Parallel analysis of transcription, integration, and sequence of single HIV-1 proviruses.

HIV-1-infected cells that persist despite antiretroviral therapy (ART) are frequently considered "transcriptionally silent," but active viral gene expression may occur in some cells, challenging the concept of viral latency. Applying an assay for profiling the transcriptional activity and the chromosomal locations of individual proviruses, we describe a global genomic and epigenetic map of transcriptionally active and silent proviral species and evaluate their longitudinal evolution in persons receiving suppressive ART. Using genome-wide epigenetic reference data, we show that proviral transcriptional activity is associated with activating epigenetic chromatin features in linear proximity of integration sites and in their inter- and intrachromosomal contact regions. Transcriptionally active proviruses were actively selected against during prolonged ART; however, this pattern was violated by large clones of virally infected cells that may outcompete negative selection forces through elevated intrinsic proliferative activity. Our results suggest that transcriptionally active proviruses are dynamically evolving under selection pressure by host factors.

Ectodomain shedding of PLA2R1 is mediated by the metalloproteases ADAM10 and ADAM17.

Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer and is the major autoantigen in membranous nephropathy, a rare but severe autoimmune kidney disease. A soluble form of PLA2R1 has been detected in mouse and human serum. It is likely produced by proteolytic shedding of membrane-bound PLA2R1 but the mechanism is unknown. Here, we show that human PLA2R1 is cleaved by A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 in HEK293 cells, mouse embryonic fibroblasts, and human podocytes. By combining site-directed mutagenesis and sequencing, we determined the exact cleavage site within the extracellular juxtamembrane stalk of human PLA2R1. Orthologs and paralogs of PLA2R1 are also shed. By using pharmacological inhibitors and genetic approaches with RNA interference and knock-out cellular models, we identified a major role of ADAM10 in the constitutive shedding of PLA2R1 and a dual role of ADAM10 and ADAM17 in the stimulated shedding. We did not observe evidence for cleavage by ß- or γ-secretase, suggesting that PLA2R1 may not be a substrate for regulated intramembrane proteolysis. PLA2R1 shedding occurs constitutively and can be triggered by the calcium ionophore ionomycin, the protein kinase C activator PMA, cytokines, and lipopolysaccharides, in vitro and in vivo. Altogether, our results show that PLA2R1 is a novel substrate for ADAM10 and ADAM17, producing a soluble form that is increased in inflammatory conditions and likely exerts various functions in physiological and pathophysiological conditions including inflammation, cancer, and membranous nephropathy.

Methiothepin downregulates SNAP-23 and inhibits degranulation of rat basophilic leukemia cells and mouse bone marrow-derived mast cells.

In the present study, we found that methiothepin (a nonselective 5-hydroxytryptamine [5-HT] receptor antagonist) inhibited antigen-induced degranulation in rat basophilic leukemia cells and mouse bone marrow-derived mast cells. Although antigen stimulation induces release of histamine and serotonin (5-HT) by exocytosis and mast cells express several types of 5-HT receptor, the detailed role of these receptors remains unclear. Here, pretreatment of cells with methiothepin attenuated increased intracellular Ca2+ concentration, phosphorylated critical upstream signaling components (Src family tyrosine kinases, Syk, and PLCγ1), and suppressed TNF-α secretion via inhibition of Akt (a Ser/Thr kinase activated by PI3K)and ERK phosphorylation. Furthermore, it inhibited PMA/ionomycin-induced degranulation; this finding suggested that methiothepin affected downstream signaling. IκB kinase ß phosphorylates synaptosomal associated protein 23, which regulates the fusion events of the secretory granule/plasma membrane after mast cell activation, resulting in degranulation. We showed that methiothepin blocked PMA/ionomycin-induced phosphorylation of synaptosomal associated protein 23 by inhibiting its interaction with IκB kinase ß. Together with the results of selective 5-HT antagonists, it is suggested that methiothepin inhibits mast cell degranulation by downregulating upstream signaling pathways and exocytotic fusion machinery through mainly 5-HT1A receptor. Our findings provide that 5-HT antagonists may be used to relieve allergic reactions.

