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Autophagy is a double-edged sword for cells; it can lead to both cell survival and death. Calcium (Ca2+) signalling plays a crucial role in regulating various cellular behaviours, including cell migration, proliferation and death. In this study, we investigated the effects of modulating cytosolic Ca2+ levels on autophagy using chemical and optogenetic methods. Our findings revealed that ionomycin and thapsigargin induce Ca2+ influx to promote autophagy, whereas the Ca2+ chelator BAPTA-AM induces Ca2+ depletion and inhibits autophagy. Furthermore, the optogenetic platform allows the manipulation of illumination parameters, including density, frequency, duty cycle and duration, to create different patterns of Ca2+ oscillations. We used the optogenetic tool Ca2+-translocating channelrhodopsin, which is activated and opened by 470 nm blue light to induce Ca2+ influx. These results demonstrated that high-frequency Ca2+ oscillations induce autophagy. In addition, autophagy induction may involve Ca2+-activated adenosine monophosphate (AMP)-activated protein kinases. In conclusion, high-frequency optogenetic Ca2+ oscillations led to cell death mediated by AMP-activated protein kinase-induced autophagy.

Abstract Maladaptive repair of acute kidney injury (AKI) is associated with a high risk of developing chronic kidney disease deemed irremediable even in present days. When AKI arises from ischemia-reperfusion injury, hypoxia usually plays a major role. Although both hypoxia-inducible factor-1α (HIF-1α) and yes-associated protein (YAP) have been proven to promote renal cell survival under hypoxia, there is a lack of research that studies the crosstalk of the two and its effect on kidney repair. In studying the crosstalk, CoCl2 was used to create a mimetic hypoxic environment. Immunoprecipitation and proximity ligation assays were performed to verify protein interactions. The results show that HIF-1α interacts with YAP and promotes nuclear translocation of YAP at a high cell density under hypoxic conditions, suggesting HIF-1α serves as a direct carrier that enables YAP nuclear translocation. This is the first study to identify HIF-1α as a crucial pathway for YAP nuclear translocation under hypoxic conditions. Once translocated into a nucleus, YAP protects cells from DNA damage and apoptosis under hypoxic conditions. Since it is unlikely for YAP to translocate into a nucleus without HIF-1α, any treatment that fosters the crosstalk between the two holds the potential to improve cell recovery from hypoxic insults.

Abstract Background Hypoxia is commonly characterized by malignant tumors that promote the aggressiveness and metastatic potential of cancer. Triple‐negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with approximately 46% capacity related to distant metastasis. Transcriptional factor yes‐associated protein (YAP), a core component of the Hippo pathway, is associated with poor prognosis and outcome in cancer metastasis. Here, we explored the effect of hypoxia‐mediated YAP activation and focal adhesions (FAs) turnover in mesenchymal TNBC cell migration. Methods We characterized the effect of hypoxia on YAP in different breast cancer cell lines using a hypoxia chamber and CoCl2. Results Hypoxia‐induced YAP nuclear translocation is significantly observed in normal breast epithelial cells, non‐TNBC cells, mesenchymal TNBC cells, but not in basal‐like TNBC cells. Functionally, we demonstrated that YAP activation was required for hypoxia to promote mesenchymal TNBC cell migration. Furthermore, hypoxia induced the localization of FAs at the leading edge of mesenchymal TNBC cells. In contrast, verteporfin (VP), a YAP inhibitor, significantly reduced the migration and the recruitment of nascent FAs at the cell periphery under hypoxia conditions, which only showed in mesenchymal TNBC cells. Conclusions Our data support the hypothesis that YAP is novel factor and positively responsible for hypoxia‐promoting mesenchymal TNBC cell migration. Our findings provide further evidence and outcomes to help prevent the progression of TNBC.

