Evaluation of microRNA expression profiles in human sperm frozen using permeable cryoprotectant-free droplet vitrification and conventional methods.

ABSTRACT: For sperm cryopreservation, the conventional method, which requires glycerol, has been used for a long time. In addition, the permeable cryoprotectant-free vitrification method has been continuously studied. Although the differences of cryopreservation effects between the two methods have being studied, differences in microRNA (miRNA) profiles between them remain unclear. In this study, we investigated the differences in miRNA expression profiles among conventional freezing sperm, droplet vitrification freezing sperm and fresh human sperm. We also analyzed the differences between these methods in terms of differentially expressed miRNAs (DEmiRs) related to early embryonic development and paternal epigenetics. Our results showed no significant differences between the cryopreservation methods in terms of sperm motility ratio, plasma membrane integrity, DNA integrity, mitochondrial membrane potential, acrosome integrity, and ultrastructural damage. However, sperm miRNA-sequencing showed differences between the two methods in terms of the numbers of DEmiRs (28 and 19 with vitrification using a nonpermeable cryoprotectant and the conventional method, respectively) in postthaw and fresh sperm specimens. DEmiRs related to early embryonic development and paternal epigenetics mainly included common DEmiRs between the groups. Our results showed that the differences between conventional freezing and droplet vitrification were minimal in terms of miRNA expression related to embryonic development and epigenetics. Changes in sperm miRNA expression due to freezing are not always detrimental to embryonic development. This study compared differences in miRNA expression profiles before and after cryopreservation between cryopreservation by conventional and vitrification methods. It offers a new perspective to evaluate various methods of sperm cryopreservation.

An apical Phe-His pair defines the Orai1-coupling site and its occlusion within STIM1.

Ca2+ signal-generation through inter-membrane junctional coupling between endoplasmic reticulum (ER) STIM proteins and plasma membrane (PM) Orai channels, remains a vital but undefined mechanism. We identify two unusual overlapping Phe-His aromatic pairs within the STIM1 apical helix, one of which (F394-H398) mediates important control over Orai1-STIM1 coupling. In resting STIM1, this locus is deeply clamped within the folded STIM1-CC1 helices, likely near to the ER surface. The clamped environment in holo-STIM1 is critical-positive charge replacing Phe-394 constitutively unclamps STIM1, mimicking store-depletion, negative charge irreversibly locks the clamped-state. In store-activated, unclamped STIM1, Phe-394 mediates binding to the Orai1 channel, but His-398 is indispensable for transducing STIM1-binding into Orai1 channel-gating, and is spatially aligned with Phe-394 in the exposed Sα2 helical apex. Thus, the Phe-His locus traverses between ER and PM surfaces and is decisive in the two critical STIM1 functions-unclamping to activate STIM1, and conformational-coupling to gate the Orai1 channel.

Identifying 2PHDO as a SOCE inhibitor from Chinese herbal extracts.

BACKGROUND: STIM- and Orai-mediated store operated calcium entry (SOCE) is a ubiquitous Ca2+ signaling process, crucial for the proper function of immune, muscle and neuronal systems. To treat SOCE-related disorder or diseases of these systems, and to mechanistically dissect activation and function of SOCE, specific SOCE inhibitors are needed. However, strategies for developing new SOCE modifiers are still limited.

Methodology: In this study, we identified a novel SOCE inhibitor named 2PHDO from a small pool of Chinese herbal extracts used for treating psoriasis. It could block SOCE and SOCE-mediated NFAT translocation in multiple types of cells with a half inhibitory concentration around 1 µM. At this concentration, 2PHDO was specific for SOCE. Mechanistically, 2PHDO didn't affect the activation of STIM1 or its physical coupling with Orai1. Rather, 2PHDO inhibited SOCE via its actions on Orai1.

Results: 2PHDO may serve as a good template for developing new medicines aiming to treat SOCE related diseases.

Conclusion: Overall, we proved the feasibility of screening and identification of novel SOCE inhibitors from active monomers of Chinese herbal medicine.

Cell-Cell Communication and Extracellular Vesicles in Cancer.

Cell-cell communication, either through direct contact or indirectly, is critical for multiple cellular processes, such as proliferation, survival, differentiation, and transdifferentiation, and it plays a fundamental role in maintaining the integrity of tissue structure and cellular environment [...].