CPHEN-017: Comprehensive phenotyping of neutrophil extracellular traps (NETs) on peripheral human neutrophils.

With the recent discovery of their ability to produce neutrophil extracellular traps (NETs), neutrophils are increasingly appreciated as active participants in infection and inflammation. NETs are characterized as large, web-like networks of DNA and proteins extruded from neutrophils, and there is considerable interest in how these structures drive disease in humans. Advancing research in this field is contingent on developing novel tools for quantifying NETosis. To this end, we have developed a 7-marker flow cytometry panel for analyzing NETosis on human peripheral neutrophils following in vitro stimulation, and in fresh circulating neutrophils under inflammatory conditions. This panel was optimized on neutrophils isolated from whole blood and analyzed fresh or in vitro stimulated with phorbol 12-myristate 13-acetate (PMA) or ionomycin, two known NET-inducing agonists. Neutrophils were identified as SSChighFSChighCD15+CD66b+. Neutrophils positive for amine residues and 7-Aminoactinomycin D (7-AAD), our DNA dye of choice, were deemed necrotic (Zombie-NIR+7-AAD+) and were removed from downstream analysis. Exclusion of Zombie-NIR and positivity for 7-AAD (Zombie-NIRdim7-AAD+) was used here as a marker of neutrophil-appendant DNA, a key feature of NETs. The presence of two NET-associated proteins - myeloperoxidase (MPO) and neutrophil elastase (NE) - were utilized to identify neutrophil-appendant NET events (SSChighFSChighCD15+CD66b+Zombie NIRdim7-AAD+MPO+NE+). We also demonstrate that NETotic neutrophils express citrullinated histone H3 (H3cit), are concentration-dependently induced by in vitro PMA and ionomycin stimulation but are disassembled with DNase treatment, and are present in both chronic and acute inflammation. This 7-color flow cytometry panel provides a novel tool for examining NETosis in humans.

Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles.

BACKGROUND: Disruption of Ca2+ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca2+ kinetics and gene expression involved in the regulation of Ca2+ signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1. METHODS: CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca2+ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca2+ regulation ([Ca2+]i) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl3, and GdCl3. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca2+ signaling through RNA sequencing. RESULTS: EA.hy926 cells, at increasing Ca2+ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca2+-dependent morphological changes in EA.hy926's actin filaments, microtubules, and cell-cell junctions. Spectrofluorometric Ca2+ kinetics showed higher amplitudes in Ca2+ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca2+]i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca2+ ions induced a significantly higher [Ca2+]i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca2+-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca2+ import and downregulated export genes in EA.hy926. CONCLUSIONS: Our finding show that significant differences in CaEP response and [Ca2+]i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca2+]i changes, which may be linked to overexpression of Ca2+-related genes and an inability to mitigate changes in [Ca2+]i. The study offers a bioinformatic basis for selecting EC models based on research objectives.

Distinct regulation of two flagella by calcium during chemotaxis of male gametes in the brown alga Mutimo cylindricus (Cutleriaceae, Tilopteridales).

