Allosteric regulation of nitrate transporter NRT via the signaling protein PII.

Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein.

Fine structure and assembly pattern of a minimal myophage Pam3.

The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 Å in diameter, connected via a three-section neck to an 840-Å-long contractile tail, ending with a three-module baseplate composed of only six protein components. This simplified baseplate consists of a central hub-spike surrounded by six wedge heterotriplexes, to which twelve tail fibers are covalently attached via disulfide bonds in alternating upward and downward configurations. In vitro reduction assays revealed a putative redox-dependent mechanism of baseplate assembly and tail sheath contraction. These findings establish a minimal myophage that might become a user-friendly chassis phage in synthetic biology.

Ficolin-A/2 Aggravates Severe Lung Injury through Neutrophil Extracellular Traps Mediated by Gasdermin D-Induced Pyroptosis.

Neutrophil extracellular traps (NETs) and pyroptosis are critical events in lung injury. This study investigated whether ficolin-A influenced NET formation through pyroptosis to exacerbate lipopolysaccharide (LPS)-induced lung injury. The expression of ficolin-A/2, NETs, and pyroptosis-related molecules was investigated in animal and cell models. Knockout and knockdown (recombinant protein) methods were used to elucidate regulatory mechanisms. The Pearson correlation coefficient was used to analyze the correlation between ficolins and pyroptosis- and NET-related markers in clinical samples. In this study, ficolin-2 (similar to ficolin-A) showed significant overexpression in patients with acute respiratory distress syndrome. In vivo, knockout of Fcna, but not Fcnb, attenuated lung inflammation and inhibited NET formation in the LPS-induced mouse model. DNase I further alleviated lung inflammation and NET formation in Fcna knockout mice. In vitro, neutrophils derived from Fcna-/- mice showed less pyroptosis and necroptosis than those from the control group after LPS stimulation. Additionally, GSDMD knockdown or Nod-like receptor protein 3 inhibitor reduced NET formation. Addition of recombinant ficolin-2 protein to human peripheral blood neutrophils promoted NET formation and pyroptosis after LPS stimulation, whereas Fcn2 knockdown had the opposite effect. Acute respiratory distress syndrome patients showed increased levels of pyroptosis- and NET-related markers, which were correlated positively with ficolin-2 levels. In conclusion, these results suggested that ficolin-A/2 exacerbated NET formation and LPS-induced lung injury via gasdermin D-mediated pyroptosis.

Causal relationship between gut microbiota, plasma metabolites, inflammatory cytokines and abdominal aortic aneurysm: a Mendelian randomization study.

BACKGROUND: Complex interconnections are evident among gut microbiota, circulating metabolites, inflammatory cytokines, and the pathogenesis of abdominal aortic aneurysms (AAA), with the causal dynamics yet to be comprehensively elucidated. The primary objective of this study was to elucidate the potential causal relationships involving gut microbiota-mediated plasma metabolites, inflammatory cytokines, and AAA. METHODS: We utilized data from genome-wide association studies predominantly comprising individuals of European ancestry, encompassing four major gut microbiota signatures, 233 plasma metabolite signatures (N = 136,016), 91 inflammatory cytokine signatures (N = 14,824), and AAA signatures (N = 1,458,875). Mendelian randomization (MR), employed in a two-sample format, was utilized as a tool to investigate the potential causal pathways from gut microbiota to the development of AAA. Additionally, a two-step MR approach was employed to dissect the impact of plasma metabolites and inflammatory cytokines on the relationship between gut microbiota and AAA and to ascertain the mediated fractions. RESULTS: Our findings indicate that five phylum or family-identical bacteria, 175 plasma metabolites, and seven inflammatory factors are causally associated with AAA. Among them, five bacterial species from the same phylum or family, identified from different GWAS data, were strongly associated with AAA. Of these, two exhibited negative causality and three exhibited positive causality. We found that the phylum Firmicutes and the families Oscillospiraceae might reduce the risk of AAA, whereas the families Prevotellaceae, Sutterellaceae, and Aminobacteriaceae might increase the risk of AAA. Further screening indicated that phylum Firmicutes id.1672 (GCST90017114) may confer a protective effect against AAA by reducing triglyceride levels in medium/small high-density lipoprotein (HDL). CONCLUSION: MR analysis has delineated a causal pathway from gut microbiota, through plasma circulating metabolites and inflammatory cytokines, to the pathogenesis of AAA. The role of intestinal flora and certain biomarkers may provide a reference for the diagnosis of AAA, and contribute to the prevention, diagnosis, and treatment of AAA disease.

