Intracellular Calcium Release Research Articles

Leishmania exploits host cAMP/EPAC/calcineurin signaling to induce an IL-33-mediated anti-inflammatory environment for the establishment of infection

Host anti-inflammatory responses are critical for the progression of visceral leishmaniasis and the pleomorphic cytokine IL-33 was found to be upregulated in infection. The underlying mechanism is not yet known. Here, we documented that IL-33 induction is a consequence of elevated cAMP-mediated EPAC/calcineurin-dependent signaling and is essential for the sustenance of infection. L. donovani-infected RAW and bone marrow-derived macrophages showed significant up-regulation of IL-33 and its neutralization by anti-IL-33 antibody resulted in decreased parasite survival and increased inflammatory responses. Infection-induced elevated cAMP was involved in IL-33 production and of its two downstream effectors PKA and EPAC, only the latter was responsible for elevated IL-33 levels. EPAC initiated Rap-dependent phospholipase C activation, which triggered the release of intracellular calcium followed by calcium/calmodulin complex formation. Screening of calmodulin-dependent enzymes affirmed the involvement of the phosphatase calcineurin in cAMP/EPAC/calcium/calmodulin signaling-induced IL-33 production and parasite survival. Activated calcineurin thereby ensured nuclear localization of the transcription factors NFATc1 and HIF-1α for IL-33 transcription and we further ascertained their role in IL-33 transcription using a ChIP assay. Administering specific inhibitors of NFATc1 and HIF-1α in a BALB/c mouse model of visceral leishmaniasis led to decreased liver and spleen parasite burden with concomitant decrease in IL-33 levels. Splenocyte supernatants of inhibitor-treated infected mice further documented an increase in TNF-α and IL-12 with simultaneous decrease of IL-10, thereby indicating an overall disease-escalating effect of IL-33. Thus, this study demonstrates that cAMP/EPAC/calcineurin signaling is crucial for the activation of IL-33 and in effect creates anti-inflammatory responses, essential for infection.

Calcium-activated chloride channels in pericytes shape capillary electrical signaling

The brain vasculature, composed of penetrating arterioles (PAs) giving rise to the dense capillary network, facilitates continuous energy substrate delivery to neurons and glia by tightly controlling blood flow. The capillaries are covered by perivascular cells known as pericytes, which exhibit varying morphological and physiological properties based on their location in relation to the terminal arteriole, and these contribute to blood flow control within the capillary network in distinct ways. Thin-strand pericytes —the focus of this study—are located in the deep capillary bed. Serendipitously, we have discovered a robust chloride current during perforated patch clamp experiments on these thin-strand pericytes. We hypothesized that this current is mediated by calcium-activated chloride channels (CaCCs), which may influence electrical signaling and thereby regulate blood flow. Our data reveal that this robust current is dependent on external calcium concentration and can be blocked by voltage-gated calcium channel blockers, depletion of endoplasmic reticulum calcium stores, and calcium-activated chloride channel blockers. This suggests a mechanism wherein calcium entry into the cytosol via voltage-gated channels and intracellular calcium release govern the activity of CaCCs in the thin-strand pericyte membrane. Using two-photon microscopy, we further observed that blocking CaCCs led to PA dilation and hyperemia. Together, our data indicate a novel role for CaCCs in thin-strand pericytes, where they modulate electrical signaling across the capillary bed in response to intracellular calcium activity. By providing a depolarizing signal countering hyperpolarizing electrical signaling, these channels contribute to the intricate regulation of blood flow and vessel dilation. This work was supported by the NIH (grants 1R01AG066645, 5R01NS115401, 1DP2NS121347-01), and the American Heart Association (19IPLOI34660108). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Senescence and Mitochondrial Dysfunction in Lung Trisomy 21 Fibroblast

