3,5-bis(styryl)pyrazole inhibits mitosis and induces cell death independent of BubR1 and p53 levels by depolymerizing microtubules.

Here, we show that 3,5-bis[(1E)-2-(2,6-dichlorophenyl)ethenyl]-1H-pyrazole 2l depolymerizes microtubules and reduces the number of growing tips of microtubules. The fluorescence recovery after photobleaching experiment in live MCF-7 cells showed that pyrazole 2l suppresses spindle microtubule dynamics. Further, the compound inhibits chromosome movements, activates the spindle assembly checkpoint and blocks mitosis in MCF-7 cells. Pyrazole 2l treatment induced cell death in a variety of pathways. Pyrazole 2l induces cell death independent of BubR1 and p53 levels of MCF-7 cells upon microtubule depolymerization. Further, pyrazole 2l increases the interaction between NF-κB and microtubules and enhances the nuclear localization of NF-κB at its half-maximal proliferation inhibitory concentration while a high concentration of the compound reduced the nuclear localization of NF-κB. Interestingly, the compound exerted significantly stronger antiproliferative effects in cancerous cells than in non-cancerous cells. The results indicated that pyrazole 2l inhibits mitosis by targeting microtubules, induces several types of cell death stimuli and suggests its potential as a lead in developing anticancer agent.

A subfraction obtained from the venom of the tarantula Poecilotheria regalis contains inhibitor cystine knot peptides and induces relaxation of rat aorta by inhibiting L-type voltage-gated calcium channels.

Venoms from tarantulas contain low molecular weight vasodilatory compounds whose biological action is conceived as part of the envenomation strategy due to its propagative effects. However, some properties of venom-induced vasodilation do not match those described by such compounds, suggesting that other toxins may cooperate with these ones to produce the observed biological effect. Owing to the distribution and function of voltage-gated ion channels in blood vessels, disulfide-rich peptides isolated from venoms of tarantulas could be conceived into potential vasodilatory compounds. However, only two peptides isolated from spider venoms have been investigated so far. This study describes for the first time a subfraction containing inhibitor cystine knot peptides, PrFr-I, obtained from the venom of the tarantula Poecilotheria regalis. This subfraction induced sustained vasodilation in rat aortic rings independent of vascular endothelium and endothelial ion channels. Furthermore, PrFr-I decreased calcium-induced contraction of rat aortic segments and reduced extracellular calcium influx to chromaffin cells by the blockade of L-type voltage-gated calcium channels. This mechanism was unrelated to the activation of potassium channels from vascular smooth muscle, since vasodilation was not affected in the presence of TEA, and PrFr-I did not modify the conductance of the voltage-gated potassium channel Kv10.1. This work proposes a new envenomating function of peptides from venoms of tarantulas, and establishes a new mechanism for venom-induced vasodilation.

Water-soluble protein from walleye pollock (Gadus chalcogrammus) suppresses lipopolysaccharide-induced inflammation by attenuating TLR4-MyD88 expression in macrophages.

Water-soluble protein (WSP) from fish meat is abundant in the waste effluent generated via the surimi manufacturing process. This study investigated the anti-inflammatory effects and mechanisms of fish WSP using primary macrophages (MΦ) and animal ingestion. MΦ were treated with digested-WSP (d-WSP, 500 µg/mL) with or without lipopolysaccharide (LPS) stimulation. For the ingestion study, male ICR mice (5 weeks old) were fed 4% WSP for 14 days following LPS administration (4 mg/kg body weight). d-WSP decreased the expression of Tlr4, an LPS receptor. Additionally, d-WSP significantly suppressed the secretion of inflammatory cytokines, phagocytic ability, and Myd88 and Il1b expressions of LPS-stimulated macrophages. Furthermore, the ingestion of 4% WSP attenuated not only LPS-induced IL-1ß secretion in the blood but also Myd88 and Il1b expressions in the liver. Thus, fish WSP decreases the expressions of the genes involved in the TLR4-MyD88 pathway in MΦ and the liver, thereby suppressing inflammation.

Voluntary running exercise modifies astrocytic population and features in the peri-infarct cortex.