Regulatory B cells protect against chronic hypoxia-induced pulmonary hypertension by modulating the Tfh/Tfr immune balance.

Hypoxia-induced pulmonary hypertension (HPH) is a progressive and lethal disease characterized by the uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) and obstructive vascular remodelling. Previous research demonstrated that Breg cells were involved in the pathogenesis of pulmonary hypertension. This work aimed to evaluate the regulatory function of Breg cells in HPH. HPH mice model were established and induced by exposing to chronic hypoxia for 21 days. Mice with HPH were treated with anti-CD22 or adoptive transferred of Breg cells. The coculture systems of Breg cells with CD4+ T cells and Breg cells with PASMCs in vitro were constructed. Lung pathology was evaluated by HE staining and immunofluorescence staining. The frequencies of Breg cells, Tfh cells and Tfr cells were analysed by flow cytometry. Serum IL-21 and IL-10 levels were determined by ELISA. Protein levels of Blimp-1, Bcl-6 and CTLA-4 were determined by western blot and RT-PCR. Proliferation rate of PASMCs was measured by EdU. Compared to the control group, mean PAP, RV/(LV + S) ratio, WA% and WT% were significantly increased in the model group. Anti-CD22 exacerbated abnormal hemodynamics, pulmonary vascular remodelling and right ventricle hypertrophy in HPH, which ameliorated by adoptive transfer of Breg cells into HPH mice. The proportion of Breg cells on day 7 induced by chronic hypoxia was significantly higher than control group, which significantly decreased on day 14 and day 21. The percentage of Tfh cells was significantly increased, while percentage of Tfr cells was significantly decreased in HPH than those of control group. Anti-CD22 treatment increased the percentage of Tfh cells and decreased the percentage of Tfr cells in HPH mice. However, Breg cells restrained the Tfh cells differentiation and expanded Tfr cells differentiation in vivo and in vitro. Additionally, Breg cells inhibited the proliferation of PASMCs under hypoxic condition in vitro. Collectively, these findings suggested that Breg cells may be a new therapeutic target for modulating the Tfh/Tfr immune balance in HPH.

Electroacupuncture may alleviate diabetic neuropathic pain by inhibiting the microglia P2X4R and neuroinflammation.

Diabetic neuropathic pain (DNP) is a common and destructive complication of diabetes mellitus. The discovery of effective therapeutic methods for DNP is vitally imperative because of the lack of effective treatments. Although 2 Hz electroacupuncture (EA) was a successful approach for relieving DNP, the mechanism underlying the effect of EA on DNP is still poorly understood. Here, we established a rat model of DNP that was induced by streptozotocin (STZ) injection. P2X4R was upregulated in the spinal cord after STZ-injection. The upregulation of P2X4R was mainly expressed on activated microglia. Intrathecal injection of a P2X4R antagonist or microglia inhibitor attenuated STZ-induced nociceptive thermal hyperalgesia and reduced the overexpression of brain-derived neurotrophic factor (BDNF), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the spinal cord. We also assessed the effects of EA treatment on the pain hypersensitivities of DNP rats, and further investigated the possible mechanism underlying the analgesic effect of EA. EA relieved the hyperalgesia of DNP. In terms of mechanism, EA reduced the upregulation of P2X4R on activated microglia and decreased BDNF, IL-1ß and TNF-α in the spinal cord. Mechanistic research of EA's analgesic impact would be beneficial in ensuring its prospective therapeutic effect on DNP as well as in extending EA's applicability.

Dorsal root ganglia P2X4 and P2X7 receptors contribute to diabetes-induced hyperalgesia and the downregulation of electroacupuncture on P2X4 and P2X7.

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate), a nonspecific P2X1-7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4-L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7.

Inhibition of phosphorylated calcium/calmodulin-dependent protein kinase IIα relieves streptozotocin-induced diabetic neuropathic pain through regulation of P2X3 receptor in dorsal root ganglia.