Intracellular calcium (Ca2+) has been reported to regulate transcription factor activity and cancer development, but how it affects the function of Forkhead box protein M1 (FOXM1), a crucial transcription factor and key oncogene participating in tumorigenesis, remains unclear. Here, we investigated the regulatory role of Ca2+ on FOXM1 and found that Ca2+ depletion caused the distribution of FOXM1 to aggregate on the nuclear envelope, which was also observed in many cell lines. Further experiments revealed that sequestrated FOXM1 colocalized with lamin B in the inner nuclear membrane (INM) and was affected by the activity of nuclear export protein exportin 1 (XPO1). To investigate how intracellular Ca2+ affects FOXM1, we found that among the posttranscriptional modifications, only SUMOylation of FOXM1 showed a pronounced increase under reduced Ca2+, and suppressed SUMOylation rescued FOXM1 sequestration. In addition, Ca2+-dependent SUMOylated FOXM1 appeared to enhance the G2/M transition of the cell cycle and decrease cell apoptosis. In conclusion, our findings provide a molecular basis for the relationship between Ca2+ signaling and FOXM1 regulation, and we look to elucidate Ca2+-dependent FOXM1 SUMOylation-related biological functions in the future.

Replication fork reversal which restrains DNA replication progression is an important protective mechanism in response to replication stress. PARP1 is recruited to stalled forks to restrain DNA replication. However, PARP1 has no helicase activity, and the mechanism through which PARP1 participates in DNA replication restraint remains unclear. Here, we found novel protein-protein interactions between PARP1 and DNA translocases, including HLTF, SHPRH, ZRANB3, and SMARCAL1, with HLTF showing the strongest interaction among these DNA translocases. Although HLTF and SHPRH share structural and functional similarity, it remains unclear whether SHPRH contains DNA translocase activity. We further identified the ability of SHPRH to restrain DNA replication upon replication stress, indicating that SHPRH itself could be a DNA translocase or a helper to facilitate DNA translocation. Although hydroxyurea (HU) and MMS induce different types of replication stress, they both induce common DNA replication restraint mechanisms independent of intra-S phase activation. Our results suggest that the PARP1 facilitates DNA translocase recruitment to damaged forks, preventing fork collapse and facilitating DNA repair.

Abstract Human HLTF participates in the lesion-bypass mechanism through the fork reversal structure, known as template switching of post-replication repair. However, the mechanism by which HLTF promotes the replication progression and fork stability of damaged forks remains unclear. Here, we identify a novel protein–protein interaction between HLTF and PARP1. The depletion of HLTF and PARP1 increases chromosome breaks, further reduces the length of replication tracks, and concomitantly increases the number of stalled forks after methyl methanesulfonate treatment according to a DNA fiber analysis. The progression of replication also depends on BARD1 in the presence of MMS treatment. By combining 5-ethynyl-2′-deoxyuridine with a proximity ligation assay, we revealed that the HLTF, PARP1, and BRCA1/BARD1/RAD51 proteins were initially recruited to damaged forks. However, prolonged stalling of damaged forks results in fork collapse. HLTF and PCNA dissociate from the collapsed forks, with increased accumulation of PARP1 and BRCA1/BARD1/RAD51 at the collapsed forks. Our results reveal that HLTF together with PARP1 and BARD1 participates in the stabilization of damaged forks, and the PARP1–BARD1 interaction is further involved in the repair of collapse forks.

Abstract Oxygen is essentially required by most eukaryotic organisms as a scavenger to remove harmful electron and hydrogen ions or as a critical substrate to ensure the proper execution of enzymatic reactions. All nucleated cells can sense oxygen concentration and respond to reduced oxygen availability (hypoxia). When oxygen delivery is disrupted or reduced, the organisms will develop numerous adaptive mechanisms to facilitate cells survived in the hypoxic condition. Normally, such hypoxic response will cease when oxygen level is restored. However, the situation becomes complicated if hypoxic stress persists (chronic hypoxia) or cyclic normoxia-hypoxia phenomenon occurs (intermittent hypoxia). A series of chain reaction-like gene expression cascade, termed hypoxia-mediated gene regulatory network, will be initiated under such prolonged or intermittent hypoxic conditions and subsequently leads to alteration of cellular function and/or behaviors. As a result, irreversible processes occur that may cause physiological disorder or even pathological consequences. A growing body of evidence implicates that hypoxia plays critical roles in the pathogenesis of major causes of mortality including cancer, myocardial ischemia, metabolic diseases, and chronic heart and kidney diseases, and in reproductive diseases such as preeclampsia and endometriosis. This review article will summarize current understandings regarding the molecular mechanism of hypoxia in these common and important diseases.