Cold sensory transduction through the STIM-Orai signaling pathway.

The STIM-Orai signaling pathway mediates Ca2+ signals vital for controlling transcription and cell growth. The Ca2+ sensing STIM proteins are activated by depletion of Ca2+ stored in the ER, and translocate into ER-PM junctions to gate PM Orai channels. STIM1 activation also results from heating STIM1 proteins, and new evidence reveals the STIM1-mediated gating of Orai1 channels is activated by noxious cooling of cells. This activation of the STIM-Orai pathway may be important in mediating vascular dilation that occurs in response to severe cold exposure.

Celastrol inhibits store operated calcium entry and suppresses psoriasis.

Introduction: Psoriasis is an inflammatory autoimmune skin disease that is hard to cure and prone to relapse. Currently available global immunosuppressive agents for psoriasis may cause severe side effects, thus it is crucial to identify new therapeutic reagents and druggable signaling pathways for psoriasis. Methods: To check the effects of SOCE inhibitors on psoriasis, we used animal models, biochemical approaches, together with various imaging techniques, including calcium, confocal and FRET imaging. Results and discussion: Store operated calcium (Ca2+) entry (SOCE), mediated by STIM1 and Orai1, is crucial for the function of keratinocytes and immune cells, the two major players in psoriasis. Here we showed that a natural compound celastrol is a novel SOCE inhibitor, and it ameliorated the skin lesion and reduced PASI scores in imiquimod-induced psoriasis-like mice. Celastrol dose- and time-dependently inhibited SOCE in HEK cells and HaCaT cells, a keratinocyte cell line. Mechanistically, celastrol inhibited SOCE via its actions both on STIM1 and Orai1. It inhibited Ca2+ entry through constitutively-active Orai1 mutants independent of STIM1. Rather than blocking the conformational switch and oligomerization of STIM1 during SOCE activation, celastrol diminished the transition from oligomerized STIM1 into aggregates, thus locking STIM1 in a partially active state. As a result, it abolished the functional coupling between STIM1 and Orai1, diminishing SOCE signals. Overall, our findings identified a new SOCE inhibitor celastrol that suppresses psoriasis, suggesting that SOCE pathway may serve as a new druggable target for treating psoriasis.

Associations of alcohol consumption with the risk and surgical outcomes of chronic rhinosinusitis in China: A case-control study.

OBJECTIVE: To investigate the associations between weekly alcohol consumption and the risk and surgical outcome of Chronic Rhinosinusitis (CRS). DESIGN: A case-control study. SETTING AND PARTICIPANTS: The study population consisted of 1095 CRS patients and 909 healthy collected from the first affiliated hospital of Zhengzhou University between May 2018 and December 2019. MAIN OUTCOME MEASURES: Unconditional multivariate logistic regression analysis and Cox proportional hazards regression analysis were performed to estimate the association of alcohol consumption with the risk and surgical outcomes of CRS. Odds ratios (OR) or hazard ratio (HR) with 95% confidence intervals (CI) were calculated separately. The Kruskal-Wallis test was used to explore the possible molecular mechanisms. RESULTS: As compared with nondrinkers, the multivariable-adjusted OR (95% CI) values of current drinkers consuming 7.5-22 drinks and >22 drinks per week were 2.158 (1.249-3.729) and 5.373 (2.912-9.911), respectively. The rate of mucosal epithelialization after CRS surgery for patients who drank 7.5-22 drinks and >22 drinks per week was lower than that of nondrinkers [HR (95% CI) = 0.487 (0.351-0.675) and 0.252 (0.184-0.346), respectively]. The association of alcohol consumption with the risk and surgical outcome of CRS was dose dependent (p < .01). Alcohol consumption increased the risk of CRS and extended the time of mucosal epithelialization after CRS surgery by possibly increasing serum IgE levels (p < .05). CONCLUSION: Higher alcohol consumption of >7.5 drinks per week was an independent risk factor for CRS and extended the time of mucosal epithelialization after surgery. As a potential underlying mechanism, alcohol consumption increases serum IgE levels.

Orai channel C-terminal peptides are key modulators of STIM-Orai coupling and calcium signal generation.