Brown algal male gametes show chemotaxis to the sex pheromone that is released from female gametes. The chemotactic behavior of the male gametes is controlled by the changes in the beating of two flagella known as the anterior and posterior flagellum. Our previous study using Mutimo cylindricus showed that the sex pheromone induced an increment in both the deflection angle of the anterior flagellum and sustained unilateral bend of the posterior flagellum, but the mechanisms regulating these two flagellar waveforms were not fully revealed. In this study, we analyzed the changes in swimming path and flagellar waveforms with a high-speed recording system under different calcium conditions. The extracellular Ca2+ concentration at 10-3 M caused an increment in the deflection angle of the anterior flagellum only when ionomycin was absent. No sustained unilateral bend of the posterior flagellum was induced either in the absence or presence of ionomycin in extracellular Ca2+ concentrations below 10-2 M. Real-time Ca2+ imaging revealed that there is a spot near the basal part of anterior flagellum showing higher Ca2+ than in the other parts of the cell. The intensity of the spot slightly decreased when male gametes were treated with the sex pheromone. These results suggest that Ca2+-dependent changes in the anterior and posterior flagellum are regulated by distinct mechanisms and that the increase in the anterior flagellar deflection angle and sustained unilateral bend of the posterior flagellum may not be primarily induced by the Ca2+ concentration.

Difference in Contractile Mechanisms between the Early and Sustained Components of Ionomycin-Induced Contraction in Rat Caudal Arterial Smooth Muscle.

We previously reported that the sustained component of contraction induced by depolarizing stimulation by high K+ concentration in rat caudal arterial smooth muscle involves a Ca2+-induced Ca2+ sensitization mechanism whereby Ca2+ entry through voltage-gated Ca2+ channels activates proline-rich tyrosine kinase 2 (Pyk2), leading to activation of RhoA/Rho-associated kinase (ROCK). In the present study, we investigated a potential role for Pyk2-mediated RhoA/ROCK activation in contraction mediated by elevation of cytosolic free Ca2+ concentration ([Ca2+]i) induced by a Ca2+ ionophore, ionomycin, rather than by depolarizing stimulation. Ionomycin (60 µM) induced slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca2+. Pre-treatment with a myosin light chain kinase (MLCK) inhibitor, ML-9 (30 µM), inhibited both the early phase (4 min) and the sustained phase (30 min) of ionomycin-induced contraction. On the other hand, a ROCK inhibitor, HA-1077 (3 µM), and Pyk2 inhibitors, sodium salicylate (10 mM) and PF-431396 (3 µM), suppressed only the sustained phase of ionomycin-induced contraction. A calmodulin (CaM) inhibitor, W-7 (150 µM), but not W-5 (150 µM), suppressed the early phase of contraction. Early or sustained increase of ionomycin-induced 20 kDa light chain of myosin (LC20) phosphorylation was inhibited by each inhibitor in a manner similar to the attenuation of contraction. These results indicate that the early phase of ionomycin-induced contraction is mediated by MLCK activation by [Ca2+]i elevation, whereas the sustained phase of ionomycin-induced contraction involves RhoA/ROCK activation and inhibition of myosin light chain phosphatase (MLCP) through CaM-independent Pyk2 activation by [Ca2+]i elevation.

Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

Dysregulation of ADAM10 shedding activity in naked mole-rat fibroblasts is due to deficient phosphatidylserine externalization.

The naked mole-rat (NMR, Heterocephalus glaber) is of significant interest to biogerontological research, rarely developing age-associated diseases, such as cancer. The transmembrane glycoprotein CD44 is upregulated in certain cancers and CD44 cleavage by a disintegrin and metalloproteinase 10 (ADAM10) regulates cellular migration. Here we provide evidence that mature ADAM10 is expressed in NMR primary skin fibroblasts (NPSF), and that ionomycin increases cell surface ADAM10 localization. However, we observed an absence of ADAM10 mediated CD44 cleavage, as well as shedding of exogenous and overexpressed betacellulin in NPSF, whereas in mouse primary skin fibroblasts ionomycin induced ADAM10-dependent cleavage of both CD44 and betacellulin. Overexpressing a hyperactive form of the Ca2+ -dependent phospholipid scramblase ANO6 in NPSF increased phosphatidylserine (PS) externalization, which rescued the ADAM10 sheddase activity and promoted cell migration in NPSF in an ADAM10-dependent manner. These findings suggest that dysregulation of ADAM10 shedding activity is due to a deficient PS externalization in NMR.

