Coronavirus Porcine Epidemic Diarrhea Virus Utilizes Chemokine Interleukin-8 to Facilitate Viral Replication by Regulating Ca2+ Flux.

Chemokine production by epithelial cells is crucial for neutrophil recruitment to sites of inflammation during viral infection. However, the effect of chemokine on epithelia and how chemokine is involved in coronavirus infection remains to be fully understood. Here, we identified an inducible chemokine interleukin-8 (CXCL8/IL-8), which could promote coronavirus porcine epidemic diarrhea virus (PEDV) infection in African green monkey kidney epithelial cells (Vero) and Lilly Laboratories cell-porcine kidney 1 epithelial cells (LLC-PK1). IL-8 deletion restrained cytosolic calcium (Ca2+), whereas IL-8 stimulation improved cytosolic Ca2+. The consumption of Ca2+ restricted PEDV infection. PEDV internalization and budding were obvious reductions when cytosolic Ca2+ was abolished in the presence of Ca2+ chelators. Further study revealed that the upregulated cytosolic Ca2+ redistributes intracellular Ca2+. Finally, we identified that G protein-coupled receptor (GPCR)-phospholipase C (PLC)-inositol trisphosphate receptor (IP3R)-store-operated Ca2+ (SOC) signaling was crucial for enhancive cytosolic Ca2+ and PEDV infection. To our knowledge, this study is the first to uncover the function of chemokine IL-8 during coronavirus PEDV infection in epithelia. PEDV induces IL-8 expression to elevate cytosolic Ca2+, promoting its infection. Our findings reveal a novel role of IL-8 in PEDV infection and suggest that targeting IL-8 could be a new approach to controlling PEDV infection. IMPORTANCE Coronavirus porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that caused severe economic losses worldwide, and more effort is needed to develop economical and efficient vaccines to control or eliminate this disease. The chemokine interleukin-8 (CXCL8/IL-8) is indispensable for the activation and trafficking of inflammatory mediators and tumor progression and metastasis. This study evaluated the effect of IL-8 on PEDV infection in epithelia. We found that IL-8 expression improved cytosolic Ca2+ in epithelia, facilitating PEDV rapid internalization and egress. G protein-coupled receptor (GPCR)-phospholipase C (PLC)-inositol trisphosphate receptor (IP3R)-SOC signaling was activated by IL-8, releasing the intracellular Ca2+ stores from endoplasmic reticulum (ER). These findings provide a better understanding of the role of IL-8 in PEDV-induced immune responses, which will help develop small-molecule drugs for coronavirus cure.

Microwaves can kill malaria parasites non-thermally.

Malaria, which infected more than 240 million people and killed around six hundred thousand only in 2021, has reclaimed territory after the SARS-CoV-2 pandemic. Together with parasite resistance and a not-yet-optimal vaccine, the need for new approaches has become critical. While earlier, limited, studies have suggested that malaria parasites are affected by electromagnetic energy, the outcomes of this affectation vary and there has not been a study that looks into the mechanism of action behind these responses. In this study, through development and implementation of custom applicators for in vitro experimentation, conditions were generated in which microwave energy (MW) killed more than 90% of the parasites, not by a thermal effect but via a MW energy-induced programmed cell death that does not seem to affect mammalian cell lines. Transmission electron microscopy points to the involvement of the haemozoin-containing food vacuole, which becomes destroyed; while several other experimental approaches demonstrate the involvement of calcium signaling pathways in the resulting effects of exposure to MW. Furthermore, parasites were protected from the effects of MW by calcium channel blockers calmodulin and phosphoinositol. The findings presented here offer a molecular insight into the elusive interactions of oscillating electromagnetic fields with P. falciparum, prove that they are not related to temperature, and present an alternative technology to combat this devastating disease.

Disinfectant dodecyl dimethyl benzyl ammonium chloride (DDBAC) disrupts gut microbiota, phospholipids, and calcium signaling in honeybees (Apis mellifera) at an environmentally relevant level.