Rationale: Lung associated diseases are one of the leading causes of death in Down syndrome/Trisomy21 (DS/T21). DS is often considered a progeroid syndrome, associated with increased cellular senescence and oxidative stress in dermal T21 fibroblasts. Senescence plays a crucial role in development and organogenesis. We recently demonstrated that T21 lung anomalies are initiated in utero. We aim to better understand the role of senescence in lung disease in T21. Methods: Fresh T21 and, age- and sex-matched non-T21 human fetal lungs were collected, dissociated, and human fetal lung fibroblast (HFLF) cultures were established using differential adhesion. Baseline senescence levels were determined in whole tissue and isolated fibroblasts using TAF staining for tissue only and RT-qPCR and immunofluorescence (IF) for both. β-galactosidase, CellROX/MitoSOX, and calcium release (FURA2/AM imaging) assays were performed on T21 and non-T21 fibroblasts, cultured in the absence or presence of an ER stress inhibitor (4-PBA, 2mM) or a mitochondriogenesis stimulator (PQQ, 20μM). Results: Whole tissue sections demonstrated increased TAF and γ-H2AFX staining in T21 vs non-T21. RT-qPCR demonstrated significantly increased expression of several senescence markers including CDKN1A, CDKN2A, TP53, CDKN2B and PAI1 (p<0.05; n=6) at baseline in T21 HFLF as compared to non-T21. IF for γ-H2AFX and P21 were also significantly increased in T21 cultures vs non-T21 (p<0.01, n=6). Upregulated senescence was further confirmed in T21 HFLF by increased β-galactosidase activity (6-fold vs non-T21, p<0.05, n=4) as well as increased ER and mitochondria stress markers (CellROX and mitoSOX, p<0.05, n=6) in T21 cultures. Fura2/AM assay revealed that T21 HFLF exhibit greater intracellular calcium release (p<0.01, n=3) than non-T21 HFLF at both baseline and post Acetylcholine (10uM) challenge, an indicator of increased ER stress. In addition, several mitochondria morphology genes ( FIS1, DNML1 and MFN1) increased in T21 HFLF (p<0.05, n=6). Both 4-PBA or PQQ treatments reversed senescence in T21 HFLF as shown by decreased β-galactosidase staining (p<0.01; n = 4). No differences were observed in non-T21 HFLF after treatment. However, gene expression analyses of CDKN1A, CDKN2A, TP53 and CDKN2B demonstrated attenuated expression in T21 HFLF only following PQQ treatment but not 4-PBA (p<0.05, n=4). Conclusion: In this study, we established that fibroblasts from T21 lungs present with both ER and mitochondria-associated senescence. However, only PQQ, the mitochondriogenesis stimulator, was able to completely reverse the senescence observed at baseline in T21 HFLF. Understanding this increase in senescence in the lungs of individuals with Down Syndrome may help address the respiratory issues that arise after birth. CIRM (EDUC4-12837) Postdoctoral Training Grant (RB) NIH/NHLBI R01HL141856 (DA); NIH/NHLBI Offce of The Director, National Institutes of Health (OD) R01HL155104 (SD); NIH/NHLBI R21HL165411 (SD and DA). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TRPM5 activation depends on a synergistic effect of calcium and PKC phosphorylation

Transient receptor potential melastatin 5 (TRPM5) is a calcium-activated monovalent-specific ion channel involved in insulin secretion and taste transduction, making it an attractive target for drug development in various pathologies. While TRPM5 activation involves ligand binding to Gq/G-protein coupled receptors (GPCR) and subsequent elevation of intracellular calcium levels, recent reports suggest the need for additional molecular determinants. Hence, the mechanism of TRPM5 activation remains to be elucidated. Here, we show that PKC phosphorylation and the elevation of intracellular Ca2+ levels are required for TRPM5 activation, with PKC phosphorylation being crucial for channel-evoked currents, primarily at physiological membrane potentials. In contrast, physiological relevant calcium levels alone only induce TRPM5 activation at positive voltages. Our findings highlight the necessity of coordinated intracellular calcium release and PKC phosphorylation for TRPM5 activation. Thus, our results suggest that regulation of PKC activity could be a promising therapeutic target for diseases associated with TRPM5 modulation.

Bitter Taste Receptor T2R14 and Autophagy Flux in Gingival Epithelial Cells.

Macroautophagy (hereafter autophagy) is a lysosomal degradation pathway that functions in nutrient recycling and as a mechanism of innate immunity. Previously, we reported a novel host-bacteria interaction between cariogenic S. mutans and bitter taste receptor (T2R14) in gingival epithelial cells (GECs), leading to an innate immune response. Further, S. mutans might be using the host immune system to inhibit other Gram-positive bacteria, such as S. aureus. To determine whether these bacteria exploit the autophagic machinery of GEC, it is first necessary to evaluate the role of T2R14 in modulating autophagic flux. So far, the role of T2R14 in the regulation of autophagy is not well characterized. Therefore, in this study, for the first time, we report that T2R14 downregulates autophagy flux in GECs, and T2R14 knockout increases acidic vacuoles. However, the treatments of GEC WT with a T2R14 agonist and antagonist did not lead to a significant change in acidic vacuole formation. Transmission electron microscopy morphometric results also suggested an increased number of autophagic vesicles in T2R14-knockout GEC. Further, our results suggest that S. mutans competence stimulating peptide CSP-1 showed robust intracellular calcium release and this effect is both T2R14- and autophagy protein 7-dependent. In this study, we provide the first evidence that T2R14 modulates autophagy flux in GEC. The results of the current study could help in identifying the impact of T2R in regulation of the immuno-microenvironment of GEC and subsequently oral health.