Assisted oocyte activation does not overcome recurrent embryo developmental problems.

STUDY QUESTION: Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? SUMMARY ANSWER: AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. WHAT IS KNOWN ALREADY: The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. STUDY DESIGN, SIZE, DURATION: This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. MAIN RESULTS AND THE ROLE OF CHANCE: We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. LIMITATIONS, REASONS FOR CAUTION: Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. WIDER IMPLICATIONS OF THE FINDINGS: Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. TRIAL REGISTRATION NUMBER: NCT03354013.

Artificial oocyte activation with ionomycin compared with A23187 among patients at risk of failed or impaired fertilization.

RESEARCH QUESTION: Do fertilization rates differ between intracytoplasmic sperm injection (ICSI) cycles treated with artificial oocyte activation (AOA) using 10 µmol/l ionomycin or commercial A23187 in women at risk of failed or impaired fertilization? DESIGN: This single-centre, 7-year retrospective cohort study included 157 couples with a history of total fertilization failure (TFF, 0%) or low fertilization (<30%) after ICSI, or with severe oligo-astheno-teratozoospermia (OAT) in the male partner. Couples and underwent 171 ICSI-AOA cycles using either 10 µmol/l ionomycin or commercial A23187. The embryological and clinical outcomes were compared. RESULTS: Fertilization rates in the ionomycin group were significantly higher than those in the A23187 group for all three subgroups (TFF, 46.9% versus 28.4%, P = 0.002; low fertilization, 67.7% versus 49.2%, P < 0.001; severe OAT, 66.4% versus 31.6%, P < 0.001). AOA with ionomycin significantly increased the day 3 cleavage rate (P = 0.009) when compared with A23187 in the low fertilization group, but not in the TFF or severe OAT group (both P > 0.05). The rates of day 3 good-quality embryos, clinical pregnancy, implantation and live birth, and the cumulative live birth, did not differ between the two groups (all P > 0.05). A total of 64 live births resulted in 72 healthy babies born. CONCLUSIONS: AOA with 10 µmol/l ionomycin may be more effective than commercial A23187 in improving oocyte activation in patients at risk of failed or impaired fertilization, especially in cases of sperm-related defects.

Blockade of IL-11 Trans-Signaling or JAK2/STAT3 Signaling Ameliorates the Profibrotic Effect of IL-11.

OBJECTIVES: Systemic sclerosis (SSc) is a rare rheumatic disease characterized by vascular damage, dysregulated immune response, and fibrosis. Interleukin-11 (IL-11) is upregulated in SSc. This study aimed to investigate the pathological and therapeutic role of the IL-11 trans-signaling pathway in SSc. METHODS: Plasma IL-11 level was evaluated in 32 patients with SSc and 15 healthy controls, while the expression levels of ADAM10, ADAM17, IL-11, IL-11 Rα, or IL-11 co-stained with CD3 or CD163 in the skin of SSc patients and healthy controls were analyzed. Fibroblasts were treated with IL-11 and ionomycin to evaluate the profibrotic effect of IL-11 trans-signaling pathway. TJ301 (sgp130Fc) and WP1066 (a JAK2/STAT3 inhibitor) intervention groups were set up to investigate the antifibrotic effect of targeting IL-11. RESULTS: Levels of plasma IL-11 were extremely low in most SSc patients and healthy controls. In contrast, levels of IL-11, IL-11 Rα, and ADAM10, but not ADAM17, were significantly elevated in the skin of SSc patients. Moreover, the numbers of IL-11+ CD3+ cells and IL-11+ CD163+ cells were increased in the skin of SSc patients. Besides, IL-11 and ADAM10 were also elevated in the skin and pulmonary of bleomycin-induced SSc mouse. Fibroblasts co-stimulated with IL-11 and ionomycin showed increased expression of COL3 and phosphorylation of STAT3, which could be inhibited by TJ301 or WP1066. TJ301 also ameliorated skin and lung fibrosis in BLM-induced SSc mouse. CONCLUSIONS: IL-11 induces fibrosis in SSc by regulating the trans-signaling pathway. Blockage of sgp130Fc or inhibition of the JAK2/STAT3 pathway could ameliorate the profibrotic effect of IL-11.

