Transcriptional regulation of intermolecular Ca2+ signaling in hibernating ground squirrel cardiomyocytes: The myocardin-junctophilin axis.

The contraction of heart cells is controlled by the intermolecular signaling between L-type Ca2+ channels (LCCs) and ryanodine receptors (RyRs), and the nanodistance between them depends on the interaction between junctophilin-2 (JPH2) in the sarcoplasmic reticulum (SR) and caveolin-3 (CAV3) in the transversal tubule (TT). In heart failure, decreased expression of JPH2 compromises LCC-RyR communication leading to deficient blood-pumping power. In the present study, we found that JPH2 and CAV3 transcription was concurrently regulated by serum response factor (SRF) and myocardin. In cardiomyocytes from torpid ground squirrels, compared with those from euthermic counterparts, myocardin expression was up-regulated, which boosted both JPH2 and CAV3 expression. Transmission electron microscopic imaging showed that the physical coupling between TTs and SRs was tightened during hibernation and after myocardin overexpression. Confocal Ca2+ imaging under the whole-cell patch clamp condition revealed that these changes enhanced the efficiency of LCC-RyR intermolecular signaling and fully compensated the adaptive down-regulation of LCCs, maintaining the power of heart contraction while avoiding the risk of calcium overload during hibernation. Our finding not only revealed an essential molecular mechanism underlying the survival of hibernating mammals, but also demonstrated a "reverse model of heart failure" at the molecular level, suggesting a strategy for treating heart diseases.

Adipose-derived mesenchymal stem cells overexpressing prion improve outcomes via the NLRP3 inflammasome/DAMP signalling after spinal cord injury in rat.

Traumatic spinal cord injury (SCI) is a highly destructive disease in human neurological functions. Adipose-derived mesenchymal stem cells (ADMSCs) have tissue regenerations and anti-inflammations, especially with prion protein overexpression (PrPcOE ). Therefore, this study tested whether PrPcOE -ADMSCs therapy offered benefits in improving outcomes via regulating nod-like-receptor-protein-3 (NLRP3) inflammasome/DAMP signalling after acute SCI in rats. Compared with ADMSCs only, the capabilities of PrPcOE -ADMSCs were significantly enhanced in cellular viability, anti-oxidative stress and migration against H2 O2 and lipopolysaccharide damages. Similarly, PrPcOE -ADMSCs significantly inhibited the inflammatory patterns of Raw264.7 cells. The SD rats (n = 32) were categorized into group 1 (Sham-operated-control), group 2 (SCI), group 3 (SCI + ADMSCs) and group 4 (SCI + PrPcOE -ADMSCs). Compared with SCI group 2, both ADMSCs and PrPcOE -ADMSCs significantly improved neurological functions. Additionally, the circulatory inflammatory cytokines levels (TNF-α/IL-6) and inflammatory cells (CD11b/c+/MPO+/Ly6G+) were highest in group 2, lowest in group 1, and significantly higher in group 3 than in group 4. By Day 3 after SCI induction, the protein expressions of inflammasome signalling (HGMB1/TLR4/MyD88/TRIF/c-caspase8/FADD/p-NF-κB/NEK7/NRLP3/ASC/c-caspase1/IL-ß) and by Day 42 the protein expressions of DAMP-inflammatory signalling (HGMB1/TLR-4/MyD88/TRIF/TRAF6/p-NF-κB/TNF-α/IL-1ß) in spinal cord tissues displayed an identical pattern as the inflammatory patterns. In conclusion, PrPcOE -ADMSCs significantly attenuated SCI in rodents that could be through suppressing the inflammatory signalling.

Lipid metabolomic signature might predict subclinical atherosclerosis in patients with active rheumatoid arthritis.

OBJECTIVES: Although 1H-nuclear magnetic resonance (NMR)-based lipid/metabolomics has been used to detect atherosclerosis, data regarding lipid/metabolomic signature in rheumatoid arthritis (RA)-related atherosclerosis are scarce. We aimed to identify the distinct lipid/metabolomic profiling and develop a prediction score model for RA patients with subclinical atherosclerosis (SA). METHODS: Serum levels of lipid metabolites were determined using 1H-NMR-based lipid/metabolomics in 65 RA patients and 12 healthy controls (HCs). The occurrence of SA was defined as the presence of carotid plaques revealed in ultrasound images. RESULTS: Compared with HC, RA patients had significantly higher levels of phenylalanine and glycoprotein acetyls (GlycA) and lower levels of leucine and isoleucine. RA patients with SA had significantly higher levels of phenylalanine, creatinine, and glycolysis_total and lower levels of total lipid in HDL(HDL_L) than RA patients without SA. The Lasso logistic regression analysis revealed that age, creatinine, HDL_L, and glycolysis_total were significant predictors for the presence of SA. The prediction scoring algorithm was built as ( -0.657 + 0.011*Age + 0.004*Creatinine -0.120*HDL_L + 0.056*glycolysis-related measures), with AUC 0.90, sensitivity 83.3%, and specificity 87.2%. Serum phenylalanine levels were significantly decreased, and the levels of HDL_L and HDL_Particle were significantly increased in 20 RA patients, paralleling the decrease in disease activity score for 28-joints. CONCLUSIONS: With 1H-NMR-based lipid/metabolomics, distinct profiling of lipid metabolites was identified between RA patients and HC or between RA patients with and without SA. We further developed a scoring model based on lipid/metabolomics profiling for predicting RA-associated SA.