A mechanism for the treatment of cardiovascular and renal disease.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality globally. CVD and kidney disease are closely related, with kidney injury increasing CVD mortality. The pathogenesis of cardiovascular and renal diseases involves complex and diverse interactions between multiple extracellular and intracellular signaling molecules, among which transient receptor potential vanilloid 1 (TRPV1)/ transient receptor potential ankyrin 1(TRPA1) channels have received increasing attention. TRPV1 belongs to the vanilloid receptor subtype family of transient receptor potential (TRP) ion channels, and TRPA1 belongs to the TRP channel superfamily. TRPV1/TRPA1 are jointly involved in the management of cardiovascular and renal diseases, and play important roles in regulating vascular tension, promoting angiogenesis, anti-fibrosis, anti-inflammation, and anti-oxidation. The mechanism of TRPV1 / TRPA1 is mainly related to regulation of intracellular calcium influx and release of nitric oxide (NO) and calcitonin gene-related peptide (CGRP). Therefore, this study takes TRPV1 / TRPA1 channel as the research object, analyzes and summarizes the process and mechanism of TRPV1 / TRPA1 affecting cardiovascular and renal diseases, and lays a foundation for the treatment of cardio-renal diseases.

Paternal preconception alcohol consumption increased Angiotensin II-mediated vasoconstriction in male offspring cerebral arteries via oxidative stress-AT1R pathway.

Alcohol consumption is popular worldwidely and closely associated with cardiovascular diseases. Influences of paternal preconception alcohol consumption on offspring cerebral arteries are largely unknown. Male rats were randomly given alcohol or water before being mated with alcohol-naive females to produce alcohol- and control-sired offspring. Middle cerebral artery (MCA) was tested with a Danish Myo Technology wire myograph, patch-clamp, IONOPTIX, immunofluorescence and quantitative PCR. Alcohol consumption enhanced angiotensin II (AngII)-mediated constriction in male offspring MCA mainly via AT1R. PD123,319 only augmented AngII-induced constriction in control offspring. AngII and Bay K8644 induced stronger intracellular calcium transient in vascular smooth muscle cells (VSMCs) from MCA of alcohol offspring. L-type voltage-dependent calcium channel (L-Ca2+ ) current at baseline and after AngII-stimulation was higher in VSMCs. Influence of large-conductance calcium-activated potassium channel (BKC a ) was lower. Caffeine induced stronger constriction and intracellular calcium release in alcohol offspring. Superoxide anion was higher in alcohol MCA than control. Tempol and thenoyltrifluoroacetone alleviated AngII-mediated contractions, while inhibition was significantly higher in alcohol group. The mitochondria were swollen in alcohol MCA. Despite lower Kcnma1 and Prkce expression, many genes expressions were higher in alcohol group. Hypoxia induced reactive oxygen species production and increased AT1R expression in control MCA and rat aorta smooth muscle cell line. In conclusion, this study firstly demonstrated paternal preconception alcohol potentiated AngII-mediated vasoconstriction in offspring MCA via ROS-AT1R. Alcohol consumption increased intracellular calcium via L-Ca2+ channel and endoplasmic reticulum and decreased BKCa function. The present study provided new information for male reproductive health and developmental origin of cerebrovascular diseases.

Optimization of CHARMM force field parameters for ryanodine receptor inhibitory drug dantrolene using FFTK and FFParam.

Ryanodine receptors (RyRs) are large intracellular ligand-gated calcium release ion channels. Mutations in human RyR1 in combination with a volatile anesthetic or muscle relaxant are known to cause leaky RyRs resulting in malignant hyperthermia (MH). This has long been primarily treated with the RyR inhibitory drug dantrolene. Alternatives to dantrolene as a RyR inhibitor may be found through computer-aided drug design. Additionally, molecular dynamics (MD) studies of dantrolene interacting with RyRs may reveal its full mechanism of action. The availability of accurate force field parameters is important for the success of both. In this study, force field parameters for dantrolene were obtained from the CHARMM General Force Field (CGenFF) program and optimized using the force field toolkit (FFTK) and FFParam programs. The obtained parameters were then validated by a comparison between calculated and experimental IR spectra and normal mode analysis, among other techniques.

Ryanodine receptor dysfunction causes senescence and fibrosis in Duchenne dilated cardiomyopathy.