One of the impacts of the Coronavirus disease 2019 (COVID-19) pandemic has been a profound increase in the application amounts of disinfectants. Dodecyl dimethyl benzyl ammonium chloride (DDBAC) is a widely used disinfectant, yet its hazards to non-target species remain largely unknown. We are unaware of any studies assessing DDBAC's impacts on honeybee, a pollinator species that is a useful indicator of environmental pollution essential for many forms of agricultural production. Here, we assessed the potentially negative effects of DDBAC on honeybees. After conducting a formal toxicity evaluation of DDBAC on honeybee mortality, we detected an accumulation of DDBAC in the honeybee midgut. We subsequently studied the midgut tissues of honeybees exposed to sub-lethal concentrations of DDBAC: histopathological examination revealed damage to midgut tissue upon DDBAC exposure, microbiome analysis showed a decreased abundance of beneficial midgut microbiota, lipidomics analysis revealed a significant reduction in cell membrane phospholipids with known functions in signal transduction, and a transcriptome analysis detected altered expression of genes involved in calcium signaling pathways (that variously function in calcium absorption, muscle contraction, and neurotransmission). Thus, our study establishes that DDBAC impacts honeybee midgut functions at multiple levels. Our study represents an early warning about the hazards of DDBAC and appeals for the proper stewardship of DDBAC to ensure the protection of our ecological environment.

OA Biology

Purpose: The field of osteoarthritis (OA) biology is rapidly evolving and brilliant progress has been made this year as well. Methods: Landmark studies of OA biology published in 2021 and early 2022 were selected through PubMed searches and classified by their molecular mechanisms, and it was largely divided into the intra-cellular mechanisms and the inter-compartment or inter-cellular interaction in OA progression. Results: The intra-cellular mechanisms involving OA progression included 1) Piezo1/TRPV4-mediated calcium signaling, 2) low grade inflammation by TLR-CD14-LBP complex and IKKβ-NFkB signaling, 3) PGRN/TNFR2/14-3-3ε/Elk-1 anabolic cascade, 4) G protein-coupled receptor (GPCR) signaling, 5) mechanical loading-cilia/Ift88-hedgehog signaling, 6) mitochondrial fission by ERK1/2-DRP1 pathway, and 7) hypoxia-DOT1L-H3K79 methylation pathway. The studies on inter-compartment or inter-cellular interaction in OA progression included the following subjects: 1) the anabolic role of Lubricin, a proteoglycan from superficial zone cells, 2) osteoclast-chondrocyte interaction via exosomal miRNA and sphingosine 1-phosphate (S1P), 3) αV integrin-mediated TGFβ activation by mechanical loading, 4) TGFβ-mediated suppression of sclerostin in osteocytes, 5) catabolic role of Flightless I as a DAMPs-triggering molecule, and 6) catabolic role of paracrine signaling from fat. Conclusions: Despite the disastrous Covid-19 pandemic situation, many outstanding studies have expanded the boundary of OA biology. They give us not only critical insight on pathophysiology, but also clue for the treatment of OA.

Potential of targeting host cell calcium dynamics to curtail SARS-CoV-2 infection and COVID-19 pathogenesis.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection and associated coronavirus disease 2019 (COVID-19) has severely impacted human well-being. Although vaccination programs have helped in reducing the severity of the disease, drug regimens for clinical management of COVID-19 are not well recognized yet. It is therefore important to identify and characterize the molecular pathways that could be therapeutically targeted to halt SARS-CoV-2 infection and COVID-19 pathogenesis. SARS-CoV-2 hijacks host cell molecular machinery for its entry, replication and egress. Interestingly, SARS-CoV-2 interacts with host cell Calcium (Ca2+) handling proteins and perturbs Ca2+ homeostasis. We here systematically review the literature that demonstrates a critical role of host cell Ca2+ dynamics in regulating SARS-CoV-2 infection and COVID-19 pathogenesis. Further, we discuss recent studies, which have reported that SARS-CoV-2 acts on several organelle-specific Ca2+ transport mechanisms. Moreover, we deliberate upon the possibility of curtailing SARS-CoV-2 infection by targeting host cell Ca2+ handling machinery. Importantly, we delve into the clinical trials that are examining the efficacy of FDA-approved small molecules acting on Ca2+ handling machinery for the management of COVID-19. Although an important role of host cell Ca2+ signaling in driving SARS-CoV-2 infection has emerged, the underlying molecular mechanisms remain poorly understood. In future, it would be important to investigate in detail the signaling cascades that connect perturbed Ca2+ dynamics to SARS-CoV-2 infection.