Combined tacrolimus and melatonin effectively protected kidney against acute ischemia-reperfusion injury.

Acute kidney injury (AKI) is commonly encountered and causes high mortality in hospitalized patients; however, effective therapies for AKI have still not been established. Accordingly, we performed a rodent model with acute renal ischemia-reperfusion (IR) and tested the hypothesis that combined tacrolimus and melatonin therapy could be superior to either one for protecting the kidney against IR injury. Adult-male SD rat (n = 30) were equally categorized into group 1 (receiving laparotomy only), group 2 (IR treated by 3.0 cc/normal-saline), group 3 [IR + tacrolimus/0.5 mg/kg by intravenous administration at 30 minutes and at days 1/2/3 after IR], group 4 (IR + melatonin/50 mg/kg by intra-peritoneal administration at 30 minutes and 25 mg/kg at days 1/2/3 after IR] and group 5 (IR + tacrolimus +melatonin). By day 3 after IR, the creatinine/BUN levels and ratio of urine protein to urine creatinine were highest in group 2, lowest in group 1 and significantly lower in group 5 than in groups 3/4 (all P < .0001), but they did not differ between the groups 3/4. The protein expressions of oxidative-stress (p47phox/NOX-1/NOX-2/NOX-4), upstream (TLR4/MAL/MyD88/TRAF6/ASK1/MKK4/MKK7/NF-κB) and downstream (IL-6/INF-γ/MMP-9/IL-1ß) inflammatory signaling, MAPK-family-signaling cascades(ERK1/2, JNK/p38/c-JUN), apoptotic/autophagic (p53/caspase 3/mitochondrial-Bax, ratio of LC3B-II/LC3B-I), and mitochondrial-damaged (cyclophilin D/cytochrome C/DRP1) biomarkers, and the expressions of inflammatory-immune cells (F4/80, CD14/CD3/CD8) as well as the kidney injured score exhibited an identical pattern of creatinine level (all P < .0001). In conclusion, combined tacrolimus and melatonin therapy was better than either single one on protecting the kidney functional and anatomical integrity against IR injury through suppressing inflammation and the generation of oxidative stress.

Design and Synthesis of Stem Cell-Laden Keratin/Glycol Chitosan Methacrylate Bioinks for 3D Bioprinting.

With the advancements in tissue engineering and three-dimensional (3D) bioprinting, physiologically relevant three-dimensional structures with suitable mechanical and bioactive properties that mimic the biological tissue can be designed and fabricated. However, the available bioinks are less than demanded. In this research, the readily available biomass sources, keratin and glycol chitosan, were selected to develop a UV-curable hydrogel that is feasible for the 3D bioprinting process. Keratin methacrylate and glycol chitosan methacrylate were synthesized, and a hybrid bioink was created by combining this protein-polysaccharide cross-linked hydrogel. While human hair keratin could provide biological functions, the other composition, glycol chitosan, could further enhance the mechanical strength of the construct. The mechanical properties, degradation profile, swelling behavior, cell viability, and proliferation were investigated with various ratios of keratin methacrylate to glycol chitosan methacrylate. The composition of 2% (w/v) keratin methacrylate and 2% (w/v) chitosan methacrylate showed a significantly higher cell number and swelling percentage than other compositions and was designated as the bioink for 3D printing afterward. The feasibility of stem cell loading in the selected formula was examined with an extrusion-based bioprinter. The cells and spheroids can be successfully printed with the synthesized bioink into a specific shape and cultured. This work provides a potential option for bioinks and delivers insights into personalization research on stem cell-laden biofabricated hydrogels in the future.

Cerebrospinal fluid metabolic profiling reveals divergent modulation of pentose phosphate pathway by midazolam, propofol and dexmedetomidine in patients with subarachnoid hemorrhage: a cohort study.