Abstract Background Among gynecological cancers, ovarian carcinoma has the highest mortality rate, and chemoresistance is highly prevalent in this cancer. Therefore, novel strategies are required to improve its poor prognosis. Formation and disassembly of focal adhesions are regulated dynamically during cell migration, which plays an essential role in cancer metastasis. Metastasis is intricately linked with resistance to chemotherapy, but the molecular basis for this link is unknown. Methods Transwell migration and wound healing migration assays were used to analyze the migration ability of ovarian cancer cells. Real-time recordings by total internal reflection fluorescence microscope (TIRFM) were performed to assess the turnover of focal adhesions with fluorescence protein-tagged focal adhesion molecules. SOCE inhibitors were used to verify the effects of SOCE on focal adhesion dynamics, cell migration, and chemoresistance in chemoresistant cells. Results We found that mesenchymal-like chemoresistant IGROV1 ovarian cancer cells have higher migration properties because of their rapid regulation of focal adhesion dynamics through FAK, paxillin, vinculin, and talin. Focal adhesions in chemoresistant cells, they were smaller and exhibited strong adhesive force, which caused the cells to migrate rapidly. Store-operated Ca2+ entry (SOCE) regulates focal adhesion turnover, and cell polarization and migration. Herein, we compared SOCE upregulation in chemoresistant ovarian cancer cells to its parental cells. SOCE inhibitors attenuated the assembly and disassembly of focal adhesions significantly. Results of wound healing and transwell assays revealed that SOCE inhibitors decreased chemoresistant cell migration. Additionally, SOCE inhibitors combined with chemotherapeutic drugs could reverse ovarian cancer drug resistance. Conclusion Our findings describe the role of SOCE in chemoresistance-mediated focal adhesion turnover, cell migration, and viability. Consequently, SOCE might be a promising therapeutic target in epithelial ovarian cancer. Graphical abstract

Background: Age-related changes affecting the ocular surface cause vision loss in the elderly. Cisd2 deficiency drives premature aging in mice as well as resulting in various ocular surface abnormalities. Here we investigate the role of CISD2 in corneal health and disease. Methods: We studied the molecular mechanism underlying the ocular phenotypes brought about by Cisd2 deficiency using both Cisd2 knockout (KO) mice and a human corneal epithelial cell (HCEC) cell line carrying a CRISPR-mediated CISD2KO background. We also develop a potential therapeutic strategy that targets the Ca2+ signaling pathway, which has been found to be dysregulated in the corneal epithelium of subjects with ocular surface disease in order to extend the mechanistic findings into a translational application. Findings: Firstly, in patients with corneal epithelial disease, CISD2 is down-regulated in their corneal epithelial cells. Secondly, using mouse cornea, Cisd2 deficiency causes a cycle of chronic injury and persistent repair resulting in exhaustion of the limbal progenitor cells. Thirdly, in human corneal epithelial cells, CISD2 deficiency disrupts intracellular Ca2+ homeostasis, impairing mitochondrial function, thereby retarding corneal repair. Fourthly, cyclosporine A and EDTA facilitate corneal epithelial wound healing in Cisd2 knockout mice. Finally, cyclosporine A treatment restores corneal epithelial erosion in patients with dry eye disease, which affects the ocular surface. Interpretation: These findings reveal that Cisd2 plays an essential role in the cornea and that Ca2+ signaling pathways are potential targets for developing therapeutics of corneal epithelial diseases. Funding: This study was supported by the Ministry of Science and Technology (MOST) and Chang Gung Medical Research Foundation, Taiwan.