Diabetic neuropathic pain (DNP) is frequent among patients with diabetes. We previously showed that P2X3 upregulation in dorsal root ganglia (DRG) plays a role in streptozotocin (STZ)-induced DNP but the underlying mechanism is unclear. Here, a rat model of DNP was established by a single injection of STZ (65 mg/kg). Fasting blood glucose was significantly elevated from the 1st to 3rd week. Paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in diabetic rats significantly reduced from the 2nd to 3rd week. Western blot analysis revealed that elevated p-CaMKIIα levels in the DRG of DNP rats were accompanied by pain-associated behaviors while CaMKIIα levels were unchanged. Immunofluorescence revealed significant increase in the proportion of p-CaMKIIα immune positive DRG neurons (stained with NeuN) in the 2nd and 3rd week and p-CaMKIIα was co-expressed with P2X3 in DNP rats. KN93, a CaMKII antagonist, significantly reduce mechanical hyperalgesia and thermal hyperalgesia and these effects varied dose-dependently, and suppressed p-CaMKIIα and P2X3 upregulation in the DRGs of DNP rats. These results revealed that the p-CaMKIIα upregulation in DRG is involved in DNP, which possibly mediated P2X3 upregulation, indicating CaMKIIα may be an effective pharmacological target for DNP management.

Analgesic effect of electroacupuncture on bone cancer pain in rat model: the role of peripheral P2X3 receptor.

Upregulation of P2X3 receptor (P2X3R) has been strongly implicated in nociceptive signaling including bone cancer pain (BCP). The present study, using rat bone cancer model, aimed to explore the role of P2X3R in regulating rat pain behavior under the intervention of electroacupuncture (EA). The BCP model was successfully established by injection with MRMT-1 breast cancer cell into the medullary cavity of left tibia for 3 × 104 cells/3 µL PBS in rats as revealed by obvious bone destruction, decreased paw withdrawal thresholds (PWTs), and reduced paw withdrawal latencies (PWLs). Western blot analyses showed that P2X3R expression was significantly upregulated in ipsilateral lumbar 4-6 (L4-6) dorsal root ganglia (DRG), but the difference not seen in spinal cord dorsal horn (SCDH). With the in-depth study of P2X3R activation, we observed that intrathecal injection of P2X3R agonist α,ß-meATP aggravated MRMT-1 induced BCP, while injection of P2X3R inhibitor A-317491 alleviated pain. Subsequently, we demonstrated that BCP induced mechanical allodynia and thermal hyperalgesia were attenuated after EA treatment. Under EA treatment, total P2X3R protein expression in ipsilateral DRGs was decreased, and it is worth mentioning that decreased expression of P2X3R membrane protein, which indicated that both the expression and membrane trafficking of P2X3R were inhibited by EA. The immunofluorescence assay showed that EA stimulation exerted functions by reducing the expression of P2X3R-positive cells in ipsilateral DRGs of BCP rats. Ca2+ imaging analysis revealed that the EA stimulation decreased the percentage of α,ß-meATP responsive neurons in DRGs and inhibited calcium influx. Notably, the inhibitory effect of EA on mechanical allodynia and nociceptive flinches was abolished by intrathecal injection of α,ß-meATP. These findings demonstrated EA stimulation ameliorated mechanical allodynia and thermal hyperalgesia in rat model of MRMT-1-induced BCP. EA exerts analgesic effect on BCP by reducing the overexpression and functional activity of P2X3R in ipsilateral DRGs of BCP rats. Our work first demonstrates the critical and overall role of P2X3R in EA's analgesia against peripheral sensitization of MRMT-1-induced BCP and further supports EA as a potential therapeutic option for cancer pain in clinic.

Rubisco accumulation factor 1 (Raf1) plays essential roles in mediating Rubisco assembly and carboxysome biogenesis.

Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, Synechococcus elongatus PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer-dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO2-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO2-fixing modules to improve photosynthesis.

Structural basis for juvenile hormone biosynthesis by the juvenile hormone acid methyltransferase.