Duchenne muscular dystrophy (DMD) is an X-linked disorder characterized by progressive muscle weakness due to the absence of functional dystrophin. DMD patients also develop dilated cardiomyopathy (DCM). We have previously shown that DMD (mdx) mice and a canine DMD model (GRMD) exhibit abnormal intracellular calcium (Ca2+) cycling related to early-stage pathological remodelling of the ryanodine receptor intracellular calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) contributing to age-dependent DCM. Here, we used hiPSC-CMs from DMD patients selected by Speckle-tracking echocardiography and canine DMD cardiac biopsies to assess key early-stage Duchenne DCM features. Dystrophin deficiency was associated with RyR2 remodelling and SR Ca2+ leak (RyR2 Po of 0.03±0.01 for HC vs. 0.16±0.01 for DMD, P<0.01), which led to early-stage defects including senescence. We observed higher levels of senescence markers including p15 (2.03±0.75 for HC vs. 13.67±5.49 for DMD, P<0.05) and p16 (1.86±0.83 for HC vs. 10.71±3.00 for DMD, P<0.01) in DMD hiPSC-CMs and in the canine DMD model. The fibrosis was increased in DMD hiPSC-CMs. We observed cardiac hypocontractility in DMD hiPSC-CMs. Stabilizing RyR2 pharmacologically by S107 prevented most of these pathological features, including the rescue of the contraction amplitude (1.65±0.06μm for DMD vs. 2.26±0.08μm for DMD+S107, P<0.01). These data were confirmed by proteomic analyses, in particular ECM remodelling and fibrosis. We identified key cellular damages that are established earlier than cardiac clinical pathology in DMD patients, with major perturbation of the cardiac ECC. Our results demonstrated that cardiac fibrosis and premature senescence are induced by RyR2 mediated SR Ca2+ leak in DMD cardiomyocytes. We revealed that RyR2 is an early biomarker of DMD-associated cardiac damages in DMD patients. The progressive and later DCM onset could be linked with the RyR2-mediated increased fibrosis and premature senescence, eventually causing cell death and further cardiac fibrosis in a vicious cycle leading to further hypocontractility as a major feature of DCM. The present study provides a novel understanding of the pathophysiological mechanisms of the DMD-induced DCM. By targeting RyR2 channels, it provides a potential pharmacological treatment.

Electrochemical cellular biosensor combined with fluorescence microscopy: An investigation of subtle changes in response of cells to a drug

AbstractThis study presents the combination of electrochemical cell based biosensor and fluorescence microscopy on a single platform to track biomolecular processes contributing to the morphology changes of the cells. Initial experiments demonstrate that using interdigitated electrodes where the gold microfingers were comparable in width and spacing to a single cell have the optimal sensitivity in the final electrochemical cell based‐sensing device. This was determined by measuring the cell index, based on the impedance analysis of bare and cell‐covered microelectrodes. The fabrication of the electrodes on a glass substrate enabled the capture of high‐resolution fluorescence microscopy images of single cells and related intracellular calcium release inside the HeLa cells via a window incorporated into the gold microelectrode design. As an illustration of the enhanced capability of the combined approach over traditional impedance cellular assay, the opto‐electric assay was utilized as a functional readout for G protein couple receptor activation. The simultaneous examinations of cells stimulated with histamine demonstrated an association between time courses of changes in cytosolic calcium concentration and reductions in cell‐cell adhesions.

#164 : In-Vitro Vitamin D Treatment on Human Sperm Improves Capacitation, Acrosome Reaction, and Protein Expression of Sperm-Oocyte Activation Factors (SOAF)

Background and Aims: Although many studies have been done to investigate the relationship between serum vitamin D and male infertility, the effect of in-vitro vitamin D treatment on sperm remains unclear, particularly the effect on sperm capacitation, hyperpolarisation, sperm-oocyte fusion and oocyte activation. This study aims to examine whether in-vitro vitamin D treatment on sperm can improve the condition of sperm parameters and further increase fertilisation and pregnancy rate. Method: Fifty men (N=50, fertile n=26; infertile men n=24, were recruited during their visit to fertility clinics for semen analysis. Each washed semen sample was aliquoted and divided into control and in-vitro vitamin D treatment groups. For in-vitro vitamin D treatment, semen samples were treated with 1nM in-vitro for 1 hour at 37°C. Intracellular calcium released, capacitation status and mitochondria membrane potential were measured after incubation in both the control and in-vitro vitamin D treatment groups. Furthermore, immunofluorescence of sperm-origin SOAF, Phospholipase C Zeta (PLC[Formula: see text]), was done to investigate fluorescence intensity. Results: Compared to the control, higher intracellular calcium release was noted following in-vitro vitamin D treatment, represented by higher Fluo-4AM staining (MD=25043, P=0.02). In the treatment group, sperm capacitation rate and CTCF were higher when Chlortetracyclin (CTC) (MD=4.7%, P=0.01) and immunofluorescence of phosphotyrosine (MD=0.03, P=0.01) were examined. More hyperpolarised mitochondria membranes in the treatment group showed by JC-1 staining (P=0.01) compared to the control group. The red-to-green, fluorescent ratio is 1.62 in the control group and 2.24 in the treatment group. Moreover, immunofluorescence of PLC[Formula: see text] shows that the treatment improves this protein’s expression (MD=8.013×10[Formula: see text], P=0.02). Thus, in-vitro vitamin D enhances the functionality of sperm by increasing intracellular calcium ions, sperm capacitation, hyperpolarisation and protein expression. Conclusion: Adding vitamin D in in-vitro fertilisation (IVF) in media culture is beneficial as capacitation, sperm membrane polarisation and SOAF protein expression were increased and therefore contributed to successful IVF.