Glioma, the most common subtype of primary brain tumor, is an aggressive and highly invasive neurologically tumor among human cancers. Interleukin-33 (IL-33) is considered as a dual functional cytokine, an alarmin upon tissue damage and a nuclear chromatin-associated protein. Despite that, IL-33 is known to foster the formation of the inflammatory tumor microenvironment and facilitate glioma progression, evidence showing nuclear IL-33 function is still poor. In this study using lentivirus-mediated IL-33 gene knockdown (IL33KD) and IL-33 overexpression (IL33oe) in rat C6 glioma cells and human glioma cell lines (U251MG and U87MG), we found that IL33oe-glioma cells had resistance to the insults of the alkylating agent, temozolomide (TMZ), possibly because of the increased expression of DNA repair genes (i.e., BRCA1, BRCA2, Rad51, FANCB, and FANCD) in IL33oe-glioma cells. Alternatively, examination of glioma nuclear shape from transmission electron microscopy (TEM) imaging analysis and immunofluorescence for histone protein H2A staining showed that IL33KD attenuated the abnormal cancerous nuclear characteristic, such as indentation, long clefts, and multiple nucleoids. Yet, IL33oe promoted the changes in glioma nuclear shapes, such as the formation of multiple lobes. We further found that histone proteins, H2A and H3, were reduced in IL33KD glioma cells. The non-histone DNA-binding nucleoproteins, the high mobility group A1 (HMGA1) and HMGA2, were also downregulated by IL33KD. In contrast, IL33oe increased H2A and H3 proteins and HMGA1 and HMGA2 in glioma cells. Altogether, the upregulation of nuclear IL-33 expression was along with an increase in the expression of DNA repair genes, contributing to the desensitization of glioma cells to DNA damaging agents. Moreover, nuclear IL-33 proteins in cooperation with chromatin-associated proteins regulate glioma nuclear structure, which might be crucial for glioma progression and malignancy.

Microglia and astrocytes are the glial cells of the central nervous system (CNS) to support neurodevelopment and neuronal function. Yet, their activation in association with CNS inflammation is involved in the initiation and progression of neurological disorders. Mild inflammation in the periphery and glial activation called as gliosis in the hypothalamic region, arcuate nucleus (ARC), are generally observed in obese individuals and animal models. Thus, reduction in peripheral and central inflammation is considered as a strategy to lessen the abnormality of obesity-associated metabolic indices. In this study, we reported that acute peripheral challenge by inflammagen lipopolysaccharide (LPS) upregulated the expression of hypothalamic dopamine type 2 receptor (D2R) mRNA, and chronic feeding by high-fat-diet (HFD) significantly caused increased levels of D2R in the ARC. The in vitro and in vivo studies indicated that an FDA-approved antipsychotic drug named trifluoperazine (TFP), a D2R inhibitor was able to suppress LPS-stimulated activation of microglia and effectively inhibited LPS-induced peripheral inflammation, as well as hypothalamic inflammation. Further findings showed daily peripheral administration intraperitoneally (i.p.) by TFP for 4 weeks was able to reduce the levels of plasma tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in accompany with lower levels of plasma glucose and insulin in obese mice receiving HFD for 16 weeks when compared those in obese mice without TFP treatment. In parallel, the activation of microglia and astrocytes in the ARC was also inhibited by peripheral administration by TFP. According to our results, TFP has the ability to suppress HFD-induced ARC gliosis and inflammation in the hypothalamus.

Abstract Background Thrombomodulin (TM), an integral membrane protein, has long been known for its anticoagulant activity. Recent studies showed that TM displays multifaceted activities, including the involvement in cell adhesion and collective cell migration in vitro. However, whether TM contributes similarly to these biological processes in vivo remains elusive. Methods We adapted zebrafish, a prominent animal model for studying molecular/cellular activity, embryonic development, diseases mechanism and drug discovery, to examine how TM functions in modulating cell migration during germ layer formation, a normal and crucial physiological process involving massive cell movement in the very early stages of life. In addition, an in vivo assay was developed to examine the anti-hemostatic activity of TM in zebrafish larva. Results We found that zebrafish TM-b, a zebrafish TM-like protein, was expressed mainly in vasculatures and displayed anti-hemostatic activity. Knocking-down TM-b led to malformation of multiple organs, including vessels, heart, blood cells and neural tissues. Delayed epiboly and incoherent movement of yolk syncytial layer were also observed in early TM-b morphants. Whole mount immunostaining revealed the co-localization of TM-b with both actin and microtubules in epibolic blastomeres. Single-cell tracking revealed impeded migration of blastomeres during epiboly in TM-b-deficient embryos. Conclusion Our results showed that TM-b is crucial to the collective migration of blastomeres during germ layer formation. The structural and functional compatibility and conservation between zebrafish TM-b and mammalian TM support the properness of using zebrafish as an in vivo platform for studying the biological significance and medical use of TM.

