The gut commensal Blautia maintains colonic mucus function under low-fiber consumption through secretion of short-chain fatty acids.

Beneficial gut bacteria are indispensable for developing colonic mucus and fully establishing its protective function against intestinal microorganisms. Low-fiber diet consumption alters the gut bacterial configuration and disturbs this microbe-mucus interaction, but the specific bacteria and microbial metabolites responsible for maintaining mucus function remain poorly understood. By using human-to-mouse microbiota transplantation and ex vivo analysis of colonic mucus function, we here show as a proof-of-concept that individuals who increase their daily dietary fiber intake can improve the capacity of their gut microbiota to prevent diet-mediated mucus defects. Mucus growth, a critical feature of intact colonic mucus, correlated with the abundance of the gut commensal Blautia, and supplementation of Blautia coccoides to mice confirmed its mucus-stimulating capacity. Mechanistically, B. coccoides stimulated mucus growth through the production of the short-chain fatty acids propionate and acetate via activation of the short-chain fatty acid receptor Ffar2, which could serve as a new target to restore mucus growth during mucus-associated lifestyle diseases.

Identification of novel pathogenic variants of CUBN in patients with isolated proteinuria.

BACKGROUND: Although proteinuria is long recognized as an independent risk factor for progressive chronic kidney diseases, not all forms of proteinuria are detrimental to kidney function, one of which is isolated proteinuria caused by cubilin (CUBN)-specific mutations. CUBN encodes an endocytic receptor, initially found to be responsible for the Imerslund-Gräsbeck syndrome (IGS; OMIM #261100) characterized by a combined phenotype of megaloblastic anemia and proteinuria. METHODS: After analyzing their clinical and pathological characterizations, next-generation sequencing for renal disease genes or whole-exome sequencing (WES) was performed on four patients with non-progressive isolated proteinuria. CUBN biallelic pathogenic variants were identified and further analyzed by cDNA-PCR sequencing, immunohistochemistry, minigene assay, and multiple in silico prediction tools, including 3D protein modeling. RESULTS: Here, we present four patients with isolated proteinuria caused by CUBN C-terminal biallelic pathogenic variants, all of which showed no typical IGS symptoms, such as anemia and vitamin B12 deficiency. Their urine protein levels fluctuated between +~++ and estimated glomerular filtration rate (eGFR) were normal or slightly higher. Mild mesangial hypercellularity was found in three children's renal biopsies. A homozygous splice-site variant of CUBN (c.6821+3 (IVS44) A>G) was proven to result in the exon 44 skipping and premature translation termination by cDNA sequencing and immunohistochemistry. Compound heterozygous mutations were identified among the other three children, including another novel splice-site variant (c.10764+1 (IVS66) G>A) causing the retention of first 4 nucleotides in intron 66 by minigene assay, two unreported missense mutations (c.4907G>A (p.R1636Q); c. 9095 A>G (p.Y3032C)), and two reported missense mutations in China (c.8938G>A (p.D2980N); c. 9287T>C (p.L3096P)), locating behind the vitamin B12-binding domain, affecting CUB11, CUB16, CUB22, CUB23, and CUB27 domains, respectively. CONCLUSION: These results demonstrate that above CUBN mutations may cause non-progressive and isolated proteinuria, expanding the variant spectrum of CUBN and benefiting our understanding of proteinuria and renal function.

Targeting hemoglobin receptors IsdH and IsdB of Staphylococcus aureus with a single VHH antibody inhibits bacterial growth.

Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.

Identification of Key Inflammation-related Genes as Potential Diagnostic Biomarkers of Sepsis.