Significant differences in efficiency between two commonly used ionophore solutions for assisted oocyte activation (AOA): a prospective comparison of ionomycin and A23187.

PURPOSE: Despite the success of ICSI in treating severe male factor infertile patients, total fertilization failure (FF) still occurs in around 1-3% of ICSI cycles. To overcome FF, the use of calcium ionophores has been proposed to induce oocyte activation and restore fertilization rates. However, assisted oocyte activation (AOA) protocols and ionophores vary between laboratories, and the morphokinetic development underlying AOA remains understudied. METHODS: A prospective single-center cohort study involving 81 in vitro matured metaphase-II oocytes from 66 oocyte donation cycles artificially activated by A23187 (GM508 CultActive, Gynemed) (n=42) or ionomycin (n=39). Parthenogenesis was induced, and morphokinetic parameters (tPNa, tPNf, t2-t8, tSB, and tB) were compared between the 2 study groups and a control group comprising 39 2PN-zygotes from standard ICSI cycles. RESULTS: Ionomycin treatment resulted in higher activation rates compared to A23187 (38.5% vs 23.8%, p=0.15). Importantly, none of the A23187-activated parthenotes formed blastocysts. When evaluating the morphokinetic dynamics between the two ionophores, we found that tPNa and tPNf were significantly delayed in the group treated by A23187 (11.84 vs 5.31, p=0.002 and 50.15 vs 29.69, p=0.005, respectively). t2 was significantly delayed in A23187-activated parthenotes when compared to the double heterologous control embryo group. In contrast, the morphokinetic development of ionomycin-activated parthenotes was comparable to control embryos (p>0.05). CONCLUSION: Our results suggest that A23187 leads to lower oocyte activation rates and profoundly affects morphokinetic timings and preimplantation development in parthenotes. Despite our limited sample size and low parthenote competence, standardization and further optimization of AOA protocols may allow wider use and improved outcomes for FF cycles.

Broadband Electrical Spectroscopy to Distinguish Single-Cell Ca2+ Changes Due to Ionomycin Treatment in a Skeletal Muscle Cell Line.

Many skeletal muscle diseases such as muscular dystrophy, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), and sarcopenia share the dysregulation of calcium (Ca2+) as a key mechanism of disease at a cellular level. Cytosolic concentrations of Ca2+ can signal dysregulation in organelles including the mitochondria, nucleus, and sarcoplasmic reticulum in skeletal muscle. In this work, a treatment is applied to mimic the Ca2+ increase associated with these atrophy-related disease states, and broadband impedance measurements are taken for single cells with and without this treatment using a microfluidic device. The resulting impedance measurements are fitted using a single-shell circuit simulation to show calculated electrical dielectric property contributions based on these Ca2+ changes. From this, similar distributions were seen in the Ca2+ from fluorescence measurements and the distribution of the S-parameter at a single frequency, identifying Ca2+ as the main contributor to the electrical differences being identified. Extracted dielectric parameters also showed different distribution patterns between the untreated and ionomycin-treated groups; however, the overall electrical parameters suggest the impact of Ca2+-induced changes at a wider range of frequencies.

Activated T cells are the cellular source of IL-22 that enhances proliferation and survival of lymphocytes in Nile tilapia.