Antioxidant uptake and regular exercise are two well-acknowledged measures used for rejuvenation and oxidative stress elimination. Previous studies have revealed that moderate exercise mildly increases intracellular signaling oxidant levels and strengthens the ability of an organism to deal with escalating oxidative stress by upregulating antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase. Antioxidant supplementation directly scavenges intracellular reactive oxygen species (ROS) to reduce oxidative stress. However, research to understand the impacts of these enzymes on mitigating oxidative stress from the perspective of simple animals is limited. Herein, we show that exercise combined with antioxidant supplementation ameliorates the physiological phenotypes and markers of aging in wild-type and SOD/CAT-deficient Caenorhabditis elegans. We discovered that treated wild-type and gene-deficient worms show better survivorship, reproduction, and motility compared with their control counterparts. Assays of biochemical indices revealed that variations in sod-3 expression under different stress levels imply an inducible enzyme response resulting from exercise training and antioxidant supplementation. In addition, induced ROS resistance obtained from any type of treatment could persist for several days even after treatment cessation, thus suggesting a potential long-term antioxidative stress effect. Our findings confirm that exercise, antioxidant supplementation, and their combination could significantly improve the ability of C. elegans to withstand adverse stress. Our observations provide promising insights into future therapies of anti-oxidative stress in higher animals.

Early dissemination is a unique characteristic and a detrimental process of pancreatic ductal adenocarcinoma (PDAC); however, the underlying mechanism remains largely unknown. Here, we investigate the role of dual-specificity phosphatase-2 (DUSP2)-vascular endothelial growth factor-C (VEGF-C) axis in mediating PDAC lymphangiogenesis and lymphovascular invasion. Expression of DUSP2 is greatly suppressed in PDAC, which results in increased aberrant expression of extracellular vesicle (EV)-associated VEGF-C secretion. EV-VEGF-C exerts paracrine effects on lymphatic endothelial cells and autocrine effects on cancer cells, resulting in the lymphovascular invasion of cancer cells. Tissue-specific knockout of Dusp2 in mouse pancreas recapitulates PDAC phenotype and lymphovascular invasion. Mechanistically, loss-of-DUSP2 enhances proprotein convertase activity and vesicle trafficking to promote the release of the mature form of EV-VEGF-C. Collectively, these findings represent a conceptual advance in understanding pancreatic cancer lymphovascular invasion and suggest that loss-of-DUSP2-mediated VEGF-C processing may play important roles in early dissemination of pancreatic cancer. Abbreviations: DUSP2: dual-specificity phosphatase-2; VEGF-C: vascular endothelial growth factor-C; EV: extracellular vesicles; PDAC: pancreatic ductal adenocarcinoma; KD: knockdown

The scanned laser pico-projection (SLPP) method and Stokes-Mueller matrix polarimetry technique are used to distinguish cancer cells with different cytoskeletal organizations and metastatic potencies. The speckle contrast of human bone osteosarcoma cancer cells (U2OS) with different cytoskeletal drug treatments is measured by the SLPP method. The results show that the contrast is directly related to the organization of the microtubules and actin fibers. The Stokes-Mueller matrix polarimetry technique is employed to measure polarization parameters of the same samples. The measurement results are found to be consistent with those obtained using the SLPP method. Finally, the Stokes-Mueller matrix polarimetry technique is used to extract the polarization parameters of cell-pairs with different metastatic potencies. It is shown that the extracted value of the linear phase retardance (β) is particularly sensitive to the organization of the microtubules and actin fibers. Furthermore, the linear phase retardance (β) and depolarization index (Δ) are both sensitive to the metastatic potency of the cancer cells. In general, both techniques provide a rapid, noninvasive and label-free approach for distinguishing cancer cells with different cytoskeleton organizations and metastatic potentials, and are thus of significant benefit in evaluating the malignancy of cancer cells.