Context: Sepsis is one of the leading causes of mortality for patients with severe infections who had been admitted to intensive care units (ICUs). Early diagnosis, accurate treatment, and management of sepsis remain extremely difficult in clinical settings, due to a lack of early biomarkers and diverse clinical manifestations. Objective: The study intended to identify the key genes and pathways associated with inflammation in sepsis-using microarray technology combined with bioinformatics and key inflammation-related genes (IRGs)-to perform an enrichment analysis and evaluate the value of those genes for the diagnosis and evaluation of prognosis for patients with sepsis. Design: The research team performed a genetic analysis. Setting: The study took place at the Center for Emergency and Critical Medicine at Jinshan Hospital of Fudan University in Jinshan District, Shanghai, China. Groups: The research team created two groups, the sepsis group, individuals with sepsis, and the control group, individuals without sepsis, using data for those groups from five microarray datasets obtained from the Gene Expression Omnibus (GEO) database. Outcome Measures: The research team: (1) downloaded the GSE57065, GSE28750, GSE9692, GSE13904, and GSE54514 datasets from the Gene Expression Omnibus (GEO) database for analysis; (2) analyzed the GSE57065, GSE28750, and GSE9692 datasets to detect the differentially expressed genes (DEGs) in the sepsis and control groups; (3) used Venn diagrams to obtain the intersection of DEGs and inflammation-related genes (IRGs); (4) mapped the protein-protein interaction (PPI) network using the Search Tool for Retrieval of Interacting Genes (STRING) database; (5) detected the hub genes using Cytoscape and cytoHubba; (6) performed an enrichment analysis of hub IRGs using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG); (7) validated the expression of hub IRGs in sepsis using the GSE13904 dataset; and (8) performed a survival analysis in sepsis using the GSE54514 dataset to explore the prognostic value of the hub IRGs. Results: The research team: (1) identified 104 upregulated DEGs and 4 downregulated DEGs; (2) after defining the intersection of DEGs and IRGs, detected nine differentially expressed IRGs (DEIRGs); and (3) identified five IRGs- haptoglobin (HP), high affinity immunoglobulin gamma Fc receptor I (FCGR1A), cluster of differentiation 163 (CD163), complement C3a receptor 1 human (C3AR1), C-type lectin domain containing 5A (CLEC5A)-that overlapped DEIRGs. The GO and KEGG pathway analyses showed that the hub IRGs became enriched during acute-phase response, acute inflammatory response, specific granule, specific granule membrane, endocytic vesicle membrane, tertiary granule, immunoglobulin G (IgG) binding, complement receptor activity, Ig binding, scavenger receptor activity, and scaffold protein binding. The DEGs also played a significant role in Staphylococcus aureus (S. aureus) infection. The ROC curves showed that HP (AUC: 0.956, 95% CI: 0.924-0.988); FCGR1A (AUC: 0.895,95% CI: 0.827-0.963); CD163 (AUC: 0.838, 95% CI: 0.774-0.901); C3AR1 (AUC: 0.953, 95% CI: 0.913-0.993); and CLEC5A (AUC: 0.951, 95% CI: 0 920-0 981) had meaningful diagnostic value for sepsis. Survival analysis showed that the sepsis and control groups had significant differences in HP (P = .043) and CLEC5A (P < .001). Conclusions: HP, FCGR1A, CD163, C3AR1, and CLEC5A have value for clinical application. Clinicians can use them as diagnostic biomarkers, and they provide research direction for treatment targets for sepsis.

Clinical and genetic characterization of children with cubilin variants.

BACKGROUND: Cubilin is one of the receptor proteins responsible for reabsorption of albumin in proximal tubules and is encoded by the CUBN gene. We aimed to evaluate clinical and genetic characterization of six patients with proteinuria who had CUBN mutations. METHODS: Patients' characteristics, serum creatinine, albumin, vitamin B12 levels, urine analysis, spot urine protein/creatinine, microalbumin/creatinine, beta-2 microglobulin/creatinine ratios, estimated glomerular filtration rates (eGFR), treatments, kidney biopsies, and genetic analyses were evaluated. RESULTS: Six patients (2 female, 4 male) with an incidental finding of proteinuria were evaluated. Mean admission age and follow-up time were 7.3 ± 2.9 and 6.5 ± 5.6 years, respectively. Serum albumin, creatinine, and eGFR were normal; urine analysis revealed no hematuria, and C3, C4, ANA, and anti-DNA were negative; kidney ultrasonography was normal for all patients. Urine protein/creatinine was 0.9 ± 0.3 mg/mg, and microalbumin was high in all patients. Serum vitamin B12 was low in two patients and normal in four. Kidney biopsy was performed in four patients, three demonstrated normal light microscopy, and there was one focal segmental glomerulosclerosis (FSGS). Genetic tests revealed four homozygous and two compound heterozygous mutations in the C-terminal part of cubilin. All patients had normal eGFR and still had non-nephrotic range proteinuria at last visit. CONCLUSIONS: CUBN gene mutations should be considered in patients with isolated non-nephrotic range proteinuria and normal kidney function. Diagnosing these patients, who are thought to have a better prognosis, is important in terms of avoiding unnecessary treatment and predicting prognosis. CUBN gene mutations may also present as FSGS which extends the spectrum of renal manifestation of these patients. A higher resolution version of the Graphical abstract is available as Supplementary information.

