Calcium-mediated Signaling Pathways Research Articles

ORAI2 is Important for the Development of Early-Stage Postirradiation Fibrosis in Salivary Glands

PurposeAlthough post-irradiation hyposalivation significantly impairs patient quality of life, the underlying mechanisms driving radiation-induced salivary gland fibrosis and hyposalivation remain poorly understood. This study aims to explore the role of calcium-mediated signaling pathways in radiation-induced salivary gland fibrosis. Materials and MethodsPrimary human submandibular gland (SG) cells and C57BL/6J female mouse SGs were exposed to irradiation to model fibrosis development. Following 15 Gy irradiation exposure, RNA sequencing and bioinformatic analysis were conducted on mouse SGs. The effects of Store-Operated Calcium Entry (SOCE) inhibition using SKF96365 and YM58483 on fibrosis markers were assessed in vitro and in vivo. Additionally, the involvement of ORAI2 protein and the newly identified JNK/NFAT1/TGF-β1 signaling axis in SG fibrosis was explored. ResultsWe identified that the calcium release-activated calcium modulator ORAI2 was important in promoting early-stage post-irradiation fibrosis in SGs. Calcium channel signaling was activated in both human patients and irradiated C57BL/6J female mice SGs. Inhibition of SOCE signaling effectively blocked fibrosis in an ORAI2-dependent manner 30 days after irradiation. Our mechanistic studies revealed a novel ORAI2/JNK/NFAT1 axis within the SOCE pathway critical in driving TGF-β1-mediated fibrogenesis. Encouragingly, pharmacological inhibition of NFAT1 significantly mitigated radiation-induced SG fibrosis and restored saliva flow to 84.61% of normal levels in treated mice 30 days after irradiation, without detectable side effects. ConclusionsOur findings highlight the significance of the ORAI2-mediated calcium signaling pathway, specifically via the ORAI2/JNK/NFAT1 axis, in promoting TGF-β1 expression and contributing to the development of early-stage salivary gland fibrosis following irradiation exposure. Targeting the ORAI2/JNK/NFAT1 axis emerges as a promising therapeutic strategy to alleviate radiation-induced hyposalivation and fibrosis, potentially improving the quality of life for patients undergoing radiotherapy.

Identification of key lncRNAs and mRNAs in muscle development pathways of Tan sheep

The study aimed to identify the long noncoding RNA (lncRNA) responsible for regulating muscle development in Tan sheep. RNA-seq analysis was conducted on longissimus dorsi samples from 1-day-old and 60-day-old Tan sheep to investigate the molecular processes involved in muscle development. A total of 5517 lncRNAs and 2885 mRNAs were found to be differentially expressed in the 60-day-old Tan sheep. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these differentially expressed lncRNAs and mRNAs were linked to pathways crucial for muscle development, such as MAPK, cAMP, and calcium-mediated signaling pathways. Key genes like CDKN1A, MAPK14, TGFB1, MEF2C, MYOD1, and CD53 were identified as significant players in muscle development. The study validated the RNA-seq results through RT-qPCR, confirming the consistency of expression levels of differentially expressed lncRNAs and mRNAs. These findings indicate that lncRNA-mRNA networks produce a remarked effect on modulating muscle development in Tan sheep, such as lncRNAs (MSTRG.12808.1/MSTRG.22662.3/MSTRG.18310.1) and mRNAs (MSTRG.10027/MSTRG.10029/MSTRG.10258/MSTRG.11011/MSTRG.10354), laying the groundwork for future research in this area.

TRPC absence induces pro-inflammatory macrophage polarization to promote obesity and exacerbate colorectal cancer.