The dysregulation of store-operated Ca2+ entry (SOCE) promotes cancer progression by changing Ca2+ levels in the cytosol or endoplasmic reticulum. Stromal interaction molecule 1 (STIM1), a component of SOCE, is upregulated in several types of cancer and responsible for cancer cell migration, invasion, and metastasis. To explore the impact of STIM1-mediated SOCE on the turnover of focal adhesion (FA) and cell migration, we overexpressed the wild-type and constitutively active or dominant negative variants of STIM1 in an osteosarcoma cell line. In this study, we hypothesized that STIM1-mediated Ca2+ elevation may increase cell migration. We found that constitutively active STIM1 dramatically increased the Ca2+ influx, calpain activity, and turnover of FA proteins, such as the focal adhesion kinase (FAK), paxillin, and vinculin, which impede the cell migration ability. In contrast, dominant negative STIM1 decreased the turnover of FA proteins as its wild-type variant compared to the cells without STIM1 overexpression while promoting cell migration. These unexpected results suggest that cancer cells need an appropriate amount of Ca2+ to control the assembly and disassembly of focal adhesions by regulating calpain activity. On the other hand, overloaded Ca2+ results in excessive calpain activity, which is not beneficial for cancer metastasis.

Abstract The Fanconi anemia pathway in coordination with homologous recombination is essential to repair interstrand crosslinks (ICLs) caused by cisplatin. TIP60 belongs to the MYST family of acetyltransferases and is involved in DNA repair and regulation of gene transcription. Although the physical interaction between the TIP60 and FANCD2 proteins has been identified that is critical for ICL repair, it is still elusive whether TIP60 regulates the expression of FA and HR genes. In this study, we found that the chemoresistant nasopharyngeal carcinoma cells, derived from chronic treatment of cisplatin, show elevated expression of TIP60. Furthermore, TIP60 binds to the promoters of FANCD2 and BRCA1 by using the chromatin immunoprecipitation experiments and promote the expression of FANCD2 and BRCA1. Importantly, the depletion of TIP60 significantly reduces sister chromatid exchange, a measurement of HR efficiency. The similar results were also shown in the FNACD2-, and BRCA1-deficient cells. Additionally, these TIP60-deficient cells encounter more frequent stalled forks, as well as more DNA double-strand breaks resulting from the collapse of stalled forks. Taken together, our results suggest that TIP60 promotes the expression of FA and HR genes that are important for ICL repair and the chemoresistant phenotype under chronic treatment with cisplatin.

Ovarian cancer is the most malignant of all gynecological cancers. A challenge that deteriorates with ovarian adenocarcinoma in neoplastic disease patients has been associated with the chemoresistance of cancer cells. Cisplatin (CP) belongs to the first-line chemotherapeutic agents and it would be beneficial to identify chemoresistance for ovarian adenocarcinoma cells, especially CP-resistance. Gray level co-occurrence matrix (GLCM) was characterized imaging from a numeric matrix and find its texture features. Serous type (OVCAR-4 and A2780), and clear cell type (IGROV1) ovarian carcinoma cell lines with CP-resistance were used to demonstrate GLCM texture feature extraction of images. Cells were cultured with cell density of 6 × 105 in a glass-bottom dish to form a uniform coverage of the glass slide to get the optical images by microscope and DVC camera. CP-resistant cells included OVCAR-4, A2780 and IGROV and had the higher contrast and entropy, lower energy, and homogeneity. Signal to noise ratio was used to evaluate the degree for chemoresistance of cell images based on GLCM texture feature extraction. The difference between wile type and CP-resistant cells was statistically significant in every case (p < 0.001). It is a promising model to achieve a rapid method with a more reliable diagnostic performance for identification of ovarian adenocarcinoma cells with CP-resistance by feature extraction of GLCM in vitro or ex vivo.