Roles of ATP and SERCA in the Regulation of Calcium Turnover in Unloaded Skeletal Muscles: Current View and Future Directions.

A decrease in skeletal muscle contractile activity or its complete cessation (muscle unloading or disuse) leads to muscle fibers' atrophy and to alterations in muscle performance. These changes negatively affect the quality of life of people who, for one reason or another, are forced to face a limitation of physical activity. One of the key regulatory events leading to the muscle disuse-induced changes is an impairment of calcium homeostasis, which leads to the excessive accumulation of calcium ions in the sarcoplasm. This review aimed to analyze the triggering mechanisms of calcium homeostasis impairment (including those associated with the accumulation of high-energy phosphates) under various types of muscle unloading. Here we proposed a hypothesis about the regulatory mechanisms of SERCA and IP3 receptors activity during muscle unloading, and about the contribution of these mechanisms to the excessive calcium ion myoplasmic accumulation and gene transcription regulation via excitation-transcription coupling.

21st ESPD Annual Meeting

The proceedings contain 169 papers. The topics discussed include: GNAQ/11 mosaicism causes aberrant calcium signaling and drives systemic hypocalcemia;pediatric obesity and skin disease (PicoSkin-study): cutaneous findings and associated quality of life in 86 children and adolescents with obesity;what gives them the shivers? two new cases of infantile transient smooth muscle contraction of the skin;dermatologic manifestations of multisystem inflammatory syndrome in children during the COVID-19 pandemic;clinical characteristics and management of cutaneous lymphangioma circumscriptum;different shades of grey! infantile black hairy tongue- a case series and review of the literature;descriptive series of cases of pediatric linear morphea in a tertiary hospital in Barcelona;the prevalence of itch in German schoolchildren: a population-based study;neurocognitive functioning, physical health, and mental health of school-aged children treated with propranolol or atenolol for infantile hemangioma;and efficacy and safety of tralokinumab in adolescents with moderate-to-severe atopic dermatitis: results of the phase 3 ECZTRA 6 trial.

SARS-CoV-2 SPIKE PROTEIN DESTABILIZES MICROVASCULAR HOMEOSTASIS

Background: SARS-CoV-2 infection can compromise respiratory function and cause thrombotic events. SARS-CoV-2 binds to and mediates downregulation of angiotensin converting enzyme 2 (ACE2) on infected cells. Diminished enzymatic activity of ACE2 could result in increased concentrations of the pro-inflammatory molecules angiotensin II and bradykinin, contributing to SARS-CoV-2 pathology. Methods: Immunofluorescence microscopy and digital image data quantification, Computer assisted molecular docking analyses, Western blot. Results: Using immunofluorescence microscopy of lung tissues from uninfected and SARS-CoV-2 infected individuals, we find evidence that ACE2 is highly expressed in the pulmonary alveolar epithelium and is significantly reduced along the alveolar lining of SARS-CoV-2 infected lungs. Ex vivo analyses indicate that ACE2 is readily detected on primary human pulmonary alveolar epithelial and primary human aortic endothelial cells (HAoECs). Exposure of these cells to recombinant SARS-CoV-2 spike protein was sufficient to reduce surface ACE2 expression. Moreover, exposure of HAoECs to spike protein induced endothelial dysfunction (increased expression of von Willebrand Factor and decreased expression of Krüppel-like Factor 2), caspase activation, and apoptosis. Exposure of HAoECs to bradykinin (BK, 10μ M) induced calcium signaling and endothelial dysfunction but did not adversely affect viability. Computer assisted analyses of molecules with potential to bind bradykinin receptor B2 (BKRB2) suggested a potential role for aspirin as a bradykinin antagonist. When tested in our in vitro model, we found that aspirin (1μM) could significantly blunt cell signaling, and endothelial dysfunction caused by bradykinin in these cells. Conclusion: SARS-CoV-2 causes complex effects on microvascular homeostasis that potentially contribute to organ dysfunction and coagulopathies. Reduced ACE2 enzymatic activity could contribute to inflammation and pathology in the lung. Our studies add to this understanding by providing evidence that spike protein alone can mediate adverse effects on vascular cells. Understanding these mechanisms of pathogenesis may provide rationale for interventions, such as interference with the interactions of spike protein or bradykinin with endothelial cells, that could limit microvascular events associated with SARS-CoV-2 infection and stabilize microvascular homeostasis in COVID-19 disease.