BACKGROUND: Agitation is common in subarachnoid hemorrhage (SAH), and sedation with midazolam, propofol and dexmedetomidine is essential in agitation management. Previous research shows the tendency of dexmedetomidine and propofol in improving long-term outcome of SAH patients, whereas midazolam might be detrimental. Brain metabolism derangement after SAH might be interfered by sedatives. However, how sedatives work and whether the drugs interfere with patient outcome by altering cerebral metabolism is unclear, and the comprehensive view of how sedatives regulate brain metabolism remains to be elucidated. METHODS: For cerebrospinal fluid (CSF) and extracellular space of the brain exchange instantly, we performed a cohort study, applying CSF of SAH patients utilizing different sedatives or no sedation to metabolomics. Baseline CSF metabolome was corrected by selecting patients of the same SAH and agitation severity. CSF components were analyzed to identify the most affected metabolic pathways and sensitive biomarkers of each sedative. Markers might represent the outcome of the patients were also investigated. RESULTS: Pentose phosphate pathway was the most significantly interfered (upregulated) pathway in midazolam (p = 0.0000107, impact = 0.35348) and propofol (p = 0.00000000000746, impact = 0.41604) groups. On the contrary, dexmedetomidine decreased levels of sedoheptulose 7-phosphate (p = 0.002) and NADP (p = 0.024), and NADP is the key metabolite and regulator in pentose phosphate pathway. Midazolam additionally augmented purine synthesis (p = 0.00175, impact = 0.13481) and propofol enhanced pyrimidine synthesis (p = 0.000203, impact = 0.20046), whereas dexmedetomidine weakened pyrimidine synthesis (p = 0.000000000594, impact = 0.24922). Reduced guanosine diphosphate (AUC of ROC 0.857, 95%CI 0.617-1, p = 0.00506) was the significant CSF biomarker for midazolam, and uridine diphosphate glucose (AUC of ROC 0.877, 95%CI 0.631-1, p = 0.00980) for propofol, and succinyl-CoA (AUC of ROC 0.923, 95%CI 0.785-1, p = 0.000810) plus adenosine triphosphate (AUC of ROC 0.908, 95%CI 0.6921, p = 0.00315) for dexmedetomidine. Down-regulated CSF succinyl-CoA was also associated with favorable outcome (AUC of ROC 0.708, 95% CI: 0.524-0.865, p = 0.029333). CONCLUSION: Pentose phosphate pathway was a crucial target for sedatives which alter brain metabolism. Midazolam and propofol enhanced the pentose phosphate pathway and nucleotide synthesis in poor-grade SAH patients, as presented in the CSF. The situation of dexmedetomidine was the opposite. The divergent modulation of cerebral metabolism might further explain sedative pharmacology and how sedatives affect the outcome of SAH patients.

Synergic Effect of Early Administration of Probiotics and Adipose-Derived Mesenchymal Stem Cells on Alleviating Inflammation-Induced Chronic Neuropathic Pain in Rodents.

This study investigated the hypothesis that probiotics enhanced the therapeutic effect of adipose-derived mesenchymal stem cells (ADMSCs) on alleviating neuropathic pain (NP) due to chronic constriction injury (CCI) mainly through regulating the microbiota in rats. SD rats (n = 50) were categorized into group 1 (sham-control), group 2 (NP), group 3 (NP + probiotics (i.e., 1.5 billion C.F.U./day/rat, orally 3 h after NP procedure, followed by QOD 30 times)), group 4 (NP + ADMSCs (3.0 × 105 cells) 3 h after CCI procedure, followed by QOD six times (i.e., seven times in total, i.e., mimic a clinical setting of drug use) and group 5 (NP + probiotics + ADMSCs (3.0 × 105 cells)) and euthanized by day 60 after NP induction. By day 28 after NP induction, flow-cytometric analysis showed circulating levels of early (AN-V+/PI−) and late (AN-V+/PI+) apoptotic, and three inflammatory (CD11b-c+, Ly6G+ and MPO+) cells were lowest in group 1 and significantly progressively reduced in groups 2 to 5 (all p < 0.0001). By days 7, 14, 21, 28, and 60 after CCI, the thresholds of thermal paw withdrawal latency (PWL) and mechanical paw withdrawal threshold (PWT) were highest in group 1 and significantly progressively increased in groups 2 to 5 (all p < 0.0001). Numbers of pain-connived cells (Nav1.8+/peripherin+, p-ERK+/peripherin+, p-p38+/peripherin+ and p-p38+/NF200+) and protein expressions of inflammatory (p-NF-κB, IL-1ß, TNF-α and MMP-9), apoptotic (cleaved-caspase-3, cleaved-PARP), oxidative-stress (NOX-1, NOX-2), DNA-damaged (γ-H2AX) and MAPK-family (p-P38, p-JNK, p-ERK1/2) biomarkers as well as the protein levels of Nav.1.3, Nav.1.8, and Nav.1.9 in L4-L5 in dorsal root ganglia displayed an opposite pattern of mechanical PWT among the groups (all p < 0.0001). In conclusion, combined probiotic and ADMSC therapy was superior to merely one for alleviating CCI-induced NP mainly through suppressing inflammation and oxidative stress.