During the past half-century, although numerous interventions for obesity have arisen, the condition's prevalence has relentlessly escalated annually. Obesity represents a substantial public health challenge, especially due to its robust correlation with co-morbidities, such as colorectal cancer (CRC), which often thrives in an inflammatory tumor milieu. Of note, individuals with obesity commonly present with calcium and vitamin D insufficiencies. Transient receptor potential canonical (TRPC) channels, a subclass within the broader TRP family, function as critical calcium transporters in calcium-mediated signaling pathways. However, the exact role of TRPC channels in both obesity and CRC pathogenesis remains poorly understood. This study set out to elucidate the part played by TRPC channels in obesity and CRC development using a mouse model lacking all seven TRPC proteins (TRPC HeptaKO mice). Relative to wild-type counterparts, TRPC HeptaKO mice manifested severe obesity, evidenced by significantly heightened body weights, augmented weights of epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT), increased hepatic lipid deposition, and raised serum levels of total cholesterol (T-CHO) and low-density lipoprotein cholesterol (LDL-C). Moreover, TRPC deficiency was accompanied by an decrease in thermogenic molecules like PGC1-α and UCP1, alongside a upsurge in inflammatory factors within adipose tissue. Mechanistically, it was revealed that pro-inflammatory factors originating from inflammatory macrophages in adipose tissue triggered lipid accumulation and exacerbated obesity-related phenotypes. Intriguingly, considering the well-established connection between obesity and disrupted gut microbiota balance, substantial changes in the gut microbiota composition were detected in TRPC HeptaKO mice, contributing to CRC development. This study provides valuable insights into the role and underlying mechanisms of TRPC deficiency in obesity and its related complication, CRC. Our findings offer a theoretical foundation for the prevention of adverse effects associated with TRPC inhibitors, potentially leading to new therapeutic strategies for obesity and CRC prevention.

Calmodulin-Domain Protein Kinase PiCDPK1 Interacts with the 14-3-3-like Protein NtGF14 to Modulate Pollen Tube Growth

Calcium-mediated signaling pathways are known to play important roles in the polar growth of pollen tubes. The calcium-dependent protein kinase, PiCDPK1, has been shown to be involved in regulating this process through interaction with a guanine dissociation inhibitor, PiRhoGDI1. To more fully understand the role of PiCDPK1 in pollen tube extension, we designed a pull-down study to identify additional substrates of this kinase. These experiments identified 123 putative interactors. Two of the identified proteins were predicted to directly interact with PiCDPK1, and this possibility was investigated in planta. The first, NtGF14, a 14-3-3-like protein, did not produce a noticeable phenotype when overexpressed in pollen alone but partially rescued the spherical tube phenotype caused by PiCDPK1 over-expression when co-over-expressed with the kinase. The second, NtREN1, a GTPase activating protein (GAP), severely inhibited pollen tube germination when over-expressed, and its co-over-expression with PiCDPK1 did not substantially affect this phenotype. These results suggest a novel in vivo interaction between NtGF14 and PiCDPK1 but do not support the direct interaction between PiCDPK1 and NtREN1. We demonstrate the utility of the methodology used to identify potential protein interactions while confirming the necessity of additional studies to confirm their validity. Finally, additional support was found for intersection between PiCDPK1 and RopGTPase pathways to control polar growth at the pollen tube tip.

DNA-Binding Activity of CAMTA3 Is Essential for Its Function: Identification of Critical Amino Acids for Its Transcriptional Activity.

Calmodulin-binding transcription activators (CAMTAs), a small family of highly conserved transcription factors, function in calcium-mediated signaling pathways. Of the six CAMTAs in Arabidopsis, CAMTA3 regulates diverse biotic and abiotic stress responses. A recent study has shown that CAMTA3 is a guardee of NLRs (Nucleotide-binding, Leucine-rich repeat Receptors) in modulating plant immunity, raising the possibility that CAMTA3 transcriptional activity is dispensable for its function. Here, we show that the DNA-binding activity of CAMTA3 is essential for its role in mediating plant immune responses. Analysis of the DNA-binding (CG-1) domain of CAMTAs in plants and animals showed strong conservation of several amino acids. We mutated six conserved amino acids in the CG-1 domain to investigate their role in CAMTA3 function. Electrophoretic mobility shift assays using these mutants with a promoter of its target gene identified critical amino acid residues necessary for DNA-binding activity. In addition, transient assays showed that these residues are essential for the CAMTA3 function in activating the Rapid Stress Response Element (RSRE)-driven reporter gene expression. In line with this, transgenic lines expressing the CG-1 mutants of CAMTA3 in the camta3 mutant failed to rescue the mutant phenotype and restore the expression of CAMTA3 downstream target genes. Collectively, our results provide biochemical and genetic evidence that the transcriptional activity of CAMTA3 is indispensable for its function.