Juvenile hormone (JH) acid methyltransferase (JHAMT) is a rate-limiting enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis in insects and thus presents an excellent target for the development of insect growth regulators or insecticides. However, the three-dimensional properties and catalytic mechanism of this enzyme are not known. Herein, we report the crystal structure of the JHAMT apoenzyme, the three-dimensional holoprotein in binary complex with its cofactor S-adenosyl-l-homocysteine, and the ternary complex with S-adenosyl-l-homocysteine and its substrate methyl farnesoate. These structures reveal the ultrafine definition of the binding patterns for JHAMT with its substrate/cofactor. Comparative structural analyses led to novel findings concerning the structural specificity of the progressive conformational changes required for binding interactions that are induced in the presence of cofactor and substrate. Importantly, structural and biochemical analyses enabled identification of one strictly conserved catalytic Gln/His pair within JHAMTs required for catalysis and further provide a molecular basis for substrate recognition and the catalytic mechanism of JHAMTs. These findings lay the foundation for the mechanistic understanding of JH biosynthesis by JHAMTs and provide a rational framework for the discovery and development of specific JHAMT inhibitors as insect growth regulators or insecticides.

Biochemical and structural characterization of the cyanophage-encoded phosphate-binding protein: implications for enhanced phosphate uptake of infected cyanobacteria.

To acquire phosphorus, cyanobacteria use the typical bacterial ABC-type phosphate transporter, which is composed of a periplasmic high-affinity phosphate-binding protein PstS and a channel formed by two transmembrane proteins PstC and PstA. A putative pstS gene was identified in the genomes of cyanophages that infect the unicellular marine cyanobacteria Prochlorococcus and Synechococcus. However, it has not been determined whether the cyanophage PstS protein is functional during infection to enhance the phosphate uptake rate of host cells. Here we showed that the cyanophage P-SSM2 PstS protein was abundant in the infected Prochlorococcus NATL2A cells and the host phosphate uptake rate was enhanced after infection. This is consistent with our biochemical and structural analyses showing that the phage PstS protein is indeed a high-affinity phosphate-binding protein. We further modelled the complex structure of phage PstS with host PstCA and revealed three putative interfaces that may facilitate the formation of a chimeric ABC transporter. Our results provide insights into the molecular mechanism by which cyanophages enhance the phosphate uptake rate of cyanobacteria. Phosphate acquisition by infected bacteria can increase the phosphorus contents of released cellular debris and virus particles, which together constitute a significant proportion of the marine dissolved organic phosphorus pool.

Capsid Structure of Anabaena Cyanophage A-1(L).

A-1(L) is a freshwater cyanophage with a contractile tail that specifically infects Anabaena sp. PCC 7120, one of the model strains for molecular studies of cyanobacteria. Although isolated for half a century, its structure remains unknown, which limits our understanding on the interplay between A-1(L) and its host. Here we report the 3.35 Å cryo-EM structure of A-1(L) capsid, representing the first near-atomic resolution structure of a phage capsid with a T number of 9. The major capsid gp4 proteins assemble into 91 capsomers, including 80 hexons: 20 at the center of the facet and 60 at the facet edge, in addition to 11 identical pentons. These capsomers further assemble into the icosahedral capsid, via gradually increasing curvatures. Different from the previously reported capsids of known-structure, A-1(L) adopts a noncovalent chainmail structure of capsid stabilized by two kinds of mortise-and-tenon inter-capsomer interactions: a three-layered interface at the pseudo 3-fold axis combined with the complementarity in shape and electrostatic potential around the 2-fold axis. This unique capsomer construction enables A-1(L) to possess a rigid capsid, which is solely composed of the major capsid proteins with an HK97 fold. IMPORTANCE Cyanobacteria are the most abundant photosynthetic bacteria, contributing significantly to the biomass production, O2 generation, and CO2 consumption on our planet. Their community structure and homeostasis in natural aquatic ecosystems are largely regulated by the corresponding cyanophages. In this study, we solved the structure of cyanophage A-1(L) capsid at near-atomic resolution and revealed a unique capsid construction. This capsid structure provides the molecular details for better understanding the assembly of A-1(L), and a structural platform for future investigation and application of A-1(L) in combination with its host Anabaena sp. PCC 7120. As the first isolated freshwater cyanophage that infects the genetically tractable model cyanobacterium, A-1(L) should become an ideal template for the genetic engineering and synthetic biology studies.

Exosome-transmitted S100A4 induces immunosuppression and non-small cell lung cancer development by activating STAT3.