Somatotopy of Mouse Spinothalamic Innervation and the Localization of a Noxious Stimulus Requires Deleted in Colorectal Carcinoma Expression by Phox2a Neurons.

Anterolateral system (AS) neurons transmit pain signals from the spinal cord to the brain. Their morphology, anatomy, and physiological properties have been extensively characterized and suggest that specific AS neurons and their brain targets are concerned with the discriminatory aspects of noxious stimuli, such as their location or intensity, and their motivational/emotive dimension. Among the recently unraveled molecular markers of AS neurons is the developmentally expressed transcription factor Phox2a, providing us with the opportunity to selectively disrupt the embryonic wiring of AS neurons to gain insights into the logic of their adult function. As mice with a spinal-cord-specific loss of the netrin-1 receptor deleted in colorectal carcinoma (DCC) have increased AS neuron innervation of ipsilateral brain targets and defective noxious stimulus localization or topognosis, we generated mice of either sex carrying a deletion of Dcc in Phox2a neurons. Such DccPhox2a mice displayed impaired topognosis along the rostrocaudal axis but with little effect on left-right discrimination and normal aversive responses. Anatomical tracing experiments in DccPhox2a mice revealed defective targeting of cervical and lumbar AS axons within the thalamus. Furthermore, genetic labeling of AS axons revealed their expression of DCC on their arrival in the brain, at a time when many of their target neurons are being born and express Ntn1 Our experiments suggest a postcommissural crossing function for netrin-1:DCC signaling during the formation of somatotopically ordered maps and are consistent with a discriminatory function of some of the Phox2a AS neurons.SIGNIFICANCE STATEMENT How nociceptive (pain) signals are relayed from the body to the brain remains an important question relevant to our understanding of the basic physiology of pain perception. Previous studies have demonstrated that the AS is a main effector of this function. It is composed of AS neurons located in the spinal cord that receive signals from nociceptive sensory neurons that detect noxious stimuli. In this study, we generate a genetic miswiring of mouse AS neurons that results in a decreased ability to perceive the location of a painful stimulus. The precise nature of this defect sheds light on the function of different kinds of AS neurons and how pain information may be organized.

Planar cell polarity and the pathogenesis of Tourette Disorder: New hypotheses and perspectives.

Planar cell polarity (PCP) signaling plays a fundamental role in shaping the development and ongoing function of the nervous system, beginning from early stages of neural tube closure and spanning the maintenance of functional synapses in adults. While mutations in core PCP signaling proteins have long been suspected to underlie neural tube closure defects in humans, recent findings also implicate their potential involvement in neurodevelopmental and neuropsychiatric disorders. Missense and loss-of-function mutations in CELSR3, a core component of PCP signaling complexes, are highly associated with Tourette Disorder. Although the functional significance of these mutations has yet to be elucidated in animal and cell models, the expression patterns of Celsr3 in mice point to alterations in cortico-striato-thalamo-cortical circuits. Here, we briefly review the known functions of Celsr3 for nervous system development. We also propose circuit models for Tourette Disorder by hypothesizing roles for Celsr3 in controlling striatal neuromodulation via effects on cholinergic interneurons, and thalamic inhibition through its functions in thalamic reticular nuclei. Testing these and related hypotheses in animal and cell models will move us closer to unraveling the neuropathogenesis of Tourette Disorder, with the ultimate goal of developing more efficacious treatments for both motor and cognitive symptoms.

