-1 (MMP-1), tissue metalloproteinase inhibitor-1 (TIMP-1) in HFL-1 cells were measured by Western blotting. RESULTS: After the macrophages were exposed to SiO2 dust suspension of different concentrations for 12 h, the survival rates of macrophages were gradually decreased with the increase of SiO2 concentration. Compared with the 0 µg/mL group, the survival rates of macrophages in the 100, 200, and 400 µg/mL groups were significantly decreased, and the concentrations of TGF-ß1 and TNF-α in the cell supernatant were obviously increased (all P<0.05). When 100 µg/mL SiO2 dust suspension was applied to macrophages, the survival rates of macrophages were decreased with the prolonged exposure time. Compared with the 0 h group, the survival rates of macrophages were significantly decreased (all P<0.05), the concentrations of TGF-ß1 and TNF-α in the cell supernatant were significantly increased, and the protein expression levels of Beclin-1 and LC3II were increased markedly in the 6, 12, 24, and 48 h groups (all P<0.05). Immunofluorescence results demonstrated that after exposure to SiO2 (100 µg/mL) dust for 12 h, LC3 exhibited punctate aggregation and significantly higher fluorescence intensity compared to the blank control group (P<0.05). Compared with the blank control group, the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated in the SiO2 group (all P<0.05). Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were down-regulated and the protein expressions of LC3II and Beclin-1 were up-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-ß1 in the cell supernatant were decreased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were down-regulated (all P<0.05) in the LY294002+SiO2 group. Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were up-regulated and the protein expressions of LC3II and Beclin-1 were down-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-ß1 in the cell supernatant were increased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated (all P<0.05) in the SC79+SiO2 group. CONCLUSIONS: Silica dust exposure inhibits the PI3K/Akt/mTOR pathway, increases autophagy and concentration of inflammatory factors in macrophages, and promotes the phenotype transformation of HFL-1 cells into myofibroblasts. The regulation of the PI3K/Akt/mTOR pathway can affect the autophagy induction and the concentration of inflammatory factors of macrophages by silica dust exposure, and then affect the phenotype transformation of HFL-1 cells into myofibroblasts induced by silica dust exposure.

The insertase YidC chaperones the polytopic membrane protein MelB inserting and folding simultaneously from both termini.

The insertion and folding of proteins into membranes is crucial for cell viability. Yet, the detailed contributions of insertases remain elusive. Here, we monitor how the insertase YidC guides the folding of the polytopic melibiose permease MelB into membranes. In vivo experiments using conditionally depleted E. coli strains show that MelB can insert in the absence of SecYEG if YidC resides in the cytoplasmic membrane. In vitro single-molecule force spectroscopy reveals that the MelB substrate itself forms two folding cores from which structural segments insert stepwise into the membrane. However, misfolding dominates, particularly in structural regions that interface the pseudo-symmetric α-helical domains of MelB. Here, YidC takes an important role in accelerating and chaperoning the stepwise insertion and folding process of both MelB folding cores. Our findings reveal a great flexibility of the chaperoning and insertase activity of YidC in the multifaceted folding processes of complex polytopic membrane proteins.

MiR-30a inhibits silica dust-induced epithelial-mesenchymal transition by targeting Snail.

The mechanism of action of MicroRNA-30a(miR-30a) and Snail, a transcription factor, in silica(SiO2) dust-induced pulmonary EMT and secondary pulmonary fibrosis remains elusive. In this study, the cellular EMT model induced by the stimulation of A549 cells with SiO2 was established. A549 cells were transfected with miR-30a mimic and miR-30a inhibitor and the SNAIL gene was silenced to examine the mechanism of miR-30a targeting Snail to regulate silica dust-induced EMT. The results showed that 50 µg/mL SiO2 stained A549 cells for 24 h could induce EMT in A549 cells. Exposure of A549 cells to SiO2 dust decreased miR-30a expression, as well as mRNA and protein expression levels of E-cad. Conversely, SiO2 exposure increased mRNA and protein expression levels of α-SMA, vimentin, and Snail. The miR-30a mimic upregulated mRNA and protein expression levels of E-cadherin in SiO2-induced A549 cells, while downregulating mRNA and protein expression levels of α-SMA, vimentin and Snail. MiR-30a inhibitors have the opposite effect. Silencing the SNAIL gene, followed by SiO2 dust-induced stimulation of A549 cells, could enhance mRNA and protein expression levels of E-cad, whereas those of α-SMA and vimentin were reduced. Altogether, we found that miR-30a directly targeted Snail and inhibited its expression, thereby delaying silica induced pulmonary EMT.