Ethnopharmacological basis and pharmacodynamics prospectives for folkloric claims of Rosa webbiana wall. Ex. Royle in diarrhea and asthma via In vitro, In vivo and In silico techniques

Ethnopharmacological relevanceRosa webbiana (Family: Rosaceae) is used by South Asian herbalists to treat gastrointestinal and respiratory disorders. Aim of the studyThis research aimed at multiple targets to verify R. webbiana for treating diarrhea and asthma. In vitro, in vivo, and in silico experiments were planned to demonstrate the antispasmodic and bronchodilator potential of R. webbiana. Materials and methodsThe bioactive compounds of R. webbiana were identified and quantified through LC ESI-MS/MS and HPLC. These compounds were predicted for muti-mechanisms of bronchodilator and antispasmodic potential in network pharmacology and molecular docking. In vitro methods (isolated rabbit trachea, bladder, and jejunum tissues) confirmed these multi-mechanisms for antispasmodic and bronchodilator effects. Antiperistalsis, antidiarrheal, and antisecretory experiments were conducted in in-vivo experiments. ResultsThe phytochemical analysis indicates the presence of rutin (742.91 μg/g), kaempferol (726.32 μg/g), and quercitrin (688.20 μg/g) in Rw. EtOH. These bioactive compounds in network pharmacology interfere with the pathogenic genes of diarrhea and asthma, which are the members of calcium-mediated signaling pathways and showed the stronger binding affinity towards voltage-gated L-type calcium channels, myosin light chain-kinase, Calcium calmodulin-dependent-kinase, Phosphodiesterase-4, and phosphoinositide phospholipase-C in molecular docking. Rw. EtOH elicited a spasmolytic response in isolated jejunum, trachea, and urine preparations by relaxing K+ (80 mM) and CCh (1 μM) spastic contractions. Additionally, it suppressed calcium concentration-response curves to the right, like verapamil. Like dicyclomine, it caused a rightward parallel shift of the CCh curves, followed by a non-parallel shift at higher concentrations with suppression of the maximal response. Like papaverine, it also caused isoprenaline-induced inhibitory CRCs to shift to the left. Verapamil did not potentiate isoprenaline-induced inhibitory CRCs, although it was more efficacious against K+ (80 mM) than CCh (1 μM)-induced contractions. R. webbiana EtOH extract exhibited complete antiperistalsis (21.55%), antidiarrheal (80.33%), and antisecretory (82.59±0.60) activities in vivo experiments at the dose of 300 mg/kg. ConclusionThus, Rw. EtOH modulated multiple pathways, produced calcium antagonistic, anticholinergic, and phosphodiesterase inhibitory actions, and had antidiarrheal and bronchodilator effects.

Downregulation of mitochondrial calcium uptake family 3 attenuates secondary brain injury after intracerebral hemorrhage in rats.