Increased Ratio of CD14++CD80+ Cells/CD14++CD163+ Cells in the Infrapatellar Fat Pad of End-Stage Arthropathy Patients.

Objectives: This study sought to identify the ratio of M1/M2 cells in the infrapatellar fat pads (IFP) and subcutaneous fat tissues (SC) of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. The clinical features of OA and RA patients treated with or without biological disease-modifying anti-rheumatic drugs (bDMARDs) were also assessed. Methods: IFP and SC were collected from patients with OA and RA who are undergoing total knee arthroplasty (TKA). CD14-positive cells were then isolated from these samples. Flow cytometry was used to determine the number of CD14++CD80+ cells and CD14++CD163+ cells. The expression levels of lipid transcription factors, such as sterol regulatory element-binding protein 1 (SREBP1) and liver X receptor alpha (LXRA), and inflammatory cytokines were also evaluated. Results: Twenty OA patients and 22 RA patients were enrolled in this study. Ten of the RA patients (45.4%) received bDAMRDs before TKA. On average, a fivefold increase in the number of CD14-positive cells and lower expression levels of SREBP1C and LXRA were observed in OA IFP relative to OA SC; however, these results were not obtained from the RA samples. The median ratio of CD14++CD80+ cells/CD14++CD163+ cells of OA IFP was 0.87 (0.76-1.09, interquartile range), which is higher to that of OA SC with a lower ratio (p = 0.05835). Conclusions: The quantity and quality of CD14-positive cells differed between IFP and SC in arthropathy patients. To our knowledge, this is the first study to characterize the ratio of M1/M2 cells in the IFP and SC of end-stage OA and RA patients. The increased ratio of CD14++CD80+ cells/CD14++CD163+ cells in the IFP from patients with OA and RA treated with bDMARDs indicated that inflammation was localized in the IFP. As adipose tissue-derived innate immune cells were revealed as one of the targets for regulating inflammation, further analysis of these cells in the IFP may reveal new therapeutic strategies for inflammatory joint diseases.

Short-Chain Fatty Acids Promote Immunotherapy by Modulating Immune Regulatory Property in B Cells.

The dysfunction of regulatory B cells (Breg) may result in immune inflammation such as allergic rhinitis (AR); the underlying mechanism is not fully understood yet. Short-chain fatty acids, such as propionic acid (PA), have immune regulatory functions. This study is aimed at testing a hypothesis that modulates PA production alleviating airway allergy through maintaining Breg functions. B cells were isolated from the blood obtained from AR patients and healthy control (HC) subjects. The stabilization of IL-10 mRNA in B cells was tested with RT-qPCR. An AR mouse model was developed to test the role of PA in stabilizing the IL-10 expression in B cells. We found that the serum PA levels were negatively correlated with the serum Th2 cytokine levels in AR patients. Serum PA levels were positively associated with peripheral CD5+ B cell frequency in AR patients; the CD5+ B cells were also IL-10+. The spontaneous IL-10 mRNA decay was observed in B cells, which was prevented by the presence of PA through activating GPR43. PA counteracted the effects of Tristetraprolin (TTP) on inducing IL-10 mRNA decay in B cells through the AKT/T-bet/granzyme B pathway. Administration of Yupinfeng San, a Chinese traditional medical formula, or indole-3-PA, induced PA production by intestinal bacteria to stabilize the IL-10 expression in B cells, which promoted the allergen specific immunotherapy, and efficiently alleviated experimental AR. In summary, the data show that CD5+ B cells produce IL-10. The serum lower PA levels are associated with the lower frequency of CD5+ B cells in AR patients. Administration with Yupinfeng San or indole-3-PA can improve Breg functions and alleviate experimental AR.

Shikonin alleviates choroidal neovascularization by inhibiting proangiogenic factor production from infiltrating macrophages.