Adjuvant immune checkpoint inhibitors associated with higher recurrence-free survival in postoperative hepatocellular carcinoma (PREVENT): a prospective, multicentric cohort study.

BACKGROUND: Adjuvant therapy may improve survival of patients with hepatocellular carcinoma (HCC) after curative resection. This study compared safety and efficacy outcomes between patients at high risk of recurrence who received different types of adjuvant therapy or no such therapy after hepatic resection for HCC. METHODS: Recurrence-free survival (RFS), overall survival, and adverse events were compared among patients who received adjuvant immune checkpoint inhibitors (ICIs) alone, ICIs with tyrosine kinase inhibitors (TKIs), or no adjuvant therapy between 13 March 2019 and 19 March 2022. This study was registered on ClinicalTrials.gov (NCT05221398). RESULTS: Of the 517 patients in final analysis, 432 (83.6%) received no adjuvant therapy, 53 (10.2%) received ICIs alone, and 32 (6.2%) received adjuvant ICIs and TKIs. During median follow-up of 34.0 months (IQR 27.8 to 41.6 months), RFS was significantly longer among patients who received either type of adjuvant therapy (25.2 months, 95%CI 16.4-34.0) than among those who received none (16.1 months, 95%CI 12.9-19.4), and this difference remained significant after propensity score matching (HR 0.52, 95%CI 0.35-0.76, P = 0.004). Overall survival was unaffected by either type of adjuvant therapy, while significant difference was observed between patients who received adjuvant therapy or not after propensity score matching (HR 0.31, 95%CI 0.17-0.59, P = 0.005). The rate of grade 3 or 4 adverse events was similar between the two types of adjuvant therapy. CONCLUSIONS: ICIs alone or with TKIs may improve RFS of patients at high risk of HCC recurrence after curative resection.

In vivo and in vitro characterizations of melibiose permease (MelB) conformation-dependent nanobodies reveal sugar-binding mechanisms.

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the Na+-coupled major facilitator superfamily transporters, which are important for the cellular uptake of molecules including sugars and small drugs. Although the symport mechanisms have been well-studied, mechanisms of substrate binding and translocation remain enigmatic. We have previously determined the sugar-binding site of outward-facing MelBSt by crystallography. To obtain other key kinetic states, here we raised camelid single-domain nanobodies (Nbs) and carried out a screening against the WT MelBSt under 4 ligand conditions. We applied an in vivo cAMP-dependent two-hybrid assay to detect interactions of Nbs with MelBSt and melibiose transport assays to determine the effects on MelBSt functions. We found that all selected Nbs showed partial to complete inhibitions of MelBSt transport activities, confirming their intracellular interactions. A group of Nbs (714, 725, and 733) was purified, and isothermal titration calorimetry measurements showed that their binding affinities were significantly inhibited by the substrate melibiose. When titrating melibiose to the MelBSt/Nb complexes, Nb also inhibited the sugar-binding. However, the Nb733/MelBSt complex retained binding to the coupling cation Na+ and also to the regulatory enzyme EIIAGlc of the glucose-specific phosphoenolpyruvate/sugar phosphotransferase system. Further, EIIAGlc/MelBSt complex also retained binding to Nb733 and formed a stable supercomplex. All data indicated that MelBSt trapped by Nbs retained its physiological functions and the trapped conformation is similar to that bound by the physiological regulator EIIAGlc. Therefore, these conformational Nbs can be useful tools for further structural, functional, and conformational analyses.

Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer.

Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components and properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, and fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteolipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP-lipid organization. Applying this method, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.

SiO2 dust induces inflammation and pulmonary fibrosis in rat lungs through activation of ASMase/ceramide pathway.

The role of ASMase/ceramide signaling pathway in the development of silicosis needs to be verified by in vivo experiments. We investigated the role of the ASMase/ceramide signaling pathway in the progression of silicosis and the effect of desipramine (DMI) (1 mg/mL) on the development of silicosis, by establishing a silica (1 mL, 50 mg/mL) dust-contaminated rat silicosis model and administering the ASMase inhibitor, DMI, to the dust-contaminated rats. The results showed that the levels of interleukin (IL)-1ß and IL-6 were increased in the lung tissues of the rats in the dust-contaminated group at the initial stage after dusting; the inflammatory cell aggregation in the lung tissue was increased. With time progression, the hydroxyproline content in the lung tissue increased, and alpha-smooth muscle actin (α-SMA), collagen I, and vimentin substantially increased, suggesting that silicosis was formed in the lung tissue of the rats 28 days after SiO2 dust treatment. Moreover, the levels of ASMase, ceramide, and sphingosine-1-phosphate (S1P) were increased in the lung tissue of rats. The expression of ß-catenin, fibronectin, and caspase-3 protein was increased, and E-cadherin protein expression was decreased in the lung tissue of the rats in the late stage of dust contamination. The ASMase and ceramide in the lung tissues of the rats in the DMI intervention group were reduced, as were the lung tissue inflammation levels, collagen expression, and lung fibrosis. These results suggest that SiO2 dust may activate the ASMase/ceramide signaling pathway in rat lung tissue, promoting pulmonary fibrosis. DMI inhibited this activation, attenuated apoptosis, blocked epithelial-mesenchymal transition, and halted silica dust-induced silicofibrosis.