Rehabilitative exercise following a brain stroke has beneficial effects on the morphological plasticity of neurons. Particularly, voluntary running exercise after focal cerebral ischemia promotes functional recovery and ameliorates ischemia-induced dendritic spine loss in the peri-infarct motor cortex layer 5. Moreover, neuronal morphology is affected by changes in the perineuronal environment. Glial cells, whose phenotypes may be altered by exercise, are known to play a pivotal role in the formation of this perineuronal environment. Herein, we investigated the effects of voluntary running exercise on glial cells after middle cerebral artery occlusion. Voluntary running exercise increased the population of glial fibrillary acidic protein-positive astrocytes born between post-operative days (POD) 0 and 3 on POD15 in the peri-infarct cortex. After exercise, transcriptomic analysis of post-ischemic astrocytes revealed 10 upregulated and 70 downregulated genes. Furthermore, gene ontology analysis showed that the 70 downregulated genes were significantly associated with neuronal morphology. In addition, exercise reduced the number of astrocytes expressing lipocalin 2, a regulator of dendritic spine density, on POD15. Our results suggest that exercise modifies the composition of astrocytic population and their phenotype.

Embelin attenuates lipopolysaccharide-induced acute kidney injury through the inhibition of M1 macrophage activation and NF-κB signaling in mice.

Septic acute kidney injury (AKI) is commonly associated with renal dysfunction and high mortality in patients. Owing to the rapid and violent occurrence of septic AKI with inflammation, there are no effective therapies to clinically treat it. Embelin, a natural product, has a potential regulatory role in immunocytes. However, the role and mechanism of embelin in septic AKI remains unknown. This study aimed to elucidate the role of embelin in macrophage regulation in lipopolysaccharide (LPS)-induced septic AKI. Embelin was intraperitoneally administered to mice after LPS injection. And bone marrow-derived macrophages (BMDMs) were subsequently isolated from the mice to explore the immunomodulatory role of embelin in macrophages. We found that embelin attenuated renal dysfunction and pathological renal damage in the LPS-induced sepsis mouse model. Molecular docking predicted that embelin could bind to phosphorylated NF-κB p65 at the ser536 site. Embelin inhibited the translocation of NF-κB p65 via phosphorylation at ser536 in LPS-induced AKI. It also reduced the secretion of IL-1ß and IL-6 and increased the secretion of IL-10 and Arg-1 of BMDMs and mice after LPS stimulation, indicating that embelin suppressed macrophage M1 activation in LPS-induced AKI. Therefore, embelin attenuated LPS-induced septic AKI by suppressing NF-κB p65 at ser536 in activated macrophages. This study preclinically suggests a therapeutic role of embelin in septic AKI.

Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver.

The deficiency of CITRIN, the liver mitochondrial aspartate-glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD+ ratio increases in hepatocytes from citrin(-/-) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD+ observed in these cells. Liver mitochondria from citrin (-/-) mice expressing liver specific transgenic aralar had a small (~ 4-6 nmoles x mg prot-1 x min-1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(-/-) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(-/-) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficiency.

Effect of disinfectants and manual wiping for processing the cell product changeover in a biosafety cabinet.

Introduction: The process of cell product changeover poses a high risk of cross-contamination. Hence, it is essential to minimize cross-contamination while processing cell products. Following its use, the surface of a biosafety cabinet is commonly disinfected by ethanol spray and manual wiping methods. However, the effectiveness of this protocol and the optimal disinfectant have not yet been evaluated. Here, we assessed the effect of various disinfectants and manual wiping methods on bacterial removal during cell processing. Methods: The hard surface carrier test was performed to evaluate the disinfectant efficacy of benzalkonium chloride with a corrosion inhibitor (BKC + I), ethanol (ETH), peracetic acid (PAA), and wiping against Bacillus subtilis endospores. Distilled water (DW) was used as the control. A pressure sensor was employed to investigate the differences in loading under dry and wet conditions. The pre-spray for wiping was monitored by eight operators using a paper that turns black when wet. Chemical properties, including residual floating proteins, and mechanical properties, such as viscosity and coefficient of friction, were examined. Results: In total, 2.02 ± 0.21-Log and 3.00 ± 0.46-Log reductions from 6-Log CFU of B. subtilis endospores were observed for BKC + I and PAA, respectively, following treatment for 5 min. Meanwhile, wiping resulted in a 0.70 ± 0.12-Log reduction under dry conditions. Under wet conditions, DW and BKC + I showed 3.20 ± 0.17-Log and 3.92 ± 0.46-Log reductions, whereas ETH caused a 1.59 ± 0.26-Log reduction. Analysis of the pressure sensor suggested that the force was not transmitted under dry conditions. Evaluation of the amount of spray by eight operators showed differences and bias in the spraying area. While ETH had the lowest ratio in the protein floating and collection assays, it exhibited the highest viscosity. BKC + I had the highest friction coefficient under 4.0-6.3 mm/s; however, that of BKC + I decreased and became similar to the friction coefficient of ETH under 39.8-63.1 mm/s. Conclusions: DW and BKC + I are effective for inducing a 3-Log reduction in bacterial abundance. Moreover, the combination of optimal wet conditions and disinfectants is essential for effective wiping in specific environments containing high-protein human sera and tissues. Given that some raw materials processed in cell products contain high protein levels, our findings suggest that a complete changeover of biosafety cabinets is necessary in terms of both cleaning and disinfection.