Intermittent theta burst stimulation (iTBS) and continuous theta burst stimulation (cTBS) are protocols used in repetitive transcranial magnetic stimulation (rTMS) or cortical electrical stimulation (CES) to facilitate or suppress corticospinal excitability. However, rTMS and CES excite all types of neuron in the target cortex probed by the coil or electrode, making it difficult to differentiate the effect of TBS on specific neural circuits involved in motor plasticity. In this study, TBS protocols were converted into an optogenetic model to achieve focalized and cell-type-specific cortical modulation. Light-sensitive channelrhodopsin-2 (ChR2) was expressed in the glutamatergic neuron in the primary motor cortex (M1) driven by the CaMKIIα promoter. A custom-made optrode comprising an optical fiber and a metal cannula electrode was fabricated to achieve optogenetic stimulation and simultaneous local field potential (LFP) recording. Single-pulse CES was delivered into M1 to elicit motor-evoked potential (MEP), which served as an indicator of motor excitability, before and after TBS intervention. Results show that both CES-iTBS and optogenetic iTBS (Opto-iTBS) can potentiate MEP activity. However, CES-cTBS suppressed MEP activity whereas Opto-cTBS enhanced it. This discrepancy may have resulted from the different neural networks targeted by the two TBS modalities, with CES-cTBS exciting all types of neuron and Opto-cTBS targeting excitatory neuron specifically. The results support the idea that intra-cortical networks determine the variation of TBS-induced neuroplasticity. This study shows that focalized and cell-type-specific brain stimulation using the optogenetic approach is viable and can be extended for further exploration of neuroplasticity.

Platelet-derived growth factor (PDGF) has mitogenic and chemotactic effects on fibroblasts. An increase in intracellular Ca2+ is one of the first events that occurs following the stimulation of PDGF receptors (PDGFRs). PDGF activates Ca2+ elevation by activating the phospholipase C gamma (PLCγ)-signaling pathway, resulting in ER Ca2+ release. Store-operated Ca2+ entry (SOCE) is the major form of extracellular Ca2+ influx following depletion of ER Ca2+ stores and stromal interaction molecule 1 (STIM1) is a key molecule in the regulation of SOCE. In this study, wild-type and STIM1 knockout mouse embryonic fibroblasts (MEF) cells were used to investigate the role of STIM1 in PDGF-induced Ca2+ oscillation and its functions in MEF cells. The unexpected findings suggest that STIM1 knockout enhances PDGFR–PLCγ–STIM2 signaling, which in turn increases PDGF-BB-induced Ca2+ elevation. Enhanced expressions of PDGFRs and PLCγ in STIM1 knockout cells induce Ca2+ release from the ER store through PLCγ–IP3 signaling. Moreover, STIM2 replaces STIM1 to act as the major ER Ca2+ sensor in activating SOCE. However, activation of PDGFRs also activate Akt, ERK, and JNK to regulate cellular functions, such as cell migration. These results suggest that alternative switchable pathways can be observed in cells, which act downstream of the growth factors that regulate Ca2+ signaling.

Vitamin D has been identified as an innate anti-hepatitis C virus (HCV) agent but the possible mechanisms for this issue remain unclear. Here, we clarified the mechanisms of calcitriol-mediated inhibition of HCV infection. Calcitriol partially inhibited HCV infection, nitric oxide (NO) release and lipid accumulation in Huh7.5 human hepatoma cells via the activation of vitamin D receptor (VDR). When cells were pretreated with the activators of peroxisome proliferator-activated receptor (PPAR)-α (Wy14643) and -γ (Ly171883), the calcitriol-mediated HCV suppression was reversed. Otherwise, three individual stimulators of PPAR-α/β/γ blocked the activation of VDR. PPAR-β (linoleic acid) reversed the inhibition of NO release, whereas PPAR-γ (Ly171883) reversed the inhibitions of NO release and lipid accumulation in the presence of calcitriol. The calcitriol-mediated viral suppression, inhibition of NO release and activation of VDR were partially blocked by an inhibitor of endoplasmic reticulum-associated degradation (ERAD), kifunensine. Furthermore, calcitriol blocked the HCV-induced expressions of apolipoprotein J and 78 kDa glucose-regulated protein, which was restored by pretreatment of kifunensine. These results indicated that the calcitriol-mediated HCV suppression was associated with the activation of VDR, interference with ERAD process, as well as blockades of PPAR, lipid accumulation and nitrative stress.