Characterization and Preclinical Evaluation of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Chronic Lymphocytic Leukemia

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors (cBTKi) have transformed treatment landscape of chronic lymphocytic leukemia (CLL). These inhibitors bind to C481 residue in the kinase domain of BTK. This is also the site for the most common mutations rendering cells resistant to cBTKi. To circumvent this limitation of the cBTKi, non-covalent BTKi (ncBTKi) such as pirtobrutinib have been developed. Recently, non-C481 BTK mutations have been reported in patients with CLL at the time of disease progression during pirtobrutinib treatment. These observations underscore the need for ncBTKi that can target C481 as well as non-C481 mutations of BTK. AS-1763 is a potent, highly selective, orally available, and ncBTKi, equipotent against both wild-type and C481S-mutated BTK when tested in biochemical assays. In vivo, AS-1763 demonstrated significant antitumor effects in OCI-LY10 tumor xenograft models harboring wild-type or C481S mutant BTK (Kawahata et al. J Med Chem 64:14129, 2021). Study Design and Methods: In the present project, first we used cell free assay systems to evaluate selectivity and potency of AS-1763 by a kinome-wide profiling and inhibitory effect of AS-1763 in enzyme assays using recombinant mutant BTK. Second, we examined dose- and time-dependent inhibition of mutant BTK that is expressed in HEK293 cell line. Third, we tested biological, biochemical, and molecular impact of AS-1763 in primary CLL cells. Fourth, we determined sensitivity of CLL cells to AS-1763 when combined with other targeted agents. Results: AS-1763 showed a highly selective profile for BTK in a panel of 291 kinase assays with &amp;gt;260-fold selectivity except 3 Tec family kinases (BMX, ITK, and TEC). We have generated a total of 17 recombinant BTK mutant proteins (C481, T474, L528 variants and other BTK mutants; Table 1) reported in the literature or predicted by single nucleotide change in the codon, and established assay methods to measure inhibitory potency of BTKi for those BTK mutants (Table 1). AS-1763 showed potent inhibitory activities for those BTK mutants while inhibitory potencies of other cBTKi and ncBTKi were diminished against some BTK mutants such as T474 and/or L528 mutations. AS-1763 exhibited dose-dependent and slow-off rate inhibitions of BTK autophosphorylation (pY223) in HEK293 cells transfected with various BTK mutants. Furthermore, the observed inhibitory effects of AS-1763 on the BTK autophosphorylation (pY223) in HEK293 cells were continued up to 24 h after washing out of AS-1763. In primary CLL samples, AS-1763, pirtobrutinib or ibrutinib induced a modest apoptosis. AS-1763 effectively inhibited the BCR signaling pathway in a dose dependent manner as evidenced by downregulation of pY223-BTK expression which was also observed in cBTKi and ncBTKi relapsed/refractory CLL samples. In vitro incubations with AS-1763 inhibited CLL cell spontaneous migration, decreased CCL3/CCL4 levels in culture supernatant and was accompanied with the inhibition of intracellular calcium release and B-cell activation, as measured by surface CD86 expression. Besides, AS-1763 incubation for 24 hours was shown to modulate the expression of BCL-2 family proteins with the downregulation of MCL-1 and BCL-xl. Interestingly, in vitro treatment of CLL patient samples with AS-1763 demonstrated a notable elevation in cellular ROS and mitochondrial superoxide levels starting at 1 µM, concurrently impacting SOD1 expression in CLL patient samples. Evaluation of drug interaction models utilizing Compusyn and Synergy Finder applications predominantly indicated additive effects between AS-1763 with BCL-2 inhibitor, venetoclax as well as p53 activator APR-246. Consistent with this data, AS-1763 and venetoclax combinations showed high-rates of apoptosis in samples that were relapsed/refractory to cBTKi and ncBTKi. Conclusions: AS-1763 is a selective ncBTKi that inhibits both wild-type and mutant BTKs listed in Table 1. In CLL cells, AS-1763 was effective in inhibiting BCR pathway signaling and sensitized cells to other agents such as venetoclax. Based on these encouraging data we have initiated a clinical trial to test AS-1763 in patients with CLL and other B cell malignancies who have failed or are intolerant to at least two prior lines of systemic therapy, including cBTKi (NCT05602363 Clinical Trials.gov).