Intracerebral hemorrhage (ICH) is one type of stroke with a high incidence and mortality. Mitochondria provide energy for various life processes and regulate calcium-mediated signaling pathways by taking up calcium ions from cytoplasm. Mitochondrial calcium uptake family 3 (MICU3) is a tissue-specific enhancer of mitochondrial calcium uptake. The effects and mechanisms of MICU3 in ICH are unknown. In this study, we aimed to explore the role of MICU3 in ICH in rats and neuronal models. First, we constructed ICH model both in vivo and in vitro and observed increased expression of MICU3. Then lentivirus was transduced to knock down MICU3. We observed that knockdown of MICU3 significantly reduced mitochondrial uptake of calcium in primary neurons. Moreover, the downregulation of MICU3 attenuated cell apoptosis and decreased the accumulation of reactive oxygen species (ROS). Recovery of neurobehavioral and cognitive function also benefited from downregulation of MICU3. The findings demonstrated that MICU3 played an important role in cell apoptosis, oxidative stress, and maintenance of mitochondrial structure and function, and promoted rehabilitation of neurobehavior. In conclusion, MICU3 is expected to be a molecular marker and a potential therapeutic target for ICH.

Comparing the infection biology and gene expression differences of Plasmodiophora brassicae primary and secondary zoospores.

Plasmodiophora brassicae (Wor.) is an obligate plant pathogen affecting Brassicae worldwide. To date, there is very little information available on the biology and molecular basis of P. brassicae primary and secondary zoospore infections. To examine their roles, we used microscope to systematically investigate the infection differences of P. brassicae between samples inoculated separately with resting spores and secondary zoospores. The obvious development of P. brassicae asynchrony that is characterized by secondary plasmodium, resting sporangial plasmodium, and resting spores was observed at 12 days in Brassica rapa inoculated with resting spores but not when inoculated with secondary zoospores at the same time. Inoculation with resting spores resulted in much more development of zoosporangia clusters than inoculation with secondary zoospores in non-host Spinacia oleracea. The results indicated that primary zoospore infection played an important role in the subsequent development. To improve our understanding of the infection mechanisms, RNA-seq analysis was performed. Among 18 effectors identified in P. brassicae, 13 effectors were induced in B. rapa seedlings inoculated with resting spores, which suggested that the pathogen and host first contacted, and more effectors were needed. Corresponding to those in B. rapa, the expression levels of most genes involved in the calcium-mediated signaling pathway and PTI pathway were higher in plants inoculated with resting spores than in those inoculated with secondary zoospores. The ETI pathway was suppressed after inoculation with secondary zoospores. The genes induced after inoculation with resting spores were suppressed in B. rapa seedlings inoculated with secondary zoospores, which might be important to allow a fully compatible interaction and contribute to a susceptible reaction in the host at the subsequent infection stage. The primary zoospores undertook an more important interaction with plants.

ITRAQ-Based Comparative Proteomic Analysis Provides Insights into Tobacco Callus Response to Carbon Nanoparticles

Carbon nanomaterials (CNMs) have attracted a great deal of research interest for their potential application in plants. Some types of CNMs have exhibited the ability on regulating plant growth of plants, which showed a promising future in agriculture. However, detailed mechanism of their regulation on plant growth is still not well characterized especially at the molecular level. In this study, our objective is to study the regulation mechanism of carbon nanoparticles (CNPs) on tobacco callus growth at protein level. During the incubation period, proteomic profiling of tobacco callus was investigated by using the isobaric tags for relative and absolute quantitation labelling (iTRAQ) coupled with 2D-LC MS/MS. The function of differentially expressed proteins was achieved by using a gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis with InterproScan, and qRT-PCR was used to support the results of the proteomic data. The results showed that the growth of tobacco callus was enhanced by CNPs, with the optimal concentration at 50 mg/L. The expression levels of 198 cellular proteins were significantly up- or down-regulated after CNPs treatment. Some differentially expressed proteins were located in mitochondria and involved in calcium-mediated signaling pathway. The proteomic profile changes provided more deeper understanding of the highly complex regulatory mechanisms in tobacco callus exposed to CNPs.