A "T.E.S.T." hydrogel bioadhesive assisted by corneal cross-linking for in situ sutureless corneal repair.

Corneal transplantation is an effective clinical treatment for corneal diseases, which, however, is limited by donor corneas. It is of great clinical value to develop bioadhesive corneal patches with functions of "Transparency" and "Epithelium & Stroma generation", as well as "Suturelessness" and "Toughness". To simultaneously meet the "T.E.S.T." requirements, a light-curable hydrogel is designed based on methacryloylated gelatin (GelMA), Pluronic F127 diacrylate (F127DA) & Aldehyded Pluronic F127 (AF127) co-assembled bi-functional micelles and collagen type I (COL I), combined with clinically applied corneal cross-linking (CXL) technology for repairing damaged cornea. The patch formed after 5 min of ultraviolet irradiation possesses transparent, highly tough, and strongly bio-adhesive performance. Multiple cross-linking makes the patch withstand deformation near 600% and exhibit a burst pressure larger than 400 mmHg, significantly higher than normal intraocular pressure (10-21 mmHg). Besides, the slower degradation than GelMA-F127DA&AF127 hydrogel without COL I makes hydrogel patch stable on stromal beds in vivo, supporting the regrowth of corneal epithelium and stroma. The hydrogel patch can replace deep corneal stromal defects and well bio-integrate into the corneal tissue in rabbit models within 4 weeks, showing great potential in surgeries for keratoconus and other corneal diseases by combining with CXL.

p15INK4B is an alternative marker of senescent tumor cells in colorectal cancer.

Senescent tumor cells are nonproliferating tumor cells which are closely related to cancer progression by secreting senescence-related molecules, called senescence-associated secreting phenotypes. Therefore, the presence of senescent tumor cells is considered a prognostic factor in various cancer types. Although senescence-associated ß-galactosidase staining is considered the best marker for detection of senescent tumor cells, it can only be performed in fresh-frozen tissues. p16INK4A, a cyclin-dependent inhibitor, has been used as an alternative marker to detect senescent tumor cells in formalin-fixed paraffin-embedded tissues. However, other reliable markers to detect senescent tumor cells is still lacking. In the present study, using public single-cell RNA-sequencing data, we found that p15INK4B, a cyclin-dependent kinase inhibitor, is a novel marker for detection of senescent tumor cells. Moreover, p15INK4B expression was positively correlated with that of p16INK4A in colorectal cancer tissues. In in vitro studies, mRNA expression of p15INK4B was increased together with that of p16INK4A in H2O2- and therapy-induced cancer senescence models. However, the mRNA level of p15INK4B did not increase in the oncogene-induced senescence model in primary colonic epithelial cells. In conclusion, p15INK4B is a potential alternative marker for detection of senescent tumor cells together with conventional markers in advanced stages of colorectal cancer.

Introduction of tenomodulin by gene transfection vectors for rat bone tissue regeneration.

Introduction: Periodontal ligament is regenerated in association with hard tissue regeneration. Tenomodulin (Tnmd) expression has been confirmed in periodontal ligament and it reportedly inhibits angiogenesis or is involved in collagen fibril maturation. The introduction of Tnmd by gene transfection in bone tissue regeneration therapy might inhibit topical hard tissue formation and induce the formation of dense fibrous tissue. Therefore, the effect of Tnmd introduction by gene transfection technique in vitro and in vivo was investigated in this study. Methods: Osteogenesis- and chondrogenesis-related gene expression levels in osteoblastic cells (MC3T3E1) and rat bone marrow derived cells were detected using qPCR three days after gene transfection with plasmid DNA (Tnmd) using non-viral gene transfection vectors: a calcium phosphate-based gene transfection vector (CaP(Tnmd)) or a cationic polymer-based reagent (JetPEI (Tnmd)). Next, an atelocollagen scaffold with or without CaP (Tnmd) or JetPEI (Tnmd) was implanted into a rat calvaria bone defect, and the remaining bone defect volume and the tissue reaction at 28 days after surgery were evaluated. Results: Runx 2 and SP7 mRNA was reduced by JetPEI (Tnmd) in both cells, but not in CaP(Tnmd). The volume of expressed Tnmd was at 9 ng/mL in both gene transfection vector. The remaining bone defect volume of JetPEI (Tnmd) was significantly bigger than that of the other groups and CaP (EGFP), and that of CaP (Tnmd) was significantly bigger than that of CaP (EGFP). Conclusions: Tnmd introduction treatment inhibits bone formation in artificial bone defect, however, the effect of that was dependent on non-viral gene transfection vector.