Junctional coupling between endoplasmic reticulum (ER) Ca2+-sensor STIM proteins and plasma membrane (PM) Orai channels mediates Ca2+ signals in most cells. We reveal that PM-tethered, fluorescently tagged C-terminal M4x (fourth transmembrane helix contains a cytoplasmic C-terminal extension) peptides from Orai channels undergo a Leu-specific signature of direct interaction with the STIM1 Orai-activating region (SOAR), exactly mimicking STIM1 binding to gate Orai channels. The 20-amino-acid Orai3-M4x peptide associates avidly with STIM1 within ER-PM junctions, functions to competitively block native Ca2+ signals, and mediates a key modification of STIM-Orai coupling induced by 2-aminoethoxydiphenyl borate. By blocking STIM-Orai coupling, the Orai3-M4x peptide reveals the critical role of Orai channels in driving Ca2+ oscillatory signals and transcriptional control through NFAT. The M4x peptides interact independently with SOAR dimers consistent with unimolecular coupling between Orai subunits and STIM1 dimers. We reveal the critical role of M4x helices in defining the coupling interface between STIM and Orai proteins to mediate store-operated Ca2+ signals.

The Intricate Coupling Between STIM Proteins and Orai Channels.

Store-operated Ca2+ entry signals are critical for cellular regulation. This intricate signaling pathway involves coupling of proteins in two different membranes: the ER-resident Ca2+-sensing STIM proteins and the highly Ca2+-selective PM Orai channels. The molecular nature of the STIM-Orai coupling interface in ER-PM junctions and consequent Orai channel gating, are processes under intense study. We describe recent developments in determining the mechanism of Orai activation through the key STIM-Orai Activating Region (SOAR) of STIM1. We describe the unexpected unimolecular coupling of STIM with Orai and explain the observed variable stoichiometry of STIM-Orai interactions. We also define the discrete C-terminal regions in Orai channels that initially latch onto STIM proteins and mediate allosteric activation of the channel. A critical "nexus" region closely associated with the STIM-activated C-terminus of Orai1, propagates the STIM-binding signal through the four tightly-associated transmembrane helices of Orai1, finally to modify the pore-forming helices and effect channel opening.

Intellectual disability-associated UNC80 mutations reveal inter-subunit interaction and dendritic function of the NALCN channel complex.

The sodium-leak channel NALCN forms a subthreshold sodium conductance that controls the resting membrane potentials of neurons. The auxiliary subunits of the channel and their functions in mammals are largely unknown. In this study, we demonstrate that two large proteins UNC80 and UNC79 are subunits of the NALCN complex. UNC80 knockout mice are neonatal lethal. The C-terminus of UNC80 contains a domain that interacts with UNC79 and overcomes a soma-retention signal to achieve dendritic localization. UNC80 lacking this domain, as found in human patients, still supports whole-cell NALCN currents but lacks dendritic localization. Our results establish the subunit composition of the NALCN complex, uncover the inter-subunit interaction domains, reveal the functional significance of regulation of dendritic membrane potential by the sodium-leak channel complex, and provide evidence supporting that genetic variations found in individuals with intellectual disability are the causes for the phenotype observed in patients.

Type 3 Inositol 1,4,5-Trisphosphate Receptor is a Crucial Regulator of Calcium Dynamics Mediated by Endoplasmic Reticulum in HEK Cells.

Being the largest the Ca2+ store in mammalian cells, endoplasmic reticulum (ER)-mediated Ca2+ signalling often involves both Ca2+ release via inositol 1, 4, 5-trisphosphate receptors (IP3R) and store operated Ca2+ entries (SOCE) through Ca2+ release activated Ca2+ (CRAC) channels on plasma membrane (PM). IP3Rs are functionally coupled with CRAC channels and other Ca2+ handling proteins. However, it still remains less well defined as to whether IP3Rs could regulate ER-mediated Ca2+ signals independent of their Ca2+ releasing ability. To address this, we generated IP3Rs triple and double knockout human embryonic kidney (HEK) cell lines (IP3Rs-TKO, IP3Rs-DKO), and systemically examined ER Ca2+ dynamics and CRAC channel activity in these cells. The results showed that the rate of ER Ca2+ leakage and refilling, as well as SOCE were all significantly reduced in IP3Rs-TKO cells. And these TKO effects could be rescued by over-expression of IP3R3. Further, results showed that the diminished SOCE was caused by NEDD4L-mediated ubiquitination of Orai1 protein. Together, our findings indicate that IP3R3 is one crucial player in coordinating ER-mediated Ca2+ signalling.