Promising protein biomarkers in the early diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) is an insidious, multifactorial disease that involves the devastation of neurons leading to cognitive impairments. Alzheimer's have compounded pathologies of diverse nature, including proteins as one important factor along with mutated genes and enzymes. Although various review articles have proposed biomarkers, still, the statistical importance of proteins is missing. Proteins associated with AD include amyloid precursor protein, glial fibrillary acidic protein, calmodulin-like skin protein, hepatocyte growth factor, matrix Metalloproteinase-2. These proteins play a crucial role in the AD hypothesis which includes the tau hypothesis, amyloid-beta (Aβ) hypothesis, cholinergic neuron damage, etc. The present review highlights the role of major proteins and their physiological functions in the early diagnosis of AD. Altered protein expression results in cognitive impairment, synaptic dysfunction, neuronal degradation, and memory loss. On the medicinal ground, efforts of making anti-amyloid, anti-tau, anti-inflammatory treatments are on the peak, having these proteins as putative targets. Few proteins, e.g.,Amyloid precursor protein results in the formation of non-soluble sticky Aβ40 and Aβ42 monomers that, over time, aggregate into plaques in the cortical and limbic brain areas and neurogranin is believed to regulate calcium-mediated signaling pathways and thus modulating synaptic plasticity are few putative and potential forthcoming targets for developing effective anti-AD therapies. These proteins may help to diagnose the disease early, bode well for the successful discovery and development of therapeutic and preventative regimens for this devasting public health problem.

Transcriptomic analysis elucidates the enhanced skeletal muscle mass, reduced fat accumulation, and metabolically benign liver in human follistatin-344 transgenic pigs

Follistatin (FST) is an important regulator of skeletal muscle growth and adipose deposition through its ability to bind to several members of the transforming growth factor-β (TGF-β) superfamily, and thus may be a good candidate for future animal breeding programs. However, the molecular mechanisms underlying the phenotypic changes have yet to be clarified in pig. We generated transgenic (TG) pigs that express human FST specifically in skeletal muscle tissues and characterized the phenotypic changes compared with the same tissues in wild-type pigs. The TG pigs showed increased skeletal muscle growth, decreased adipose deposition, and improved metabolism status (P<0.05). Transcriptome analysis detected important roles of the PIK3–AKT signaling pathway, calcium-mediated signaling pathway, and amino acid metabolism pathway in FST-induced skeletal muscle hypertrophy, and depot-specific oxidative metabolism changes in psoas major muscle. Furthermore, the lipid metabolism-related process was changed in adipose tissue in the TG pigs. Gene set enrichment analysis revealed that genes related to lipid synthesis, lipid catabolism, and lipid storage were down-regulated (P<0.01) in the TG pigs for subcutaneous fat, whereas genes related to lipid catabolism were significantly up-regulated (P<0.05) in the TG pigs for retroperitoneal fat compared with their expression levels in wild-type pigs. In liver, genes related to the TGF-β signaling pathway were over-represented in the TG pigs, which is consistent with the inhibitory role of FST in regulating TGF-β signaling. Together, these results provide new insights into the molecular mechanisms underlying the phenotypic changes in pig.

Cucumis sativus L. Seeds Ameliorate Muscular Spasm-Induced Gastrointestinal and Respiratory Disorders by Simultaneously Inhibiting Calcium Mediated Signaling Pathway.

Cucumis sativus L. is globally cultivated as an edible vegetable. Besides its nutritional benefits, it is used in traditional medicines against various ailments. The current study was designed to elucidate the multi-target mechanisms of a C. sativus seeds extract against asthma and diarrhea using network pharmacology along with a molecular docking approach. Furthermore, in-vitro and in-vivo experiments were conducted to verify the mechanistic insight of in silico studies. LC-ESI-MS/MS was performed to identify the bioactive compounds in the extract; later, some compounds were quantified by HPLC. C. sativus seed. EtOH has kaempferol in higher concentration 783.02 µg/g, followed by quercetin (693.83 µg/g) and luteolin (617.17 µg/g). In silico studies showed that bioactive compounds interfered with asthma and diarrhea-associated target genes, which are members of calcium-mediated signaling to exert a calcium channel blocker activity. The seeds extract exerted a concentration-dependent spasmolytic response on isolated jejunum, trachea, and urinary bladder preparations and caused relaxation of spastic contraction of K+ (80 mM) with suppressed calcium concentration-response curves at dose 0.3 and 1 mg/mL. It also showed antiperistalsis, antidiarrheal and antisecretory activity in animal models. Thus, C. sativus seeds have therapeutic effects by regulating the contractile response through a calcium-mediated signaling pathway.