Knockout of KLF10 Ameliorated Diabetic Renal Fibrosis via Downregulation of DKK-1.

Diabetes-induced chronic kidney disease leads to mortality and morbidity and thus poses a great health burden worldwide. Krüppel-like factor 10 (KLF10), a zinc finger-containing transcription factor, regulates numerous cellular functions, such as proliferation, differentiation, and apoptosis. In this study, we explored the effects of KLF10 on diabetes-induced renal disease by using a KLF10 knockout mice model. Knockout of KLF10 obviously diminished diabetes-induced tumor growth factor-ß (TGF-ß), fibronectin, and type IV collagen expression, as evidenced by immunohistochemical staining. KLF10 knockout also repressed the expression of Dickkopf-1 (DKK-1) and phosphorylated ß-catenin in diabetic mice, as evidenced by immunohistochemical staining and Western blot analysis. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that significantly decreased type IV collagen, fibronectin, and DKK-1 existed in KLF10 knockout diabetic mice compared with control diabetic mice. Moreover, knockout of KLF10 reduced the renal fibrosis, as shown by Masson's Trichrome analysis. Overall, the results indicate that depletion of KLF10 ameliorated diabetic renal fibrosis via the downregulation of DKK-1 expression and inhibited TGF-ß1 and phosphorylated ß-catenin expression. Our findings suggest that KLF10 may be a promising therapeutic choice for the treatment of diabetes-induced renal fibrosis.

Overexpression of miR-19a and miR-20a in iPS-MSCs preserves renal function of chronic kidney disease with acute ischaemia-reperfusion injury in rat.

This study tested the hypothesis that therapy with double overexpression of miR-19a-3p and miR-20a-5p (miRDOE ) to human inducible pluripotent stem cell-derived mesenchymal stem cells (iPS-MSCs) was superior to iPS-MSCs alone for preserving renal function in rat with pre-existing chronic kidney disease (CKD), followed by ischaemia-reperfusion (IR) injury. In vitro study demonstrated that the protein expressions of oxidative stress (NOX-1/NOX-2/NOX4/oxidized protein/p22phox), inflammatory downstream signalling (TLR2&4/MyD88/TRAF6/IKK-ß/p-NFκB/IL-1ß/IL-6/MMP-9) and cell apoptosis/death signalling (cleaved caspase-3/mitochondrial Bax/p-ERKs/p-JNK/p-p38) at time-points of 24-hour/48-hour cell cultures were significantly increased in p-Cresol-treated NRK-52E cells than in the control that was significantly reversed by miR-19a-3p-transfected iPS-MSC (all P < .001). Animals were categorized into group 1 (sham-operated control), group 2 (CKD-IR), group 3 (CKD-IR + oligo-miRDOE of iPS-MSCs/6.0 ×105 /intra-renal artery transfusion/3 hours after IR procedure), group 4 (CKD-IR + iPS-MSCs) and group 5 (CKD-IR + miRDOE of iPS-MSCs/6.0 ×105/ intra-renal artery transfusion/3 hour after IR procedure). By day 35, the creatinine/BUN levels were lowest in group 1, highest in group 2 and significantly lower in group 5 than in groups 3 and 4 (all P < .0001) but they showed no difference between the latter two groups. The protein expressions of oxidative stress, inflammatory downstream signalling and cell apoptosis/death signalling exhibited an identical pattern of creatinine level among the five groups (all P < .00001). Also, the microscopic findings demonstrated that the kidney injury score/fibrotic area/number of inflammatory cells (CD14+/CD68+) exhibited an identical pattern of creatine level (all P < .0001). The miRDOE of iPS-MSCs was superior to iPS-MSCs for preserving the residual kidney function and architecture in CKD-IR rat.

Targeting HR Repair as a Synthetic Lethal Approach to Increase DNA Damage Sensitivity by a RAD52 Inhibitor in BRCA2-Deficient Cancer Cells.

BRCA mutation, one of the most common types of mutations in breast and ovarian cancer, has been suggested to be synthetically lethal with depletion of RAD52. Pharmacologically inhibiting RAD52 specifically eradicates BRCA-deficient cancer cells. In this study, we demonstrated that curcumin, a plant polyphenol, sensitizes BRCA2-deficient cells to CPT-11 by impairing RAD52 recombinase in MCF7 cells. More specifically, in MCF7-siBRCA2 cells, curcumin reduced homologous recombination, resulting in tumor growth suppression. Furthermore, a BRCA2-deficient cell line, Capan1, became resistant to CPT-11 when BRCA2 was reintroduced. In vivo, xenograft model studies showed that curcumin combined with CPT-11 reduced the growth of BRCA2-knockout MCF7 tumors but not MCF7 tumors. In conclusion, our data indicate that curcumin, which has RAD52 inhibitor activity, is a promising candidate for sensitizing BRCA2-deficient cells to DNA damage-based cancer therapies.