A Frog-Derived Immunomodulatory Peptide Promotes Cutaneous Wound Healing by Regulating Cellular Response.

Wound healing-promoting peptides exhibit excellent therapeutic potential in regenerative medicine. However, amphibian-derived wound healing-promoting peptides and their mechanism of action remain to be further elucidated. We hereby characterized a wound healing-promoting peptide, Ot-WHP, derived from Chinese concave-eared frog Odorrana tormota. It efficiently promoted wound healing in a mouse model of full-thickness wounds. Ot-WHP significantly increased the number of neutrophils in wounds, and modestly promoted neutrophil phagocytosis and phorbol myristate acetate (PMA)-induced neutrophil extracellular trap formation. Ot-WHP also significantly increased the number of macrophages in wound sites, and directly induced chemokine, cytokine and growth factor production in macrophages by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways. Of note, Ot-WHP did not act as a chemoattractant for neutrophils and macrophages, suggesting its chemotactic activity depends on inducing chemoattractant production in macrophages. Besides, Ot-WHP directly promoted keratinocyte migration by enhancing integrin expression and cell adhesion. In addition, Ot-WHP significantly enhanced the cross-talk between macrophages and keratinocytes/fibroblasts by promoting keratinocyte/fibroblast proliferation, and fibroblast-to-myofibroblast transition despite having no direct effects on keratinocyte/fibroblast proliferation, and fibroblast differentiation. Collectively, Ot-WHP directly elicited the production of regulatory factors in macrophages, consequently initiated and accelerated the inflammatory phase by recruiting neutrophils and macrophages to wounds, and in turn enhanced the cross-talk between macrophages and keratinocytes/fibroblasts, additionally promoted keratinocyte migration, and finally promoted cutaneous wound healing. Our findings provide a promising immunomodulator for acute wound management and new clues for understanding the mechanism of action of amphibian-derived wound healing-promoting peptides.

The remote allosteric control of Orai channel gating.

Calcium signals drive an endless array of cellular responses including secretion, contraction, transcription, cell division, and growth. The ubiquitously expressed Orai family of plasma membrane (PM) ion channels mediate Ca2+ entry signals triggered by the Ca2+ sensor Stromal Interaction Molecule (STIM) proteins of the endoplasmic reticulum (ER). The 2 proteins interact within curiously obscure ER-PM junctions, driving an allosteric gating mechanism for the Orai channel. Although key to Ca2+ signal generation, molecular understanding of this activation process remain obscure. Crystallographic structural analyses reveal much about the exquisite hexameric core structure of Orai channels. But how STIM proteins bind to the channel periphery and remotely control opening of the central pore, has eluded such analysis. Recent studies apply both crystallography and single-particle cryogenic electron microscopy (cryo-EM) analyses to probe the structure of Orai mutants that mimic activation by STIM. The results provide new understanding on the open state of the channel and how STIM proteins may exert remote allosteric control of channel gating.

Cross-talk between N-terminal and C-terminal domains in stromal interaction molecule 2 (STIM2) determines enhanced STIM2 sensitivity.

Store-operated Ca2+ entry (SOCE) is a ubiquitous pathway for Ca2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca2+-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1-/-, STIM2-/-, and STIM1/2-/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.

AMPKα2 knockout enhances tumour inflammation through exacerbated liver injury and energy deprivation-associated AMPKα1 activation.

Tissue damage and its associated-inflammation act as tumour initiators or propagators. AMP-activated protein kinase (AMPK) is activated by environmental or nutritional stress factors, such as hypoxia, glucose deprivation, and other cell injury factors, to regulate cell energy balance and differentiation. We previously have reported that AMPKα2 deficiency resulted in the energy deprivation in tumour-bearing liver and the enhanced-hepatocyte death. In this study, AMPKα2 knockout mice and the liver metastasis model of colon cancer cells were used to address the role of AMPKα isoforms in tumour inflammation. First, we found that the AMPKα2 deficiency exacerbated the liver injury and recruitment of macrophages. Meanwhile, although compensatory expression of AMPKα1 was not significant after AMPKα2 knockout, AMPKα1 phosphorylation was elevated in remnant liver in AMPKα2 knockout mice, which was positively associated with the enhanced energy deprivation in the AMPKα2 deficient mice. Furthermore, the activated AMPKα1 in macrophage contributed to its polarizing to tumour-associated phenotype. Thus, the enhanced tumour-associated inflammation and activation of AMPKα1 in the AMPKα2 deficient mice may exacerbate the tumour development by affecting the tumour inflammatory microenvironment. Our study suggests that the two isoforms of AMPKα, AMPKα1 and AMPKα2 play different roles in controlling tumour development.