Tris(hydroxymethyl)aminomethane Linker-Bearing Triazine-Based Triglucosides for Solubilization and Stabilization of Membrane Proteins.

High-resolution membrane protein structures are essential for a fundamental understanding of the molecular basis of diverse cellular processes and for drug discovery. Detergents are widely used to extract membrane-spanning proteins from membranes and maintain them in a functional state for downstream characterization. Due to limited long-term stability of membrane proteins encapsulated in conventional detergents, development of novel agents is required to facilitate membrane protein structural study. In the current study, we designed and synthesized tris(hydroxymethyl)aminomethane linker-bearing triazine-based triglucosides (TTGs) for solubilization and stabilization of membrane proteins. When these glucoside detergents were evaluated for four membrane proteins including two G protein-coupled receptors, a few TTGs including TTG-C10 and TTG-C11 displayed markedly enhanced behaviors toward membrane protein stability relative to two maltoside detergents [DDM (n-dodecyl-ß-d-maltoside) and LMNG (lauryl maltose neopentyl glycol)]. This is a notable feature of the TTGs as glucoside detergents tend to be inferior to maltoside detergents at stabilizing membrane proteins. The favorable behavior of the TTGs for membrane protein stability is likely due to the high hydrophobicity of the lipophilic groups, an optimal range of hydrophilic-lipophilic balance, and the absence of cis-trans isomerism.

Mfsd2a utilizes a flippase mechanism to mediate omega-3 fatty acid lysolipid transport.

Major Facilitator Superfamily Domain containing 2a (Mfsd2a) is a sodium-dependent lysophosphatidylcholine (LPC) transporter expressed at the blood-brain barrier that constitutes the main pathway by which the brain obtains omega-3 fatty acids, such as docosahexanoic acid. Mfsd2a deficiency in humans results in severe microcephaly, underscoring the importance of LPC transport by Mfsd2a for brain development. Biochemical studies and recent cryo-electron microscopy (cryo-EM) structures of Mfsd2a bound to LPC suggest that Mfsd2a transports LPC via an alternating access mechanism between outward-facing and inward-facing conformational states in which the LPC inverts during transport between the outer and inner leaflet of a membrane. However, direct biochemical evidence of flippase activity by Mfsd2a has not been demonstrated and it is not understood how Mfsd2a could invert LPC between the outer and inner leaflet of the membrane in a sodium-dependent manner. Here, we established a unique in vitro assay using recombinant Mfsd2a reconstituted in liposomes that exploits the ability of Mfsd2a to transport lysophosphatidylserine (LPS) coupled with a small molecule LPS binding fluorophore that allowed for monitoring of directional flipping of the LPS headgroup from the outer to the inner liposome membrane. Using this assay, we demonstrate that Mfsd2a flips LPS from the outer to the inner leaflet of a membrane bilayer in a sodium-dependent manner. Furthermore, using cryo-EM structures as guides together with mutagenesis and a cell-based transport assay, we identify amino acid residues important for Mfsd2a activity that likely constitute substrate interaction domains. These studies provide direct biochemical evidence that Mfsd2a functions as a lysolipid flippase.

STK25: a viable therapeutic target for cancer treatments?

Serine/threonine protein kinase 25 (STK25) is a critical regulator of ectopic lipid storage, glucose and insulin homeostasis, fibrosis, and meta-inflammation. More and more studies have revealed a strong correlation between STK25 and human diseases. On the one hand, STK25 can affect glucose and fatty acid metabolism in normal cells or tumors. On the other hand, STK25 participates in autophagy, cell polarity, cell apoptosis, and cell migration by activating various signaling pathways. This article reviews the composition and function of STK25, the energy metabolism and potential drugs that may target STK25, and the research progress of STK25 in the occurrence and development of tumors, to provide a reference for the clinical treatment of tumors.