Choroidal neovascularization (CNV), a feature of neovasular age-related macular degeneration (AMD), acts as a leading cause of vision loss in the elderly. Shikonin (SHI), a natural bioactive compound extracted from Chinese herb radix arnebiae, exerts anti-inflammatory and anti-angiogenic roles and also acts as a potential pyruvate kinase M2 (PKM2) inhibitor in macrophages. The major immune cells macrophages infiltrate the CNV lesions, where the production of pro-angiognic cytokines from macrophage facilitates the development of CNV. PKM2 contributes to the neovascular diseases. In this study, we found that SHI oral gavage alleviated the leakage, area and volume of mouse laser-induced CNV lesion and inhibited macrophage infiltration without ocular cytotoxicity. Moreover, SHI inhibited the secretion of pro-angiogenic cytokine, including basic fibroblast growth factor (FGF2), insulin-like growth factor-1 (IGF1), chemokine (C-C motif) ligand 2 (CCL2), placental growth factor and vascular endothelial growth factor (VEGF), from primary human macrophages by down-regulating PKM2/STAT3/CD163 pathway, indicating a novel potential therapy strategy for CNV.

Curcumin mediates repulsive guidance molecule B (RGMb) in the treatment mechanism of renal fibrosis induced by unilateral ureteral obstruction.

In this study, we explored the role and mechanism of repulsive guidance molecule B (RGMb, also known as Dragon) in the protective effects of curcumin against renal fibrosis and verified Dragon's effect on renal tubular epithelial cell apoptosis and cell programmability. Unilateral ureteral obstruction (UUO) was surgically induced in rats to establish a model of renal interstitial fibrosis (RIF). The rats were then treated with curcumin. Curcumin prominently decreased the serum creatinine (SCr) and blood urea nitrogen (BUN) levels, and also improved the tubular injury in the UUO-induced rats. Curcumin significantly downregulated the TGF-ß1, P-Smad2/3, cleaved caspase-3, cleaved caspase-8 and Dragon levels. Dragon knockdown also markedly reduced the TGF-ß1, P-Smad2/3, Smad2/3, cleaved caspase-3, cleaved caspase-8, fibronectin, collagen I, collagen IV, vimentin, and α-SMA expression levels. Conversely, Dragon overexpression caused higher expression levels of these proteins, and curcumin reversed this effect. Furthermore, Dragon knockdown increased the E-cadherin levels, whereas Dragon overexpression decreased these levels. Overexpressing Dragon significantly decreased the cell viability, and curcumin reversed this effect. In conclusion, curcumin acted on Dragon and attenuated RIF in UUO rat models. Curcumin downregulated the TGF-ß1/Smad signaling pathway and inhibited Dragon and fibrogenic molecules in both rats and HK-2 cells.

Agonistic Activation of Cytosolic DNA Sensing Receptors in Woodchuck Hepatocyte Cultures and Liver for Inducing Antiviral Effects.

Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-ß and interleukins 1ß and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB.

Multiple ligand recognition sites in free fatty acid receptor 2 (FFA2R) direct distinct neutrophil activation patterns.

The allosteric modulating free fatty acid receptor 2 ligands Cmp58 and AZ1729, increased the activity induced by orthosteric receptor agonists mediating a rise in intracellular calcium ions and activation of the neutrophil NADPH-oxidase. Together, the two modulators triggered an orthosteric-agonist-independent activation of the oxidase without any rise in the concentration of intracellular calcium ions. In this study, structurally diverse compounds presumed to be ligands for free fatty acid receptor 2 were used to gain additional insights into receptor-modulation/signaling. We identified two molecules that activate neutrophils on their own and we classified one as allosteric agonist and the other as orthosteric agonist. Ten compounds were classified as allosteric FFA2R modulators. Of these, one activated neutrophils when combined with AZ1729; the nine remaining compounds activated neutrophils solely when combined with Cmp58. The activation signals were primarily biased when stimulated by two allosteric modulators interacting with different binding sites, such that two complementary modulators together triggered an activation of the NADPH-oxidase but no increase in the intracellular concentration of calcium ions. No neutrophil activation was induced when allosteric receptor modulators suggested to be recognized by the same binding site were combined, results in agreement with our proposed model for activation, in which the receptor has two different sites that selectively bind allosteric modulators. The down-stream signaling mediated by cross-sensitizing allosteric receptor modulators, occurring independent of any orthosteric agonist, represent a new mechanism for activation of the neutrophil NADPH oxidase.

Tadalafil enhances the therapeutic efficacy of BET inhibitors in hepatocellular carcinoma through activating Hippo pathway.