Anti-inflammatory activities of Aedes aegypti cecropins and their protection against murine endotoxin shock.

BACKGROUND: Mosquitoes are armed with physiologically active compounds to suppress the host immunity including host inflammatory reaction. However, the specific anti-inflammatory components in mosquitoes remain unknown. RESULTS: By searching for the immunomodulatory molecules from the mosquito Aedes aegypti (Diptera: Culicidae) at NCBI for anti-inflammatory function, five cecropins (for short in this study: AeaeCec1, 2, 3, 4 and 5) were selected. AeaeCec1-5 efficiently inhibited the expression of inducible nitric oxide synthase (iNOS), nitrite, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and human peripheral blood mononuclear cells (PBMCs) with low toxicity to mammalian cells. Among the five analogues, AeaeCec5 had the strongest anti-inflammatory activity, and generated an additive effect with other AeaeCec peptides. In a mouse model of endotoxin shock, AeaeCec1-5 effectively reduced TNF-α, IL-1ß and IL-6 expression in lungs, serum and peritoneal lavage and correspondingly reduced lung damage and edema, with AeaeCec5 showing the best protection. In mice infected with Escherichia coli or Pseudomonas aeruginosa, administration of AeaeCec5 reduced the production of TNF-α, IL-1ß and IL-6 and correspondingly reduced lung tissue damage. These effects of Ae. aegypti AeaeCec1-5 were attributed to an efficient inhibition of the activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signaling pathways, as well as partial neutralization of LPS. CONCLUSIONS: The current work characterized the specific anti-inflammatory agents in Ae. aegypti and provided AeaeCec5 as a potent anti-endotoxin peptide that could serve as the basis for the development of anti-inflammatory therapy.

Pore properties of Orai1 calcium channel dimers and their activation by the STIM1 ER calcium sensor.

Store-operated Ca2+ entry signals are mediated by plasma membrane Orai channels activated through intermembrane coupling with Ca2+-sensing STIM proteins in the endoplasmic reticulum (ER). The nature of this elaborate Orai-gating mechanism has remained enigmatic. Based on the Drosophila Orai structure, mammalian Orai1 channels are hexamers comprising three dimeric subunit pairs. We utilized concatenated Orai1 dimers to probe the function of key domains within the channel pore and gating regions. The Orai1-E106Q selectivity-filter mutant, widely considered a dominant pore blocker, was surprisingly nondominant within concatenated heterodimers with Orai1-WT. The Orai1-E106Q/WT heterodimer formed STIM1-activated nonselective cation channels with significantly enlarged apparent pore diameter. Other Glu-106 substitutions entirely blocked the function of heterodimers with Orai1-WT. The hydrophobic pore-lining mutation V102C, which constitutively opens channels, was suppressed by Orai1-WT in the heterodimer. In contrast, the naturally occurring R91W pore-lining mutation associated with human immunodeficiency was completely dominant-negative over Orai-WT in heterodimers. Heterodimers containing the inhibitory K85E mutation extending outward from the pore helix gave an interesting partial effect on both channel activation and STIM1 binding, indicating an important allosteric link between the cytosolic Orai1 domains. The Orai1 C-terminal STIM1-binding domain mutation L273D powerfully blocked STIM1-induced channel activation. The Orai1-L273D/WT heterodimer had drastically impaired STIM1-induced channel gating but, unexpectedly, retained full STIM1 binding. This reveals the critical role of Leu-273 in transducing the STIM1-binding signal into the allosteric conformational change that initiates channel gating. Overall, our results provide important new insights into the role of key functional domains that mediate STIM1-induced gating of the Orai1 channel.