Enhanced osteogenic differentiation of mesenchymal stem cells on P(VDF‐TrFE) layer coated microelectrodes

Electrical stimulation has been proved to be critical to regulate cell behavior. But, cell behavior is also susceptible to multiple parameters of the adverse interferences such as surface current, electrochemical reaction products, and non-uniform compositions, which often occur during direct electric stimulation. To effectively prevent the adverse interferences, a novel piezoelectric poly(vinylidene fluoride-trfluoroethylene)(P(VDF-TrFE)) layer was designed to coat onto the indium tin oxide (ITO) planar microelectrode. We found the electrical stimulation was able to regulate the osteogenic differentiation of mesenchymal stem cells (MSCs) through calcium-mediated PKC signaling pathway. Meanwhile, the surface charge of the designed P(VDF-TrFE) coating layer could be easily controlled by the pre-polarization process, which was demonstrated to trigger integrin-mediated FAK signaling pathway, finally up-regulating the osteogenic differentiation of MSCs. Strikingly, the crosstalk in the downstream of the two signaling cascades further strengthened the ERK pathway activation for osteogenic differentiation of MSCs. This P(VDF-TrFE) layer coated electrical stimulation microelectrodes therefore provide a distinct strategy to manipulate multiple-elements of biomaterial surface to regulate stem cell fate commitment.

Red and Blue Light-Emitting Diodes Significantly Improve in vitro Tuberization of Potato (Solanum tuberosum L.)

Abstract The tuberization phenomenon in potatoes (Solanum tuberosum L.) is a highly synchronized morphophysiological process occurring on the underground stolons under the influence of various intrinsic and extrinsic factors. This involves the participation of the phytochrome sensory system, transport of sucrose from source to stolon, and several regulatory pathways including tuber-inducing hormone biosynthetic lipoxygenase (StLOX1) gene and major calcium-mediated signaling pathway genes (StCDPK and StCaM1). This study was aimed to explore the impacts of distinctive qualities of the light-emitting diode (LED) light with a specific wavelength on in vitro tuberization of potato. Single nodal segments of potato ‘Kufri Jyoti’ were incubated in vitro on the growth medium at a temperature of 22 ± 2 °C and exposed to various combinations of red (R) and blue (B) LED light. The results showed that the combination of 30% red + 70% blue LED light (R30B70) significantly shortened the tuber induction period, increased the number of tubers, and their yield compared with these parameters in the tissues exposed to the white light (W100). The induction of the in vitro tuberization correlated with the enhanced expression of the major tuberizing pathway genes, including the lipoxygenase (StLOX1), calmodulin (CaM1), and calcium-dependent protein kinase (StCDPK). The current study indicated that the combination of red and blue LED lights at R30B70 is the best spectrum for effective in vitro tuberization of potatoes.

Strategies for Neuroprotection in Multiple Sclerosis and the Role of Calcium.