Non-small cell lung cancer (NSCLC) is the primary reason of tumor morbidity and mortality worldwide. We aimed to study the transfer process of S100A4 between cells and whether it affected NSCLC development by affecting STAT3 expression. First, S100A4 expression in NSCLC cells was measured. The exosomes in MRC-5, A549, and H1299 cells were isolated and identified. We constructed si-S100A4 and si-PD-L1 to transfect A549 cells and oe-S100A4 to transfect H1299 cells, and tested the transfection efficiency. Cell function experiments were performed to assess cell proliferation, clone number, apoptosis, cell cycle, migration, and invasion abilities. In addition, ChIP was applied to determine the targeting relationship between S100A4 and STAT3. Next, we explored NSCLC cell-derived exosomes role in NSCLC progress by transmitting S100A4. Finally, we verified the function of exosome-transmitted S100A4 in NSCLC in vivo. High expression of S100A4 was secreted by exosomes. After knocking down S100A4, cell proliferation ability was decreased, clones number was decreased, apoptosis was increased, G1 phase was increased, S phase was repressed, and migration and invasion abilities were also decreased. ChIP validated STAT3 and PD-L1 interaction. After knocking down S100A4, PD-L1 expression was decreased, while ov-STAT3 reversed the effect of S100A4 on PD-L1 expression. Meanwhile, S100A4 inhibited T-cell immune activity by activating STAT3. In addition, knockdown of PD-L1 inhibited cell proliferation, migration, and invasion. NSCLC cell-derived exosomes promoted cancer progression by transmitting S100A4 to activate STAT3 pathway. Finally, in vivo experiments further verified that exosome-transmitted S100A4 promoted NSCLC progression. Exosome-transmitted S100A4 induces immunosuppression and the development of NSCLC by activating STAT3.

NAADP-induced intracellular calcium ion is mediated by the TPCs (two-pore channels) in hypoxia-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a form of obstructive vascular disease. Chronic hypoxic exposure leads to excessive proliferation of pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. This condition can potentially be aggravated by [Ca2+ ] i mobilization. In the present study, hypoxia exposure of rat's model was established. Two-pore segment channels (TPCs) silencing was achieved in rats' models by injecting Lsh-TPC1 or Lsh-TPC2. The effects of TPC1/2 silencing on PAH were evaluated by H&E staining detecting pulmonary artery wall thickness and ELISA assay kit detecting NAADP concentrations in lung tissues. TPC1/2 silencing was achieved in PASMCs and PAECs, and cell proliferation was detected by MTT and BrdU incorporation assays. As the results shown, NAADP-activated [Ca2+ ]i shows to be mediated via two-pore segment channels (TPCs) in PASMCs, with TPC1 being the dominant subtype. NAADP generation and TPC1/2 mRNA and protein levels were elevated in the hypoxia-induced rat PAH model; NAADP was positively correlated with TPC1 and TPC2 expression, respectively. In vivo, Lsh-TPC1 or Lsh-TPC2 infection significantly improved the mean pulmonary artery pressure and PAH morphology. In vitro, TPC1 silencing inhibited NAADP-AM-induced PASMC proliferation and [Ca2+ ]i in PASMCs, whereas TPC2 silencing had minor effects during this process; TPC2 silencing attenuated NAADP-AM- induced [Ca2+ ]i and ECM in endothelial cells, whereas TPC1 silencing barely ensued any physiological changes. In conclusion, TPC1/2 might provide a unifying mechanism within pulmonary arterial hypertension, which can potentially be regarded as a therapeutic target.

Chronic hypoxia promoted pulmonary arterial smooth muscle cells proliferation through upregulated calcium-sensing receptorcanonical transient receptor potential 1/6 pathway.