Bromodomain and extraterminal (BET) proteins are promising therapeutic targets for hematological and solid tumors. However, BET inhibitor monotherapy did not show a significant therapeutic benefit for hepatocellular carcinoma (HCC) in preclinical trials. Here, we identified YAP/TAZ genes, as determinants for sensitivity to BET inhibitors. YAP/TAZ expression, especially TAZ, promote resistance to BET inhibitor. In addition, we analyzed that the mRNA level of PDE5 was positively correlated with YAP/TAZ based on TCGA database and demonstrated tadalafil, a PDE5 inhibitor, could block YAP/TAZ protein expression by activating Hippo pathway. Cotreatment with tadalafil and JQ-1 synergistically reduced YAP/TAZ protein expression, suppressed proliferation and induced G0-G1 arrest of cultured HCC cells. JQ-1 alone does not show significant benefits in a mouse model of HCC induced by c-Myc/N-Ras plasmids. In contrast, the combination, tadalafil and JQ-1, successfully suppressed tumor progression, enhanced antitumor immunity by improving the ratio of activated CD8 and extended the survival time of mice. Our data define the key role of YAP/TAZ in mediating resistance to BET inhibitor, described the PDE5/PKG/Hippo/YAP/TAZ axis and identified a common clinical drug that can be developed as an effective combined strategy to overcome BET inhibitor resistance in MYC/Ras-driven HCC.

Structural basis for Ca2+ activation of the heteromeric PKD1L3/PKD2L1 channel.

The heteromeric complex between PKD1L3, a member of the polycystic kidney disease (PKD) protein family, and PKD2L1, also known as TRPP2 or TRPP3, has been a prototype for mechanistic characterization of heterotetrametric TRP-like channels. Here we show that a truncated PKD1L3/PKD2L1 complex with the C-terminal TRP-fold fragment of PKD1L3 retains both Ca2+ and acid-induced channel activities. Cryo-EM structures of this core heterocomplex with or without supplemented Ca2+ were determined at resolutions of 3.1 Å and 3.4 Å, respectively. The heterotetramer, with a pseudo-symmetric TRP architecture of 1:3 stoichiometry, has an asymmetric selectivity filter (SF) guarded by Lys2069 from PKD1L3 and Asp523 from the three PKD2L1 subunits. Ca2+-entrance to the SF vestibule is accompanied by a swing motion of Lys2069 on PKD1L3. The S6 of PKD1L3 is pushed inward by the S4-S5 linker of the nearby PKD2L1 (PKD2L1-III), resulting in an elongated intracellular gate which seals the pore domain. Comparison of the apo and Ca2+-loaded complexes unveils an unprecedented Ca2+ binding site in the extracellular cleft of the voltage-sensing domain (VSD) of PKD2L1-III, but not the other three VSDs. Structure-guided mutagenic studies support this unconventional site to be responsible for Ca2+-induced channel activation through an allosteric mechanism.

Gibberellin Signaling Promotes the Secondary Growth of Storage Roots in Panax ginseng.

Gibberellins (GAs) are an important group of phytohormones associated with diverse growth and developmental processes, including cell elongation, seed germination, and secondary growth. Recent genomic and genetic analyses have advanced our knowledge of GA signaling pathways and related genes in model plant species. However, functional genomics analyses of GA signaling pathways in Panax ginseng, a perennial herb, have rarely been carried out, despite its well-known economical and medicinal importance. Here, we conducted functional characterization of GA receptors and investigated their physiological roles in the secondary growth of P. ginseng storage roots. We found that the physiological and genetic functions of P. ginseng gibberellin-insensitive dwarf1s (PgGID1s) have been evolutionarily conserved. Additionally, the essential domains and residues in the primary protein structure for interaction with active GAs and DELLA proteins are well-conserved. Overexpression of PgGID1s in Arabidopsis completely restored the GA deficient phenotype of the Arabidopsis gid1a gid1c (atgid1a/c) double mutant. Exogenous GA treatment greatly enhanced the secondary growth of tap roots; however, paclobutrazol (PCZ), a GA biosynthetic inhibitor, reduced root growth in P. ginseng. Transcriptome profiling of P. ginseng roots revealed that GA-induced root secondary growth is closely associated with cell wall biogenesis, the cell cycle, the jasmonic acid (JA) response, and nitrate assimilation, suggesting that a transcriptional network regulate root secondary growth in P. ginseng. These results provide novel insights into the mechanism controlling secondary root growth in P. ginseng.