Calcium ions are vital for maintaining the physiological and biochemical processes inside cells. The central nervous system (CNS) is particularly dependent on calcium homeostasis and its dysregulation has been associated with several neurodegenerative disorders including Parkinson’s disease (PD), Alzheimer’s disease (AD) and Huntington’s disease (HD), as well as with multiple sclerosis (MS). Hence, the modulation of calcium influx into the cells and the targeting of calcium-mediated signaling pathways may present a promising therapeutic approach for these diseases. This review provides an overview on calcium channels in neurons and glial cells. Special emphasis is put on MS, a chronic autoimmune disease of the CNS. While the initial relapsing-remitting stage of MS can be treated effectively with immune modulatory and immunosuppressive drugs, the subsequent progressive stage has remained largely untreatable. Here we summarize several approaches that have been and are currently being tested for their neuroprotective capacities in MS and we discuss which role calcium could play in this regard.

Permethrin, a pyrethroid insecticide, regulates ERK1/2 activation through membrane depolarization-mediated pathway in HepG2 hepatocytes

Permethrin is a pyrethroid insecticide that acts thru membrane depolarization and is known to disrupt calcium levels in neurons. Disrupted calcium homeostasis is linked to oxidative stress as well as many other cellular mis-functions and permethrin has been reported to disrupt lipid and glucose metabolism in animals and mammalian cell models. It is not known, however, if permethrin influences calcium levels and its associated cellular mechanisms in liver cells. Thus, the goal of the current study was to investigate the mechanisms of permethrin on calcium-mediated cellular signaling pathway, particularly on activation of extracellular signal-related kinase (ERK1/2 or p42/p44) using human hepatocytes, HepG2. The current results showed that permethrin treatment induced oxidative stress and phosphorylation of ERK1/2, which were dependent upon voltage-sensitive sodium channels (VSSC). It was further determined that permethrin-induced ERK1/2 activation was mediated by the metabotropic glutamate receptors (mGluRs)-phosphoinositide phospholipase C (PLC)-protein kinase C (PKC) pathway, but not by changes of intracellular calcium or ER stress-mediated mechanisms.

Proteomics and Phosphoproteomics of Heat Stress-Responsive Mechanisms in Spinach.

Elevated temperatures limit plant growth and reproduction and pose a growing threat to agriculture. Plant heat stress response is highly conserved and fine-tuned in multiple pathways. Spinach (Spinacia oleracea L.) is a cold tolerant but heat sensitive green leafy vegetable. In this study, heat adaptation mechanisms in a spinach sibling inbred heat-tolerant line Sp75 were investigated using physiological, proteomic, and phosphoproteomic approaches. The abundance patterns of 911 heat stress-responsive proteins, and phosphorylation level changes of 45 phosphoproteins indicated heat-induced calcium-mediated signaling, ROS homeostasis, endomembrane trafficking, and cross-membrane transport pathways, as well as more than 15 transcription regulation factors. Although photosynthesis was inhibited, diverse primary and secondary metabolic pathways were employed for defense against heat stress, such as glycolysis, pentose phosphate pathway, amino acid metabolism, fatty acid metabolism, nucleotide metabolism, vitamin metabolism, and isoprenoid biosynthesis. These data constitute a heat stress-responsive metabolic atlas in spinach, which will springboard further investigations into the sophisticated molecular mechanisms of plant heat adaptation and inform spinach molecular breeding initiatives.

Glucose-Dependent Insulinotropic Polypeptide Suppresses Peripheral Arterial Remodeling in Male Mice.