Human Umbilical Cord-Derived Mesenchymal Stem Cells for Acute Respiratory Distress Syndrome.

OBJECTIVES: To investigate the safety, feasibility, and possible adverse events of single-dose human umbilical cord-derived mesenchymal stem cells in patients with moderate-to-severe acute respiratory distress syndrome. DESIGN: Prospective phase I clinical trial. SETTING: Medical center in Kaohsiung, Taiwan. PATIENTS: Moderate-to-severe acute respiratory distress syndrome with a PaO2/FIO2 ratio less than 200. INTERVENTIONS: Scaling for doses was required by Taiwan Food and Drug Administration as follows: the first three patients received low-dose human umbilical cord-derived mesenchymal stem cells (1.0 × 10 cells/kg), the next three patients with intermediate dose (5.0 × 10 cells/kg), and the final three patients with high dose (1.0 × 10 cells/kg) between December 2017 and August 2019. MEASUREMENTS AND MAIN RESULTS: Nine consecutive patients were enrolled into the study. In-hospital mortality was 33.3% (3/9), including two with recurrent septic shock and one with ventilator-induced severe pneumomediastinum and subcutaneous emphysema. No serious prespecified cell infusion-associated or treatment-related adverse events was identified in any patient. Serial flow-cytometric analyses of circulating inflammatory biomarkers (CD14CD33/CD11b+CD16+/CD16+MPO+/CD11b+MPO+/CD14CD33+) and mesenchymal stem cell markers (CD26+CD45-/CD29+CD45-/CD34+CD45-/CD44+CD45-/CD73+CD45-/CD90+CD45-/CD105+CD45-/CD26+CD45-) were notably progressively reduced (p for trend < 0.001), whereas the immune cell markers (Helper-T-cell/Cytotoxity-T-cell/Regulatory-T-cell) were notably increased (p for trend < 0.001) after cell infusion. CONCLUSIONS: The result of this phase I clinical trial showed that a single-dose IV infusion of human umbilical cord-derived mesenchymal stem cells was safe with favorable outcome in nine acute respiratory distress syndrome patients.

A self-assembled layer-by-layer surface modification to fabricate the neuron-rich model from neural stem/precursor cells.

BACKGROUND/PURPOSE: In vitro neural cell-based models have been widely used to mimic the in vivo neural tissue environments and quantitatively understand the effects of pharmaceutical molecules on neural diseases. Recently, several biomimetic neural tissue models have been widely developed by using biomaterials or surface modification. However, the complex protocols of material synthesis or surface modification lack an easy execution to fabricate the neuron favorite environment. METHODS: In this study, we utilized a layer-by-layer technique as a surface modification method for regulating the behaviors of neural stem/precursor cells (NSPCs) on material surfaces. Polyelectrolyte multilayers (PEMs) via alternate deposition of poly (allylamine hydrochloride) (PAH) and poly (sodium-4-styrenesulfonate) (PSS) were used to culture NSPCs. After incubation for 7 days, the neuronal differentiation of NSPCs and synapse function of differentiated neurons were identified by immunocytochemistry for lineage specific markers. RESULTS: Compared with the only PAH film, the PSS-ending film (neuron-rich model) was shown to significantly promote differentiation of NSPCs into neurons (more than 50%), form a neuronal network structure; and differentiated neurons exhibiting functional synaptic activity. CONCLUSION: This study shows that the PEMs provided an easily alternative approach to modify the surface properties; and might be a method to obtain a neuron-rich model for the biological/pharmaceutical applications.

Circulatory Rejuvenated EPCs Derived from PAOD Patients Treated by CD34+ Cells and Hyperbaric Oxygen Therapy Salvaged the Nude Mouse Limb against Critical Ischemia.