Dopaminergic PC12 cells can synthesize and release dopamine, providing a good cellular model for investigating dopamine regulation. Optogenetic stimulation of channelrhodopsin-2 provides high spatial and temporal precision for selective stimulation as a powerful neuromodulation tool for neuroscience studies. The aim of this study is to measure dopamine release from dopaminergic PC12 cells under optogenetic stimulation using electrochemical recording of self-assembled monolayers modified microelectrode with amperometric measurement in real time. The activation of PC12 cells under various optogenetic stimulation schemes are characterized by measuring single-cell Ca(2+) imaging. After 10 seconds of optogenetic stimulation, the evoked intracellular Ca(2+) level and dopamine current of channelrhodopsin-2-transfected PC12 cells were 1.6- and 3.5-fold higher than those of the control cells. The optogenetic stimulation effects on Ca(2+) influx and dopamine release were 81% and 63% inhibition by using a Ca(2+) channel antagonist Nifedipine. The results indicate that optogenetic stimulation can evoke voltage-gated Ca(2+) channel-dependent dopamine exocytosis from PC12 cells in a cell specific, temporally precise and dose-dependent manner. This proposed dopamine recording system can be developed to be a good cell model for dopamine regulation and drug screening in vitro, or dopaminergic cell implantation therapy in vivo using optogenetic stimulation in a precise and convenient way.

The primary cilium is an antenna-like organelle protruding from the cell surface that can detect physical and chemical stimuli in the extracellular space to activate specific signaling pathways and downstream gene expressions. Calcium ion (Ca2+) signaling regulates a wide spectrum of cellular processes, including fertilization, proliferation, differentiation, muscle contraction, migration, and death. This study investigated the effects of the regulation of cytosolic Ca2+ levels on ciliogenesis using chemical, genetic, and optogenetic approaches. We found that ionomycin-induced Ca2+ influx inhibited ciliogenesis and Ca2+ chelator BATPA-AM-induced Ca2+ depletion promoted ciliogenesis. In addition, store-operated Ca2+ entry and the endoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) negatively regulated ciliogenesis. Moreover, an optogenetic platform was used to create different Ca2+ oscillation patterns by manipulating lighting parameters, including density, frequency, exposure time, and duration. Light-activated Ca2+-translocating channelrhodopsin (CatCh) is activated by 470-nm blue light to induce Ca2+ influx. Our results show that highfrequency Ca2+ oscillations decrease ciliogenesis. Furthermore, the inhibition of cilia formation induced by Ca2+ may occur via the activation of Aurora kinase A. Cilia not only induce Ca2+ signaling but also regulate cilia formation by Ca2+ signaling. [ABSTRACT FROM AUTHOR]

Mitochondrial dynamics regulate the quality and morphology of mitochondria. Calcium (Ca2+) plays an important role in regulating mitochondrial function. Here, we investigated the effects of optogenetically engineered Ca2+ signaling on mitochondrial dynamics. More specifically, customized illumination conditions could trigger unique Ca2+ oscillation waves to trigger specific signaling pathways. In this study, we found that modulating Ca2+ oscillations by increasing the light frequency, intensity and exposure time could drive mitochondria toward the fission state, mitochondrial dysfunction, autophagy and cell death. Moreover, illumination triggered phosphorylation at the Ser616 residue but not the Ser637 residue of the mitochondrial fission protein, dynamin-related protein 1 (DRP1, encoded by DNM1L), via the activation of Ca2+-dependent kinases CaMKII, ERK and CDK1. However, optogenetically engineered Ca2+ signaling did not activate calcineurin phosphatase to dephosphorylate DRP1 atSer637. In addition, light illumination had no effect on the expression levels of the mitochondrial fusion proteins mitofusin 1 (MFN1) and 2 (MFN2). Overall, this study provides an effective and innovative approach to altering Ca2+ signaling for controlling mitochondrial fission with a more precise resolution than pharmacological approaches in the temporal dimension. [ABSTRACT FROM AUTHOR]