Biodegradable nanocarrier of gemcitabine and tocopherol succinate synergistically ameliorates anti-proliferative response in MIA PaCa-2 cells

Gemcitabine (GEM) is an important chemotherapeutic agent used alone or in combination with other anticancer agents for the treatment of various solid tumors. In this study, the potential of a dietary supplement, α-tocopherol succinate (TOS) was investigated in combination with GEM by utilizing human serum albumin-based nanoparticles (HSA NPs). The developed nanoparticles were characterized using DLS, SEM and FTIR and evaluated in a panel of cell lines to inspect cytotoxic efficacy. The ratio metric selected combination of the NPs was further investigated in human pancreatic cancer cell line (MIA PaCa-2 cells) to assess the cellular death mechanism via a myriad of biochemical and bio-analytical assays including nuclear morphometric analysis by DAPI staining, ROS generation, MMP loss, intracellular calcium release, in vitro clonogenic assay, cell migration assay, cell cycle analysis, immunocytochemical staining followed by western blotting, Annexin V-FITC and cellular uptake studies. The desolvation-crosslinking method was used to prepare the NPs. The average size of TOS-HSA NPs and GEM-HSA NPs was found to be 189.47 ± 5 nm and 143.42 ± 7.4 nm, respectively. In combination, the developed nanoparticles exhibited synergism by enhancing cytotoxicity in a fixed molar ratio. The selected combination also significantly triggered ROS generation and mitochondrial destabilization, alleviated cell migration potential and clonogenic cell survival in MIA PaCa-2 cells. Further, cell cycle analysis, Annexin-V FITC assay and caspase-3 activation, up regulation of Bax and down regulation of Bcl-2 protein confirmed the occurrence of apoptotic event coupled with the G0/G1 phase arrest. Nanocarriers based this combination also offered approximately 14-folds dose reduction of GEM. Overall, the combined administration of TOS-HSA NPs and GEM-HSA NPs showed synergistic cytotoxicity accompanied with dose reduction of the gemcitabine. These encouraging findings could have implication in designing micronutrient based-combination therapy with gemcitabine and demands further investigation.

In vitro cannabinoid activity profiling of generic ban-evading brominated synthetic cannabinoid receptor agonists and their analogs.

Following the enactment of a generic ban in China in 2021, the synthetic cannabinoid market has been evolving, now encompassing even wider structural diversity. Compounds carrying a brominated core such as ADB-5'Br-BUTINACA (ADMB-B-5Br-INACA) and tail-less analogs, such as ADB-5'Br-INACA (ADMB-5Br-INACA), MDMB-5'Br-INACA, and ADB-INACA (ADMB-INACA), have been detected since late 2021. This study investigated the cannabinoid receptor (CB) activation potential of synthesized (S)-enantiomers of these substances, as well as of two predicted analogs MDMB-5'Br-BUTINACA (MDMB-B-5Br-INACA) and ADB-5'F-BUTINACA (ADMB-B-5F-INACA), using CB1 and CB2 β-arrestin 2 recruitment assays and a CB1 intracellular calcium release assay. Surprisingly, the tail-less (S)-ADB-5'Br-INACA and (S)-MDMB-5'Br-INACA retained CB activity, albeit with a decreased potency compared to their tailed counterparts (S)-ADB-5'Br-BUTINACA and (S)-MDMB-5'Br-BUTINACA, respectively, which were potent and efficacious CB1 agonists. Also, at CB2, tail-less analogs showed a lower potency but increased efficacy. Removing the bromine substitution ((S)-ADB-INACA) resulted in a reduced activity at CB1; however, this effect was less prominent at CB2. Looking at tailed analogs, replacing the bromine with a fluorine substitution ((S)-ADB-5'F-BUTINACA) resulted in an increased potency and efficacy at both receptors. Furthermore, as ADB-5'Br-INACA and MDMB-5'Br-INACA have been frequently detected together in Scottish prisons, this study also evaluated the CB1 receptor activation potential of different mixtures of their respective reference standards, showing no unexpected cannabimimetic effect of combining both substances. Lastly, two powders seized by Belgian Customs and confirmed to contain ADB-5'Br-INACA and MDMB-5'Br-INACA, respectively, were assessed for CB activity. Based on the comparison with their reference standards, varying degrees of purity were suspected.

Beyond reproduction: Exploring the Non-Canonical roles of the Kisspeptin System in Diverse Biological Systems

G protein-coupled receptors are integral membrane proteins in cell signaling processes. Activation of G protein-coupled receptors by specific agonists promotes the activation of different G-proteins, activating different intracellular signaling pathways, including adenylate cyclase activation and intracellular calcium release. One of the G protein-coupled receptors studied is the kisspeptin receptor, which regulates reproduction and gonadotropin secretion. However, recent studies have shown that kisspeptin and its receptor have non-canonical roles in cell signaling and several biological systems. In the present review, we will present these different functions exerted by the kisspeptin system in different biological systems, such as the central nervous system, the cardiovascular system, and the immune system, as well as the role of this system in pathologies such as preeclampsia, diabetes, and cancer. Understanding their non-canonical roles in cell signaling may have important implications in developing new therapies for various diseases. Keywords: Kisspeptin-1 Receptor, Kisspeptins, G-protein coupled receptor, Signal Transduction, Cancer, Diabetes Mellitus, Preeclampsia.

Splenectomy improves erythrocyte functionality in spherocytosis based on septin abundance, but not maturation defects.