This study tested whether circulatory endothelial progenitor cells (EPCs) derived from peripheral arterial occlusive disease (PAOD) patients after receiving combined autologous CD34+ cell and hyperbaric oxygen (HBO) therapy (defined as rejuvenated EPCs) would salvage nude mouse limbs against critical limb ischemia (CLI). Adult-male nude mice (n = 40) were equally categorized into group 1 (sham-operated control), group 2 (CLI), group 3 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood prior to CD34+ cell and HBO treatment (EPCPr-T) by intramuscular injection at 3 h after CLI induction) and group 4 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood after CD34+ cell and HBO treatment (EPCAf-T) by the identical injection method). By 2, 7 and 14 days after the CLI procedure, the ischemic to normal blood flow (INBF) ratio was highest in group 1, lowest in group 2 and significantly lower in group 4 than in group 3 (p < 0.0001). The protein levels of endothelial functional integrity (CD31/von Willebrand factor (vWF)/endothelial nitric-oxide synthase (eNOS)) expressed a similar pattern to that of INBF. In contrast, apoptotic/mitochondrial-damaged (mitochondrial-Bax/caspase-3/PARP/cytosolic-cytochrome-C) biomarkers and fibrosis (Smad3/TGF-ß) exhibited an opposite pattern, whereas the protein expressions of anti-fibrosis (Smad1/5 and BMP-2) and mitochondrial integrity (mitochondrial-cytochrome-C) showed an identical pattern of INBF (all p < 0.0001). The protein expressions of angiogenesis biomarkers (VEGF/SDF-1α/HIF-1α) were progressively increased from groups 1 to 3 (all p < 0.0010). The number of small vessels and endothelial cell surface markers (CD31+/vWF+) in the CLI area displayed an identical pattern of INBF (all p < 0.0001). CLI automatic amputation was higher in group 2 than in other groups (all p < 0.001). In conclusion, EPCs from HBO-C34+ cell therapy significantly restored the blood flow and salvaged the CLI in nude mice.

Long-term Therapeutic Effects of Extracorporeal Shock Wave-Assisted Melatonin Therapy on Mononeuropathic Pain in Rats.

We evaluated the ability of extracorporeal shock wave (ECSW)-assisted melatonin (Mel) therapy to offer an additional benefit for alleviating the neuropathic pain (NP) in rats. Left sciatic nerve was subjected to chronic constriction injury (CCI) to induce NP. Animals (n = 30) were randomized into group 1 (sham-operated control), group 2 (CCI only), group 3 (CCI + ECSW), group 4 (CCI + Mel) and group 5 (CCI + ECSW + Mel). By days 15, 22 and 29 after CCI, the thermal paw withdrawal latency (TPWL) and mechanical paw withdrawal threshold (MPWT) were highest in group 1, lowest in group 2, significantly higher in group 5 than in groups 3 and 4, but they showed no difference between the later two groups (all p < 0.0001). The protein expressions of inflammatory (TNF-α, NF-κB, MMP-9, IL-1ß), oxidative-stress (NOXs-1, -2, -4, oxidized protein), apoptotic (cleaved-caspase3, cleaved-PARP), DNA/mitochondrial-damaged (γ-H2AX/cytosolic-cytochrome C), microglia/astrocyte activation (ox42/GFAP), and MAPKs [phosphorylated (p)-p38, p-JNK, p-ERK] biomarkers in dorsal root ganglia neurons (DRGs) and in spinal dorsal horn were exhibited an opposite pattern of TPWL among the five groups (all p < 0.0001). Additionally, protein expressions of Nav.1.3, Nav.1.8 and Nav.1.9 in sciatic nerve exhibited an identical pattern to inflammation among the five groups (all p < 0.0001). The numbers of cellular expressions of MAPKs (p-ERK1/2+/peripherin + cells, p-ERK1/2+/NF200 + cells and p-JNK+/peripherin + cells, p-JNK+/NF200 + cells) and voltage-gated sodium channels (Nav.1.8+/peripherin + cells, Nav.1.8+/NF200 + cells, Nav.1.9+/peripherin + cells, Nav.1.9+/NF200 + cells) in small and large DRGs displayed an identical pattern to inflammation among the five groups (all p < 0.0001). In conclusion, the synergistic effect of combined ECSW-Mel therapy is superior to either one alone for long-term improvement of mononeuropathic pain-induced by CCI in rats.

The Five-Year Clinical and Angiographic Follow-Up Outcomes of Intracoronary Transfusion of Circulation-Derived CD34+ Cells for Patients With End-Stage Diffuse Coronary Artery Disease Unsuitable for Coronary Intervention-Phase I Clinical Trial.