The impact of thiamine deficiency and benfotiamine treatment on Nod-like receptor protein-3 inflammasome in microglia.

Thiamine-dependent processes are critical in cerebral glucose metabolism, it is abnormity induces oxidative stress, inflammation and neurodegeneration. Nod-like receptor protein-3 (NLRP3) inflammasome-mediated inflammation is closely related to neurologic diseases and can be activated by oxidative stress. However, the impact of thiamine deficiency on NLRP3 inflammasome activation remains unknown. In this study, we found that NLRP3 inflammasomes were significantly activated in the microglia of thiamine deficiency mice model. In contrast, benfotiamine dampened inflammation NLRP3 mediated in BV2 cells stimulated with LPS and ATP through reducing mitochondrial reactive oxygen species levels and mitigating autophagy flux defect. These data identify an important role of thiamine metabolism in NLRP3 inflammasome activation, and correcting thiamine metabolism through benfotiamine provides a new therapeutic strategy for NLRP3 inflammasome related neurological, metabolic, and inflammatory diseases.

Active components in Ephedra sinica stapf disrupt the interaction between ACE2 and SARS-CoV-2 RBD: Potent COVID-19 therapeutic agents.

ETHNOPHARMACOLOGICAL RELEVANCE: Ephedra sinica Stapf is a widely used folk medicine in Asia to treat lung diseases, such as cold, cough and asthma. Many efforts have revealed that some traditional Chinese medicine (TCM) prescriptions containing Ephedra sinica could effectively alleviate the symptoms and prevent the fatal deterioration of COVID-19. AIM OF THE STUDY: The present study aims to discover active compounds in Ephedra sinica disrupting the interaction between angiotensin-converting enzyme 2 (ACE2) and the SARS-CoV-2 spike protein receptor-binding domain (SARS-CoV-2 RBD) to inhibit SARS-CoV-2 virus infection. MATERIALS AND METHODS: The ethanol extracts of Ephedra sinica were prepared. Activity guided isolation of constituents was carried out by measuring the inhibitory activity on ACE2-RBD interaction. The structures of active compounds were identified by HPLC-Q-TOF-MS/MS and NMR. To testify the contribution of main components for the inhibitory activity, different samples were prepared by components knock-out strategy. The mechanism of compounds inhibiting protein-protein interaction (PPI) was explored by competition inhibition assays, surface plasmon resonance (SPR) assays and molecular docking. SARS-CoV-2 S protein-pseudoviruses were used to observe the viropexis effect in cells. RESULTS: Ephedra sinica extracts (ESE) could effectively inhibit the interaction between ACE2 and SARS-CoV-2 RBD (IC50 = 95.01 µg/mL). Three active compounds, 4,6-dihydroxyquinoline-2-carboxylic acid, 4-hydroxyquinoline-2-carboxylic acid and 4-hydroxy-6-methoxyquinoline-2-carboxylic acid were identified to inhibit ACE2-RBD interaction (IC50 = 0.58 µM, 0.07 µM and 0.15 µM respectively). And knock-out the three components could eliminate the inhibitory activity of ESE. Molecular docking calculations indicated that the hydrogen bond was the major intermolecular force. Finally, our results also showed that these compounds could inhibit the infectivity of SARS-CoV-2 S protein-pseudoviruses to 293T-ACE2 (IC50 = 0.44-1.09 µM) and Calu-3 cells. CONCLUSION: These findings suggested that quinoline-2-carboxylic acids in Ephedra sinica could be considered as potential therapeutic agents for COVID-19. Further, this study provided some justification for the ethnomedicinal use of Ephedra sinica for COVID-19.

(Pro)renin Receptor Knockdown Attenuates Liver Fibrosis Through Inactivation of ERK/TGF-ß1/SMAD3 Pathway.