Glucose-dependent insulinotropic polypeptide (GIP) exhibits direct cardiovascular actions in addition to its well-known insulinotropic effect. However, the role of GIP in peripheral artery disease remains unclear. In this study, we evaluated the effects of GIP against peripheral arterial remodeling in mouse models. The genetic deletion of GIP receptor (GIPR) led to exaggerated neointimal hyperplasia after transluminal femoral artery wire injury. Conversely, chronic GIP infusion suppressed neointimal hyperplasia and facilitated endothelial regeneration. The beneficial effects of GIP were abrogated by inhibiting nitric oxide (NO) synthase, suggesting a possible mechanism mediated by NO. In cultured human umbilical vein endothelial cells (HUVECs), GIP elevated cytosolic calcium levels without affecting intracellular cAMP levels. Furthermore, GIP dose-dependently increased NO production, whereas this effect was abolished by inhibiting AMP-activated protein kinase (AMPK). GIP induced AMPK phosphorylation, which was abrogated by inhibiting phospholipase C and calcium-calmodulin-dependent protein kinase kinase but not by adenylate cyclase or liver kinase B1, suggesting the existence of a calcium-mediated GIPR signaling pathway. These effects of GIP were retained in severe hyperglycemic Leprdb/ Leprdb mice and in high-glucose-cultured HUVECs. Overall, we demonstrated the protective effects of GIP against peripheral arterial remodeling as well as the involvement of a calcium-mediated GIPR signaling pathway in vascular endothelial cells. Our findings imply the potential vascular benefits of multiple agonists targeting G protein-coupled receptors, including GIPR, which are under development for the treatment of type 2 diabetes.

Differentially Expressed MicroRNAs in Conservatively Treated Nontraumatic Osteonecrosis Compared with Healthy Controls.

Deregulation of microRNAs (miRNAs) contributes to nontraumatic osteonecrosis of the femoral head (ONFH-N), but the differentially expressed circulating miRNAs in patients with ONFH-N receiving nonsurgical therapy are unknown. This study aimed to determine the miRNAs expression profile of patients with ONFH-N receiving conservative treatments. This was a case-control prospective study of 43 patients with ONFH-N and 43 participants without ONFH-N, enrolled from 10/2014 to 10/2016 at the Department of Orthopedics of the Linyi People's Hospital (China). The two groups were matched for age, gender, and living area. Microarray analysis and quantitative RT-PCR were used to examine the differentially expressed miRNAs. Bioinformatics was used to predict miRNA target genes and signaling pathways. Microarray and quantitative RT-PCR revealed that nine miRNAs were downregulated and five miRNAs were upregulated in ONFH-N (n = 3) compared with controls (n = 3). Bioinformatics showed that calcium-mediated signaling pathway, regulation of calcium ion transmembrane transporter activity, cytoskeletal protein binding, and caveolae macromolecular signaling complex were probably regulated by the identified differentially expressed miRNAs. In the remaining 80 subjects (n = 40/group), miR-335-5p was downregulated (P = 0.01) and miR-100-5p was upregulated (P = 0.02) in ONFH-N compared with controls. In conclusion, some miRNAs are differentially expressed in conservatively treated ONFH-N compared with controls. Those miRNAs could contribute to the pathogenesis of ONFH-N.

The rice cold-responsive calcium-dependent protein kinase OsCPK17 is regulated by alternative splicing and post-translational modifications

Plant calcium-dependent protein kinases (CDPKs) are key proteins implicated in calcium-mediated signaling pathways of a wide range of biological events in the organism. The action of each particular CDPK is strictly regulated by many mechanisms in order to ensure an accurate signal translation and the activation of the adequate response processes. In this work, we investigated the regulation of a CDPK involved in rice cold stress response, OsCPK17, to better understand its mode of action. We identified two new alternative splicing (AS) mRNA forms of OsCPK17 encoding truncated versions of the protein, missing the CDPK activation domain. We analyzed the expression patterns of all AS variants in rice tissues and examined their subcellular localization in onion epidermal cells. The results indicate that the AS of OsCPK17 putatively originates truncated forms of the protein with distinct functions, and different subcellular and tissue distributions. Additionally, we addressed the regulation of OsCPK17 by post-translational modifications in several in vitro experiments. Our analysis indicated that OsCPK17 activity depends on its structural rearrangement induced by calcium binding, and that the protein can be autophosphorylated. The identified phosphorylation sites mostly populate the OsCPK17 N-terminal domain. Exceptions are phosphosites T107 and S136 in the kinase domain and S558 in the C-terminal domain. These phosphosites seem conserved in CDPKs and may reflect a common regulatory mechanism for this protein family.