OBJECTIVES: This study investigated the clinical and angiographic long-term outcomes of intracoronary transfusion of circulation-derived CD34+ cells for patients with end-stage diffuse coronary artery disease unsuitable for coronary intervention. DESIGN AND SETTING: A single-center prospective randomized double-blinded phase I clinical trial. Thirty-eight patients undergoing CD34+ cell therapy were allocated into groups 1 (1.0 × 10 cells/each vessel; n = 18) and 2 (3.0 × 10 cells/each vessel; n = 20). PATIENTS: Those with end-stage diffuse coronary artery disease were unsuitable for percutaneous and surgical coronary revascularization. INTERVENTIONS: Intracoronary delivery of circulation-derived CD34+ cells. MEASUREMENTS AND MAIN RESULTS: We prospectively evaluated long-term clinical and echocardiographic/angiographic outcomes between survivors and nonsurvivors. By the end of 5-year follow-up, the survival rate and major adverse cardio/cerebrovascular event were 78.9% (30/38) and 36.8% (14/38), respectively. During follow-up period, 31.6% patients (12/38) received coronary stenting for reason of sufficient target vessel size grown-up after the treatment. Endothelial function was significantly reduced in the nonsurvivors than the survivors (p = 0.039). Wimasis image analysis of angiographic findings showed that the angiogenesis was significantly and progressively increased from baseline to 1 and 5 years (all p < 0.001). The 3D echocardiography showed left ventricular ejection fraction increased from baseline to 1 year and then remained stable up to 5 years, whereas left ventricular chamber diameter exhibited an opposite pattern to left ventricular ejection fraction among the survivors. The clinical scores for angina and heart failure were significantly progressively reduced from baseline to 1 and 5 years (all p < 0.001). CONCLUSIONS: CD34+ cell therapy for end-stage diffuse coronary artery disease patients might contribute to persistently long-term effects on improvement of left ventricular function, angina/heart failure, and amelioration of left ventricular remodeling.

Daily melatonin protects the endothelial lineage and functional integrity against the aging process, oxidative stress, and toxic environment and restores blood flow in critical limb ischemia area in mice.

We tested the hypothesis that daily melatonin treatment protects endothelial lineage and functional integrity against the aging process, oxidative stress/endothelial denudation (ED), and toxic environment and restored blood flow in murine critical limb ischemia (CLI). In vitro study using HUVECs, in vivo models (ie, CLI through left femoral artery ligation and ED through carotid artery wire injury), and model of lipopolysaccharide-induced aortic injury in young (3 months old) and aged (8 months old) mice were used to elucidate effects of melatonin treatment on vascular endothelial integrity. In vitro study showed that menadione-induced oxidative stress (NOX-1/NOX-2), inflammation (TNF-α/NF-kB), apoptosis (cleaved caspase-3/PARP), and mitochondrial damage (cytosolic cytochrome c) in HUVECs were suppressed by melatonin but reversed by SIRT3-siRNA (all P < .001). In vivo, reduced numbers of circulating endothelial progenitor cells (EPCs) (C-kit/CD31+/Sca-1/KDR+/CXCR4/CD34+), and angiogenesis (Matrigel assay of bone marrow-derived EPC and ex vivo aortic ring cultures) in older (compared with younger) mice were significantly reversed through daily melatonin administration (20 mg/kg/d, ip) (all P < .001). Aortic vasorelaxation and nitric oxide release were impaired in older mice and reversed in age-match mice receiving melatonin (all P < .01). ED-induced intimal/medial hyperplasia, reduced blood flow to ischemic limb, and angiogenesis (reduced CD31+/vWF+ cells/small vessel number) were improved after daily melatonin treatment (all P < .0001). Lipopolysaccharide-induced aortic endothelial cell detachment, which was more severe in aged mice, was also alleviated after daily melatonin treatment (P < .0001). Daily melatonin treatment protected both structural and functional integrity of vascular endothelium against aging-, oxidative stress-, lipopolysaccharide-, and ischemia-induced damage probably through upregulating the SIRT signaling pathway.

Shock Wave Therapy Enhances Mitochondrial Delivery into Target Cells and Protects against Acute Respiratory Distress Syndrome.

This study tested the hypothesis that shock wave therapy (SW) enhances mitochondrial uptake into the lung epithelial and parenchymal cells to attenuate lung injury from acute respiratory distress syndrome (ARDS). ARDS was induced in rats through continuous inhalation of 100% oxygen for 48 h, while SW entailed application 0.15 mJ/mm2 for 200 impulses at 6 Hz per left/right lung field. In vitro and ex vivo studies showed that SW enhances mitochondrial uptake into lung epithelial and parenchyma cells (all p < 0.001). Flow cytometry demonstrated that albumin levels and numbers of inflammatory cells (Ly6G+/CD14+/CD68+/CD11b/c+) in bronchoalveolar lavage fluid were the highest in untreated ARDS, were progressively reduced across SW, Mito, and SW + Mito (all p < 0.0001), and were the lowest in sham controls. The same profile was also seen for fibrosis/collagen deposition, levels of biomarkers of oxidative stress (NOX-1/NOX-2/oxidized protein), inflammation (MMP-9/TNF-α/NF-κB/IL-1ß/ICAM-1), apoptosis (cleaved caspase 3/PARP), fibrosis (Smad3/TGF-ß), mitochondrial damage (cytosolic cytochrome c) (all p < 0.0001), and DNA damage (γ-H2AX+), and numbers of parenchymal inflammatory cells (CD11+/CD14+/CD40L+/F4/80+) (p < 0.0001). These results suggest that SW-assisted Mito therapy effectively protects the lung parenchyma from ARDS-induced injury.