BACKGROUND & AIMS: Activation of the (pro)renin receptor (PRR) up-regulates the expression of profibrotic genes in the kidney and heart. We aimed to investigate the role of PRR in hepatic fibrogenesis. METHODS: Human hepatic PRR levels were measured in patients with or without liver fibrosis. PRR expression was analyzed in primary mouse hepatic stellate cells (HSCs). Experimental fibrosis was studied in thioacetamide (TAA)-treated or methionine choline-deficient (MCD) diet-fed C57BL/6 mice. Lentivirus-mediated PRR short hairpin RNA was used to knockdown hepatic PRR expression. Lentiviral vectors expressing PRR short hairpin RNA or complementary DNA from the α-smooth muscle actin promoter were used for myofibroblast-specific gene knockdown or overexpression. RESULTS: PRR is up-regulated in human and mouse fibrotic livers, and in activated HSCs. Hepatic PRR knockdown reduced liver fibrosis by suppressing the activation of HSCs and expression of profibrotic genes in TAA or MCD diet-injured mice without significant changes in hepatic inflammation. Renin and prorenin increased the expression of PRR and production of TGF-ß1 in human activated HSC Lieming Xu-2 cells, and knockdown of PRR inactivated Lieming Xu-2 cells with decreased production of transforming growth factor (TGF)-ß1 and Mothers against decapentaplegic homolog 3 (Smad3) phosphorylation. Myofibroblast-specific PRR knockdown also attenuated liver fibrosis in TAA or MCD diet-injured mice. Mice with both myofibroblast-specific and whole-liver PRR knockdown showed down-regulation of the hepatic extracellular signal-regulated kinase (ERK)/TGF-ß1/Smad3 pathway. Myofibroblast-specific PRR overexpression worsened TAA-induced liver fibrosis by up-regulating the ERK/TGF-ß1/Smad3 pathway. CONCLUSIONS: PRR contributes to liver fibrosis and HSC activation, and its down-regulation attenuates liver fibrosis through inactivation of the ERK/TGF-ß1/Smad3 pathway. Therefore, PRR is a promising therapeutic target for liver fibrosis.

Ultrasensitive detection by maxillary palp neurons allows non-host recognition without consumption of harmful allelochemicals.

Most lepidopteran insect larvae exhibit stepwise feeding behaviors, such as palpation using the maxillary palps (MPs) followed by test biting and persistent biting. However, the purpose of palpation has been unclear. In particular, nothing is known about the neurons in the MP and their mode of recognition of undesired plants, although such neurons have been suggested to exist. In this study, we used larvae of the stenophagous insect Bombyx mori and compared the roles of palpation and test biting in the selection of feeding behavior. When the larvae were given non-host plant leaves, they did not initiate test biting, indicating that non-host plant leaves were recognized via palpation without biting, and that this behavior resulted in a lack of persistent biting, as the leaves were judged non-suitable for consumption. Surface extracts of inedible leaves significantly suppressed test biting of mulberry leaves, a host plant of B. mori, suggesting that secondary metabolites on the leaf surface of inedible leaves function as test biting suppressors, even when another conditions are suitable for test biting. The allelochemical coumarin, which is found in the inedible leaves of cherry, Cerasus speciosa, significantly suppressed test biting of mulberry leaves, suggesting that coumarin is a possible deterrent to the eating of cherry leaves. Using the electrophysiological method of tip recording and a leaf-surface extract as the test material, leaf-surface compound-responsive neurons were identified in the MP. In addition, several neurons that respond to coumarin in the attomolar range were identified, suggesting that the larvae use ultrasensitive neurons in the MP to recognize inedible leaves. In the HEK293T cell heterologous expression system, the B. mori gustatory receptors BmGr53 and BmGr19, which were previously found to be expressed in the MP and to respond to coumarin in the attomolar range, responded to a leaf-surface extract of C. speciosa, suggesting that these receptors may be present on the inedible-leaf-recognizing neurons of the MP. These findings suggest that ultrasensitive plant secondary metabolite-recognizing neurons in the MP allow for the recognition of non-host plants via palpation without risking damage caused by ingesting harmful allelochemicals.