OBJECTIVES: Although both calcium-sensing receptor (CaSR) and canonical transient receptor potential (TRPC) proteins contribute to chronic hypoxia (CH)-induced pulmonary arterial smooth muscle cells (PASMCs) proliferation, the relationship between CaSR and TRPC in hypoxic PASMCs proliferation remains poorly understood. The goal of this study was to identify that CH promotes PASMCs proliferation through CaSR-TRPC pathway. METHODS: Rat PASMCs were isolated and treated with CH. Cell proliferation was assessed by cell counting, CCK-8 assay, and EdU incorporation. CaSR and TRPC expressions were determined by qPCR and Western blotting. Store-operated Ca2+ entry (SOCE) was assessed by extracellular Ca2+ restoration. RESULTS: In PASMCs, CH enhanced the cell number, cell viability and DNA synthesis, which is accompanied by upregulated expression of CaSR, TRPC1 and TRPC6. Negative CaSR modulators (NPS2143, NPS2390) inhibited, whereas positive modulators (spermine, R568) enhanced, the CH-induced increases in cell number, cell viability and DNA synthesis in PASMCs. Knockdown of CaSR by siRNA inhibited the CH-induced upregulation of TRPC1 and TRPC6 and enhancement of SOCE and attenuated the CH-induced enhancements of cell number, cell viability and DNA synthesis in PASMCs. However, neither siTRPC1 nor siTRPC6 had an effect on the CH-induced CaSR upregulation, although both significantly attenuated the CH-induced enhancements of cell number, cell viability and DNA synthesis in PASMCs. CONCLUSION: These results demonstrate that upregulated CaSR-TRPC1/6 pathway mediating PASMCs proliferation is an important pathogenic mechanism under hypoxic conditions.

Transcriptome profiling of long noncoding RNAs and mRNAs in spinal cord of a rat model of paclitaxel-induced peripheral neuropathy identifies potential mechanisms mediating neuroinflammation and pain.

BACKGROUND: Paclitaxel is a widely prescribed chemotherapy drug for treating solid tumors. However, paclitaxel-induced peripheral neuropathy (PIPN) is a common adverse effect during paclitaxel treatment, which results in sensory abnormalities and neuropathic pain among patients. Unfortunately, the mechanisms underlying PIPN still remain poorly understood. Long noncoding RNAs (lncRNAs) are novel and promising targets for chronic pain treatment, but their involvement in PIPN still remains unexplored. METHODS: We established a rat PIPN model by repetitive paclitaxel application. Immunostaining, RNA sequencing (RNA-Seq) and bioinformatics analysis were performed to study glia cell activation and explore lncRNA/mRNA expression profiles in spinal cord dorsal horn (SCDH) of PIPN model rats. qPCR and protein assay were used for further validation. RESULTS: PIPN model rats developed long-lasting mechanical and thermal pain hypersensitivities in hind paws, accompanied with astrocyte and microglia activation in SCDH. RNA-Seq identified a total of 814 differentially expressed mRNAs (DEmRNA) (including 467 upregulated and 347 downregulated) and 412 DElncRNAs (including 145 upregulated and 267 downregulated) in SCDH of PIPN model rats vs. control rats. Functional analysis of DEmRNAs and DElncRNAs identified that the most significantly enriched pathways include immune/inflammatory responses and neurotrophin signaling pathways, which are all important mechanisms mediating neuroinflammation, central sensitization, and chronic pain. We further compared our dataset with other published datasets of neuropathic pain and identified a core set of immune response-related genes extensively involved in PIPN and other neuropathic pain conditions. Lastly, a competing RNA network analysis of DElncRNAs and DEmRNAs was performed to identify potential regulatory networks of lncRNAs on mRNA through miRNA sponging. CONCLUSIONS: Our study provided the transcriptome profiling of DElncRNAs and DEmRNAs and uncovered immune and inflammatory responses were predominant biological events in SCDH of the rat PIPN model. Thus, our study may help to identify promising genes or signaling pathways for PIPN therapeutics.

Resistin-like molecule ß acts as a mitogenic factor in hypoxic pulmonary hypertension via the Ca2+-dependent PI3K/Akt/mTOR and PKC/MAPK signaling pathways.