Splenectomy improves the clinical parameters of patients with hereditary spherocytosis, but its potential benefit to red blood cell (RBC) functionality and the mechanism behind this benefit remain largely overlooked. Here, we compared 7 nonsplenectomized and 13 splenectomized patients with mutations in the β-spectrin or the ankyrin gene. We showed that hematological parameters, spherocyte abundance, osmotic fragility, intracellular calcium, and extracellular vesicle release were largely but not completely restored by splenectomy, whereas cryohemolysis was not. Affected RBCs exhibited decreases in β-spectrin and/or ankyrin contents and slight alterations in spectrin membrane distribution, depending on the mutation. These modifications were found in both splenectomized and nonsplenectomized patients and poorly correlated with RBC functionality alteration, suggesting additional impairments. Accordingly, we found an increased abundance of septins, small guanosine triphosphate-binding cytoskeletal proteins. Septins-2, -7, and -8 but not -11 were less abundant upon splenectomy and correlated with the disease severity. Septin-2 membrane association was confirmed by immunolabeling. Except for cryohemolysis, all parameters of RBC morphology and functionality correlated with septin abundance. The increased septin content might result from RBC maturation defects, as evidenced by (1) the decreased protein 4.2 and Rh-associated glycoprotein content in all patient RBCs, (2) increased endoplasmic reticulum remnants and endocytosis proteins in nonsplenectomized patients, and (3) increased lysosomal and mitochondrial remnants in splenectomized patients. Our study paves the way for a better understanding of the involvement of septins in RBC membrane biophysical properties. In addition, the lack of restoration of septin-independent cryohemolysis by splenectomy may call into question its recommendation in specific cases.

Electrophilic Agonists Modulate the Transient Receptor Potential Ankyrin-1 Channels Mediated by Insulin and Glucagon-like Peptide-1 Secretion for Glucose Homeostasis.

This pre-clinical study investigated the transient receptor potential ankyrin-1 (TRPA1) channels on modulating targets for glucose homeostasis using agonists: the electrophilic agonists, cinnamaldehyde (CIN) and allyl isothiocyanate (AITC), and the non-electrophilic agonist, carvacrol (CRV). A glucose tolerance test was performed on rats. CIN and AITC (5, 10 and 20 mg/kg) or CRV (25, 100, 300, and 600 mg/kg) were administered intraperitoneally (i.p.), and glycemia was measured. In the intestine, Glucagon-like peptide-1 (GLP-1) and disaccharidase activity were evaluated (in vivo and in vitro, respectively). Furthermore, in vivo and in vitro insulin secretion was determined. Islets were used to measure insulin secretion and calcium influx. CIN and AITC improved glucose tolerance and increased insulin secretion in vivo and in vitro. CRV was unable to reduce glycemia. Electrophilic agonists, CIN and AITC, inhibited disaccharidases and acted as secretagogues in the intestine by inducing GLP-1 release in vivo and in vitro and contributed to insulin secretion and glycemia. The effect of CIN on calcium influx in pancreatic islets (insulin secretion) involves voltage-dependent calcium channels and calcium from stores. TRPA1 triggers calcium influx and potentiates intracellular calcium release to induce insulin secretion, suggesting that electrophilic agonists mediate this signaling transduction for the control of glycemia.

Assessing mitochondria-targeted acyl hydroquinones on the mitochondrial platelet function and cytotoxic activity: Role of the linker length

IntroductionThe use of triphenylphosphonium cation (TPP+) linked to phenolic compounds by alkyl chains has a significant relevance as a mitochondrial delivery strategy in biomedicine because it affects mitochondrial bioenergetics in models of noncommunicable diseases such as cancer and cardiovascular-related conditions. Studies indicate that a long alkyl chain (10–12 carbon) increases the mitochondrial accumulation of TPP+-linked drugs. In contrast, other studies show that these compounds are consistently toxic to micromolar concentrations (as observed in platelets). In the present study, we evaluated the in vitro effect of three series of triphenylphosphonium-linked acyl hydroquinones derivates on the metabolism and function of human platelets using 3–9 carbons for the alkyl linker. Those were assessed to determine the role of the length of the alkyl chain linker on platelet toxicity. MethodsHuman platelets were exposed in vitro to different concentrations (2–40 μM) of every compound; cellular viability, phosphatidylserine exposition, mitochondrial membrane potential (ΔΨm), intracellular calcium release, and intracellular ROS generation were assessed by flow cytometry. An in silico energetic profile was generated with Umbrella sampling molecular dynamics (MD). Results and conclusionsThere was an increase in cytotoxic activity directly related to the length of the acyl chain and lipophilicity, as seen by three techniques, and this was consistent with a decrease in ΔΨm. The in silico energetic profiles point out that the permeability of the mitochondrial membrane may be involved in the cytotoxicity of phosphonium salts. This information may be relevant for the design of new TPP+ -based drugs with a safe cardiovascular profile.