Dynamic Ca2+ sensitivity stimulates the evolved SARS-CoV-2 spike strain-mediated membrane fusion for enhanced entry.

Mutations in the spike protein generated a highly infectious and transmissible D614G variant, which is present in newly evolved fast-spreading variants. The D614G, Alpha, Beta, and Delta spike variants of SARS-CoV-2 appear to expedite membrane fusion process for entry, but the mechanism of spike-mediated fusion is unknown. Here, we reconstituted an in vitro pseudovirus-liposome fusion reaction and report that SARS-CoV-2 wild-type spike is a dynamic Ca2+ sensor, and D614G mutation enhances dynamic calcium sensitivity of spike protein for facilitating membrane fusion. This dynamic calcium sensitivity for fusion is found to be higher in Alpha and Beta variants and highest in Delta spike variant. We find that efficient fusion is dependent on Ca2+ concentration at low pH, and the fusion activity of spike dropped as the Ca2+ level rose beyond physiological levels. Thus, evolved spike variants may control the high fusion probability for entry by increasing Ca2+ sensing ability.

Calcium Signaling Pathway Is Involved in the Shedding of ACE2 Catalytic Ectodomain: New Insights for Clinical and Therapeutic Applications of ACE2 for COVID-19.

The angiotensin-converting enzyme 2 (ACE2) is a type I integral membrane that exists in two forms: the first is a transmembrane protein; the second is a soluble catalytic ectodomain of ACE2. The catalytic ectodomain of ACE2 undergoes shedding by a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), in which calmodulin mediates the calcium signaling pathway that is involved in ACE2 release, resulting in a soluble catalytic ectodomain of ACE2 that can be measured as soluble ACE2 plasma activity. The shedding of the ACE2 catalytic ectodomain plays a role in cardiac remodeling and endothelial dysfunction and is a predictor of all-cause mortality, including cardiovascular mortality. Moreover, considerable evidence supports that the ACE2 catalytic ectodomain is an essential entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Additionally, endotoxins and the pro-inflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor-alpha (TNFα) all enhanced soluble catalytic ectodomain ACE2 shedding from the airway epithelia, suggesting that the shedding of ACE2 may represent a mechanism by which viral entry and infection may be controlled such as some types of betacoronavirus. In this regard, ACE2 plays an important role in inflammation and thrombotic response, and its down-regulation may aggravate COVID-19 via the renin-angiotensin system, including by promoting pathological changes in lung injury. Soluble forms of ACE2 have recently been shown to inhibit SARS-CoV-2 infection. Furthermore, given that vitamin D enhanced the shedding of ACE2, some studies reported that vitamin D treatment is associated with prognosis improvement in COVID-19. This is an updated review on the evidence, clinical, and therapeutic applications of ACE2 for COVID-19.

The effect of canakinumab on COVID19-associated cardiomyocyte contractile dysfunction and arrhythmias