BACKGROUND: Pulmonary arterial smooth muscle cell (PASMC) proliferation plays a crucial role in hypoxia-induced pulmonary hypertension (HPH). Previous studies have found that resistin-like molecule ß (RELM-ß) is upregulated de novo in response to hypoxia in cultured human PASMCs (hPASMCs). RELM-ß has been reported to promote hPASMC proliferation and is involved in pulmonary vascular remodeling in patients with PAH. However, the expression pattern, effects, and mechanisms of action of RELM-ß in HPH remain unclear. METHODS: We assessed the expression pattern, mitogenetic effect, and mechanism of action of RELM-ß in a rat HPH model and in hPASMCs. RESULTS: Overexpression of RELM-ß caused hemodynamic changes in a rat model of HPH similar to those induced by chronic hypoxia, including increased mean right ventricular systolic pressure (mRVSP), right ventricular hypertrophy index (RVHI) and thickening of small pulmonary arterioles. Knockdown of RELM-ß partially blocked the increases in mRVSP, RVHI, and vascular remodeling induced by hypoxia. The phosphorylation levels of the PI3K, Akt, mTOR, PKC, and MAPK proteins were significantly up- or downregulated by RELM-ß gene overexpression or silencing, respectively. Recombinant RELM-ß protein increased the intracellular Ca2+ concentration in primary cultured hPASMCs and promoted hPASMC proliferation. The mitogenic effects of RELM-ß on hPASMCs and the phosphorylation of PI3K, Akt, mTOR, PKC, and MAPK were suppressed by a Ca2+ inhibitor. CONCLUSIONS: Our findings suggest that RELM-ß acts as a cytokine-like growth factor in the development of HPH and that the effects of RELM-ß are likely to be mediated by the Ca2+-dependent PI3K/Akt/mTOR and PKC/MAPK pathways.

SNI and CFA induce similar changes in TRPV1 and P2X3 expressions in the acute phase but not in the chronic phase of pain.

Peripheral inflammation and nerve injury usually accompany each other. However, whether inflammatory and neuropathic pain share similar mechanisms at all stages is unknown. TRPV1 and P2X3 are two major ion channels in dorsal root ganglia (DRGs) and are involved in chronic pain. Here, their function and expression in DRGs at different phases of the two types of pain were investigated. Both the paw withdrawal threshold (PWT) and paw withdrawal latency were decreased in rats injected with complete Freud's adjuvant (CFA). However, only the PWT was decreased in rats with spared nerve injury (SNI). CFA increased the magnitude of the TRPV1-mediated Ca2+ response but not the P2X3-mediated Ca2+ response 14 days after injection. Consistent with this result, the P2X3 expression level in CFA rats was increased only at 3 days after injection. SNI surgery increased the magnitudes of the TRPV1- and P2X3-mediated Ca2+ responses and upregulated both TRPV1 and P2X3 expression in lumbar DRGs. The distributions of TRPV1 and P2X3 in DRGs after modeling were observed, and TRPV1 was found to be highly expressed mainly in the L4-L5 DRGs in CFA rats and in the L5-L6 DRGs in SNI rats. P2X3 was highly expressed in the L4-L6 DRGs in CFA rats 3 days after injection but was only highly expressed in the L4 DRG 14 days after modeling. On the other hand, SNI promoted the P2X3 expression L4-L5 DRGs 3 days after surgery, but only L6 DRG 14 days after modeling. All the results indicate that P2X3 and TPRV1 are involved in inflammatory and neuropathic pain by different expression levels and distributions in the lumbar DRG in the chronic stage.

Coordinating carbon and nitrogen metabolic signaling through the cyanobacterial global repressor NdhR.

The coordination of carbon and nitrogen metabolism is essential for bacteria to adapt to nutritional variations in the environment, but the underlying mechanism remains poorly understood. In autotrophic cyanobacteria, high CO2 levels favor the carboxylase activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) to produce 3-phosphoglycerate, whereas low CO2 levels promote the oxygenase activity of RuBisCO, leading to 2-phosphoglycolate (2-PG) production. Thus, the 2-PG level is reversely correlated with that of 2-oxoglutarate (2-OG), which accumulates under a high carbon/nitrogen ratio and acts as a nitrogen-starvation signal. The LysR-type transcriptional repressor NAD(P)H dehydrogenase regulator (NdhR) controls the expression of genes related to carbon metabolism. Based on genetic and biochemical studies, we report here that 2-PG is an inducer of NdhR, while 2-OG is a corepressor, as found previously. Furthermore, structural analyses indicate that binding of 2-OG at the interface between the two regulatory domains (RD) allows the NdhR tetramer to adopt a repressor conformation, whereas 2-PG binding to an intradomain cleft of each RD triggers drastic conformational changes leading to the dissociation of NdhR from its target DNA. We further confirmed the effect of 2-PG or 2-OG levels on the transcription of the NdhR regulon. Together with previous findings, we propose that NdhR can sense 2-OG from the Krebs cycle and 2-PG from photorespiration, two key metabolites that function together as indicators of intracellular carbon/nitrogen status, thus representing a fine sensor for the coordination of carbon and nitrogen metabolism in cyanobacteria.