As a pleiotropic cytokine mainly secreted by CD4+ T cells, interleukin (IL)-22 plays an important role in immune regulation and infection elimination. Despite IL-22 homologues have been identified in non-mammal, whether and how IL-22 participates in the adaptive immune response of early vertebrates have not been fully addressed. In this study, we identified an evolutionarily conserved IL-22 from Nile tilapia Oreochromis niloticus (defined as OnIL-22), proved by its properties regarding sequence, gene structure, functional domain, tertiary structure and phylogeny. IL-22 was broadly expressed in lymphoid-related tissues of tilapia, and with relatively higher levels in skin, gill, intestine and liver. The expression of OnIL-22 in spleen lymphocytes was markedly induced at the adaptive immune stage after Streptococcus agalactiae infection. Moreover, once lymphocytes were activated by PMA plus ionomycin or T-cell specific mitogen PHA in vitro, OnIL-22 expression was obviously up-regulated at both mRNA and protein levels. These results thus suggest that activated T cells produce IL-22 to take part in the adaptive immune response of tilapia. Furthermore, treatment of lymphocytes with recombinant OnIL-22 increased the expression of genes related to proliferation and survival, and further promoted the proliferation and reduced the apoptosis of lymphocytes during bacterial infection or T-cell activation. These cellular effects of IL-22 seem to be associated with JAK1/STAT3 axis downstream of IL-22, because IL-22 application not only elevated the mRNA expression of JAK1 and STAT3, but also enhanced their phosphorylation in lymphocytes. Altogether, we suggest that activated T cells produce IL-22 to promote lymphocyte proliferation and survival probability via JAK1/STAT3 signaling pathway, thus participating in adaptive immune response of Nile tilapia. Our study therefore provides helpful perspective for understanding the function and mechanism of adaptive immune system in teleost.

Design, synthesis and in vitro biological evaluation of marine phidianidine derivatives as potential anti-inflammatory agents.

Phidianidines A and B are novel marine indole alkaloids with various biological activities. Based on their potential anti-inflammatory properties, a series of phidianidine derivatives were designed, synthesized, and tested for their effects on IL-17A production in PMA/ionomycin-stimulated T-cell-lymphoma EL-4 cells. Compounds 9a and 22c exhibited excellent anti-inflammatory activity and low toxicity, with IC50 values of 7.7 µM and 5.3 µM for IL-17A production in PMA/ionomycin-stimulated EL-4 cells, respectively. Further mechanistic study showed that 9a could decrease the STAT3 phosphorylation at Y705 to inhibit IL-17A production in EL-4 cells, indicating its ability of preventing the differentiation of Th17 cells and their possible function. This research may give an insight for the discovery of marine indole alkaloid derived anti-inflammatory drug leads for the treatment of T cell-mediated diseases.

Baseline T-lymphocyte and cytokine indices in sheep peripheral blood.

BACKGROUND: Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune indicators, especially T-lymphocyte parameters, is necessary for research on sheep diseases and vaccines, to better understand the immune response to bacteria and viruses for reducing the use of antibiotics and improving the welfare of sheep. We randomly selected 36 sheep of similar ages to analyze cell-related immune indicators in peripheral blood mononuclear cells (PBMCs). The proportions of CD4+ and CD8+ T cells in PBMCs were detected by flow cytometry. We used Concanavalin A (Con A) and Phorbol-12-myristate-13-acetate (PMA)/Ionomycin to stimulate PBMCs, and measured the expression of IFN-γ, IL-4, and IL-17A using enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISpot). Simultaneously, PMA/Ionomycin/brefeldin A (BFA) was added to PBMCs, then the expression of IFN-γ, IL-4, and IL-17A was detected by flow cytometry after 4 h of culturing. In addition, we observed the proliferation of PBMCs stimulated with Con A for 3, 4, and 5 days. RESULTS: The proportions of CD4+ T lymphocytes (18.70 ± 4.21%) and CD8+ T lymphocytes (8.70 ± 3.65%) were generally consistent among individuals, with a CD4/CD8 ratio of 2.40 ± 0.79. PBMCs produced high levels of IFN-γ, IL-4, and IL-17A after stimulation with PMA/Ionomycin and Con A. Furthermore, PMA/Ionomycin stimulation of PBMC yielded significantly higher cytokine levels than Con A stimulation. Flow cytometry showed that the level of IFN-γ (51.49 ± 11.54%) in CD8+ T lymphocytes was significantly (p < 0.001) higher than that in CD4+ T lymphocytes (14.29 ± 3.26%); IL-4 (16.13 ± 6.81%) in CD4+ T lymphocytes was significantly (p < 0.001) higher than that in CD8+ T lymphocytes (1.84 ± 1.33%), There was no difference in IL-17A between CD4+ (2.83 ± 0.98%) and CD8+ T lymphocytes (1.34 ± 0.67%). The proliferation of total lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes continued to increase between days 3 and 5; however, there were no significant differences in proliferation between the cell types during the stimulation period. CONCLUSIONS: Evaluating primary sheep immune indicators, especially T lymphocytes, is significant for studying cellular immunity. This study provided valuable data and theoretical support for assessing the immune response of sheep to pathogens and improving sheep welfare.

Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 µM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

Characterization of Citrullination Sites in Neutrophils and Mast Cells Activated by Ionomycin via Integration of Mass Spectrometry and Machine Learning.

Citrullination is an important post-translational modification implicated in many diseases including rheumatoid arthritis (RA), Alzheimer's disease, and cancer. Neutrophil and mast cells have different expression profiles for protein-arginine deiminases (PADs), and ionomycin-induced activation makes them an ideal cellular model to study proteins susceptible to citrullination. We performed high-resolution mass spectrometry and stringent data filtration to identify citrullination sites in neutrophil and mast cells treated with and without ionomycin. We identified a total of 833 validated citrullination sites on 395 proteins. Several of these citrullinated proteins are important components of pathways involved in innate immune responses. Using this benchmark primary sequence data set, we developed machine learning models to predict citrullination in neutrophil and mast cell proteins. We show that our models predict citrullination likelihood with 0.735 and 0.766 AUCs (area under the receiver operating characteristic curves), respectively, on independent validation sets. In summary, this study provides the largest number of validated citrullination sites in neutrophil and mast cell proteins. The use of our novel motif analysis approach to predict citrullination sites will facilitate the discovery of novel protein substrates of protein-arginine deiminases (PADs), which may be key to understanding immunopathologies of various diseases.

Identification of a selective manganese ionophore that enables nonlethal quantification of cellular manganese.

Available assays for measuring cellular manganese (Mn) levels require cell lysis, restricting longitudinal experiments and multiplexed outcome measures. Conducting a screen of small molecules known to alter cellular Mn levels, we report here that one of these chemicals induces rapid Mn efflux. We describe this activity and the development and implementation of an assay centered on this small molecule, named manganese-extracting small molecule (MESM). Using inductively-coupled plasma-MS, we validated that this assay, termed here "manganese-extracting small molecule estimation route" (MESMER), can accurately assess Mn in mammalian cells. Furthermore, we found evidence that MESM acts as a Mn-selective ionophore, and we observed that it has increased rates of Mn membrane transport, reduced cytotoxicity, and increased selectivity for Mn over calcium compared with two established Mn ionophores, calcimycin (A23187) and ionomycin. Finally, we applied MESMER to test whether prior Mn exposures subsequently affect cellular Mn levels. We found that cells receiving continuous, elevated extracellular Mn accumulate less Mn than cells receiving equally-elevated Mn for the first time for 24 h, indicating a compensatory cellular homeostatic response. Use of the MESMER assay versus a comparable detergent lysis-based assay, cellular Fura-2 Mn extraction assay, reduced the number of cells and materials required for performing a similar but cell lethality-based experiment to 25% of the normally required sample size. We conclude that MESMER can accurately quantify cellular Mn levels in two independent cells lines through an ionophore-based mechanism, maintaining cell viability and enabling longitudinal assessment within the same cultures.