Endophilin B is required for the Drosophila oocyte to endocytose yolk downstream of Oskar.

The nutritional environment is crucial for Drosophila oogenesis in terms of controlling hormonal conditions that regulate yolk production and the progress of vitellogenesis. Here, we discovered that Drosophila Endophilin B (D-EndoB), a member of the endophilin family, is required for yolk endocytosis as it regulates membrane dynamics in developing egg chambers. Loss of D-EndoB leads to yolk content reduction, similar to that seen in yolkless mutants, and also causes poor fecundity. In addition, mutant egg chambers exhibit an arrest at the previtellogenic stage. D-EndoB displayed a crescent localization at the oocyte posterior pole in an Oskar-dependent manner; however, it did not contribute to pole plasm assembly. D-EndoB was found to partially colocalize with Long Oskar and Yolkless at the endocytic membranes in ultrastructure analysis. Using an FM4-64 dye incorporation assay, D-EndoB was also found to promote endocytosis in the oocyte. When expressing the full-length D-endoB(FL) or D-endoB(ΔSH3) mutant transgenes in oocytes, the blockage of vitellogenesis and the defect in fecundity in D-endoB mutants was restored. By contrast, a truncated N-BAR domain of the D-EndoB only partially rescued these defects. Taken together, these results allow us to conclude that D-EndoB contributes to the endocytic activity downstream of Oskar by facilitating membrane dynamics through its N-BAR domain in the yolk uptake process, thereby leading to normal progression of vitellogenesis.

Gold Nanocomposite Bioink for Printing 3D Cardiac Constructs.

Bioprinting is the most convenient microfabrication method to create biomimetic three-dimensional (3D) cardiac tissue constructs, which can be used to regenerate damaged tissue and provide platforms for drug screening. However, existing bioinks, which are usually composed of polymeric biomaterials, are poorly conductive and delay efficient electrical coupling between adjacent cardiac cells. To solve this problem, we developed a gold nanorod (GNR) incorporated gelatin methacryloyl (GelMA)-based bioink for printing 3D functional cardiac tissue constructs. The GNR concentration was adjusted to create a proper microenvironment for the spreading and organization of cardiac cells. At optimized concentration of GNR, the nanocomposite bioink had a low viscosity, similar to pristine inks, which allowed for the easy integration of cells at high densities. As a result, rapid deposition of cell-laden fibers at a high resolution was possible, while reducing shear stress on the encapsulated cells. In the printed GNR constructs, cardiac cells showed improved cell adhesion and organization when compared to the constructs without GNRs. Furthermore, the incorporated GNRs bridged the electrically resistant pore walls of polymers, improved the cell-to-cell coupling, and promoted synchronized contraction of the bioprinted constructs. Given its advantageous properties, this gold nanocomposite bioink may find wide application in cardiac tissue engineering.

Proteomics Analysis Reveals Involvement of Krt17 in Areca Nut-Induced Oral Carcinogenesis.

The areca nut is a known carcinogen that causes oral cancer in individuals in Southeast Asia, but the molecular mechanism that leads to this malignancy is still unclear. To mimic the habit of areca nut chewing, our laboratory has established four oral cancer cell sublines (SAS, OECM1, K2, C9), which have been chronically exposed to areca nut extract (ANE). To elucidate the molecular basis of areca nut-induced oral carcinogenesis, the differential proteomes between oral cancer cells and the ANE-treated sublines were determined using isobaric mass tag (iTRAQ) labeling and multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). Over 1000 proteins were identified in four sublines, and 196 proteins were found to be differentially expressed in at least two ANE-treated sublines. A bioinformatic analysis revealed that these proteins participate in several pathways, and one of the most prominent pathways was the regulation of epithelial to mesenchymal transition (EMT). In all, 24 proteins including Krt17 were confirmed to be differentially expressed in the ANE-treated sublines. To reveal additional information on the mechanism of ANE-induced carcinogenesis, Krt17 was further investigated. Krt17 knockdown significantly suppressed ANE-induced cell migration and invasion and modulated the EMT process. Furthermore, in a murine model of carcinogen-induced (arecoline cocktail, an active compound of ANE) oral cancer, Krt17 was significantly up-regulated in all hyperplastic tissues and in carcinoma tissues (p < 0.001). In conclusion, we have identified a proteome of oral cancer cells that is associated with chronic areca nut exposure. Krt17 was demonstrated to contribute to areca nut-induced oral malignancy. The results of this study contribute to risk assessment, disease prevention and other clinical applications associated with areca nut-induced oral cancer.