Background: Cardiac injury associated with cytokine release occurs in almost 20% of SARS-CoV-2 positive patients during hospitalization and mortality is particularly high in these patients. Cardiac enzyme (e.g. troponin or creatinine kinase (CK)) elevations are a frequently reported finding, indicating myocardial damage and arrhythmias are the cause for ICU transfer in up to 12% of patients. However, the mechanistic role of COVID19 associated cytokine-storm for the concomitant cardiac dysfunction and associated arrhythmias is unclear. In addition, the role of anti-inflammatory therapy approaches to mitigate this cardiac dysfunction remains elusive. Methods: We investigated the effects of COVID19-associated inflammatory response on cardiac cellular function as well as its cardiac arrhythmogenic potential in rat and induced pluripotent stem cell derived cardiomyocytes (iPSc-CM). Moreover, we evaluated the therapeutic potential of the IL1-beta antagonist Canakinumab using state of the art in-vitro confocal and ratiometric high-throughput microscopy. Results: Isolated rat ventricular cardiomyocytes were exposed to control or COVID19 plasma from intensive care unit patients with severe ARDS and impaired cardiac function (LVEF 41±5%;1/3 of patients on veno-venous extracorporeal membrane oxygenation;CK 154±43 U/l). Cardiomyocytes showed decreased Ca2+ transient amplitudes and altered baseline Ca2+ concentrations leading to impaired cellular contractile function upon electrical field-stimulation and exposure to patient plasma (n=276 control and 359 COVID19 cells;Fura). In addition, we used iPSc-CM to explore the long-term effect of patient plasma on cardiac electrical and mechanical function in a translational setting (24h incubation;Fluo). In iPSc, spontaneous Ca2+ release events (i.e. Ca2+ waves and Ca2+ sparks) were more likely to occur upon incubation with COVID19 plasma and nuclear as well as cytosolic Ca2+ release were altered. Co-incubation with Canakinumab had no effect on pro-arrhythmogenic Ca2+ release or Ca2+ signaling during excitation-contraction coupling but influenced cellular automaticity upon prolonged electrical stimulation. Conclusion: Plasma derived from COVID19 patients exerts acute cardiodepressant and chronic pro-arrhythmogenic effects in rat and iPS-derived cardiomyocytes. Chronic co-incubation with Canakinumab had no beneficial effect on cellular Ca2+ signaling during excitation-contraction coupling.

MicroRNAs and Calcium Signaling in Heart Disease.

In hearts, calcium (Ca2+) signaling is a crucial regulatory mechanism of muscle contraction and electrical signals that determine heart rhythm and control cell growth. Ca2+ signals must be tightly controlled for a healthy heart, and the impairment of Ca2+ handling proteins is a key hallmark of heart disease. The discovery of microRNA (miRNAs) as a new class of gene regulators has greatly expanded our understanding of the controlling module of cardiac Ca2+ cycling. Furthermore, many studies have explored the involvement of miRNAs in heart diseases. In this review, we aim to summarize cardiac Ca2+ signaling and Ca2+-related miRNAs in pathological conditions, including cardiac hypertrophy, heart failure, myocardial infarction, and atrial fibrillation. We also discuss the therapeutic potential of Ca2+-related miRNAs as a new target for the treatment of heart diseases.

Direct Targets and Subsequent Pathways for Molecular Hydrogen to Exert Multiple Functions: Focusing on Interventions in Radical Reactions.

Molecular hydrogen (H2) was long regarded as non-functional in mammalian cells. We overturned the concept by demonstrating that H2 exhibits antioxidant effects and protects cells against oxidative stress. Subsequently, it has been revealed that H2 has multiple functions in addition to antioxidant effects, including antiinflammatory, anti-allergic functions, and as cell death and autophagy regulation. Additionally, H2 stimulates energy metabolism. As H2 does not readily react with most biomolecules without a catalyst, it is essential to identify the primary targets with which H2 reacts or interacts directly. As a first event, H2 may react directly with strong oxidants, such as hydroxyl radicals (•OH) in vivo. This review addresses the key issues related to this in vivo reaction. •OH may have a physiological role because it triggers a free radical chain reaction and may be involved in the regulation of Ca2+- or mitochondrial ATP-dependent K+-channeling. In the subsequent pathway, H2 suppressed a free radical chain reaction, leading to decreases in lipid peroxide and its end products. Derived from the peroxides, 4-hydroxy-2-nonenal functions as a mediator that up-regulates multiple functional PGC-1α. As the other direct target in vitro and in vivo, H2 intervenes in the free radical chain reaction to modify oxidized phospholipids, which may act as an antagonist of Ca2+-channels. The resulting suppression of Ca2+-signaling inactivates multiple functional NFAT and CREB transcription factors, which may explain H2 multi-functionality. This review also addresses the involvement of NFAT in the beneficial role of H2 in COVID-19, Alzheimer's disease and advanced cancer. We discuss some unsolved issues of H2 action on lipopolysaccharide signaling, MAPK and NF-κB pathways and the Nrf2 paradox. Finally, as a novel idea for the direct targeting of H2, this review introduces the possibility that H2 causes structural changes in proteins via hydrate water changes.