Pepper fruit (Dennettia tripetala [annonaceae]) seed inhibits uterine contractions via blockade of intracellular calcium release and extracellular calcium influx in ex vivo studies

Dennettia tripetala (DT) is a tree found in rain forest belt of Nigeria, Cameroon and Ivory Coast. The seed is commonly used in preparing food for mothers after delivery because it is believed to play a role in postpartum uterine contraction. There is however no scientific evidence validating this claim. Hence, the study aimed to investigate the effects of DT seed on non-pregnant and pregnant uterus using mouse model. The dried seeds of DT were macerated in methanol and the effects of DT extract were investigated on uterus tissues isolated from non-pregnant and pregnant mice. DT extract was examined on spontaneous uterine contraction, contractions induced by oxytocin, and high potassium chloride (KCl)-induced depolarization. Its effect was also evaluated on calcium ion (Ca2+) release from internal stores. The DT extract suppressed spontaneous uterine contractility in both pregnant and non-pregnant uterus in a concentration-dependent manner. It significantly inhibited uterine contractions elicited by high KCl and oxytocin-induced contractions in both pregnant and non-pregnant uterus. The extract also inhibited uterine contractility induced by oxytocin in the Ca2+-free medium in pregnant and non-pregnant uterus. The study has provided scientific evidence that DT seed extract has uterine relaxing effect in both pregnant and non-pregnant mouse uterus possibly by blocking intracellular Ca2+ released and influx of extracellular Ca2+. The result has shown that DT has no benefit in reducing the risk of post-partum hemorrhage, however, it may have potential benefits in management of dysmenorrhea and preterm labour in non-gravid and gravid uteri respectively.

P-074 In-vitro vitamin D treatment on human sperm improves sperm motility, capacitation, and oocyte activation

Abstract Study question Does in-vitro vitamin D treatment on fertile and infertile human sperm improves sperm motility and alter protein markers involved in capacitation and oocyte-activation. Summary answer In-vitro vitamin D treatment enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm motility and capacitation status. What is known already Many studies have been done to investigate the relationship between vitamin D and male infertility. It is known that low serum vitamin D in human contribute to male infertility. However, the effect of in-vitro vitamin D treatment on sperm remains unclear, particularly on the effect of vitamin D following ejaculation into female reproductive tract. This includes processes involved in fertilisation such as capacitation, hyperpolarization, oocyte activation and sperm-oocyte fusion. Therefore, we examined whether in-vitro vitamin D treatment on sperm can improve the sperm parameters, hence will then improve fertilisation and pregnancy. Study design, size, duration Randomised controlled trial with prospective interventions (in-vitro vitamin D treatment). Based on power analysis, 40 subjects both fertile and infertile men who came for semen analysis were recruited at University Malaya Medical Centre from January 2022 to January 2023. All men were consented to participate in this study. Each washed semen sample were aliquoted to serve as control and receive in-vitro vitamin D treatment. Participants/materials, setting, methods Total of 40 samples were included for final analysis. All semen samples were washed and treated with 1nM vitamin D in-vitro for 45 minutes at 37ºC. Motility, intracellular calcium released, capacitation status, mitochondria membrane potential and other basic semen parameters were measured after in-vitro vitamin D treatment for fertile and infertile samples. Furthermore, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ), was done to investigate fluorescence intensity per cell using Image J. Main results and the role of chance In both fertile and infertile sample group, total motility is increased after in-vitro vitamin D treatment (MD = 8.727%, P &amp;lt; 0.001). Fluo-4 AM stain shows there are increase of intracellular calcium release after in-vitro vitamin D, with mean difference of Corrected Total Cell Fluorescent (CTCF) for Fluo-4 AM (MD = 25043, P = 0.0228. Chlortetracyclin (CTC) test for capacitation status and immunofluorescence of phosphotyrosine shows there are increase CTCF (MD = 0.02595, P = 0.0142) after in-vitro vitamin D treatment. Staining of JC-1 shows there are more hyperpolarised mitochondria membrane in in-vitro vitamin D treatment group compare to the control group. Ratio of red to green fluorescent is 0.639 in control group and 2.22 in in-vitro vitamin D treatment group. Moreover, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ) show in-vitro vitamin D improve the expression of these protein. Mean difference of CTCF between control and vitamin D treatment group for PLCζ is 8.013x 10-3, P = 0.0226. Thus, in-vitro vitamin D enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm capacitation status and sperm membrane hyperpolarization. Limitations, reasons for caution Although power analysis is used to calculate sample size, however bigger sample size always needed to increase the accuracy. Moreover, patients’ sample are examined and treated after 3 hours due to limited collection room in the facility. Wider implications of the findings Vitamin D can be used as a tool in in-vitro fertilisation (IVF) procedure and can be added to sperm preparation medium due to its implication in increasing capacitation and hyperpolarised sperm membrane to increase chance of successful IVF. Trial registration number not applicable