Modified method for differentiation of myeloid-derived suppressor cells in vitro enhances immunosuppressive ability via glutathione metabolism.

Myeloid-derived suppressor cells (MDSCs), which accumulate in tumor bearers, are known to suppress anti-tumor immunity and thus promote tumor progression. MDSCs are considered a major cause of resistance against immune checkpoint inhibitors in patients with cancer. Therefore, MDSCs are potential targets in cancer immunotherapy. In this study, we modified an in vitro method of MDSC differentiation. Upon stimulating bone marrow (BM) cells with granulocyte-macrophage colony-stimulating factor in vitro, we obtained both lymphocyte antigen 6G positive (Ly-6G+) and negative (Ly-6G-) MDSCs (collectively, hereafter referred to as conventional MDSCs), which were non-immunosuppressive and immunosuppressive, respectively. We then found that MDSCs differentiated from Ly-6G- BM (hereafter called 6G- BM-MDSC) suppressed T-cell proliferation more strongly than conventional MDSCs, whereas the cells differentiated from Ly-6G+ BM (hereafter called 6G+ BM-MDSC) were non-immunosuppressive. In line with this, conventional MDSCs or 6G- BM-MDSC, but not 6G+ BM-MDSC, promoted tumor progression in tumor-bearing mice. Moreover, we identified that activated glutathione metabolism was responsible for the enhanced immunosuppressive ability of 6G- BM-MDSC. Finally, we showed that Ly-6G+ cells in 6G- BM-MDSC, which exhibited weak immunosuppression, expressed higher levels of Cybb mRNA, an immunosuppressive gene of MDSCs, than 6G+ BM-MDSC. Together, these data suggest that the depletion of Ly-6G+ cells from the BM cells leads to differentiation of immunosuppressive Ly-6G+ MDSCs. In summary, we propose a better method for MDSC differentiation in vitro. Moreover, our findings contribute to the understanding of MDSC subpopulations and provide a basis for further research on MDSCs.

Reversing the imbalance in bone homeostasis via sustained release of SIRT-1 agonist to promote bone healing under osteoporotic condition.

The imbalance of bone homeostasis is the root cause of osteoporosis. However current therapeutic approaches mainly focus on either anabolic or catabolic pathways, which often fail to turn the imbalanced bone metabolism around. Herein we reported that a SIRT-1 agonist mediated molecular therapeutic strategy to reverse the imbalance in bone homeostasis by simultaneously regulating osteogenesis and osteoclastogenesis via locally sustained release of SRT2104 from mineral coated acellular matrix microparticles. Immobilization of SRT2104 on mineral coating (MAM/SRT) harnessing their electrostatic interactions resulted in sustained release of SIRT-1 agonist for over 30 days. MAM/SRT not only enhanced osteogenic differentiation and mineralization, but also attenuated the formation and function of excessive osteoclasts via integrating multiple vital upstream signals (ß-catenin, FoxOs, Runx2, NFATc1, etc.) in vitro. Osteoporosis animal model also validated that it accelerated osteoporotic bone healing and improved osseointegration of the surrounding bone. Overall, our work proposes a promising strategy to treat osteoporotic bone defects by reversing the imbalance in bone homeostasis using designated small molecule drug delivery systems.

Comparison of the yields and properties of dedifferentiated fat cells and mesenchymal stem cells derived from infrapatellar fat pads.

Introduction: Infrapatellar fat pad (IFP)-derived mesenchymal stem cells (MSCs) have high chondrogenic potential and are attractive cell sources for cartilage regeneration. During ceiling culture to acquire the characteristics of MSCs, mature adipocytes from fat tissue are known to undergo dedifferentiation, generating dedifferentiated fat (DFAT) cells. The purpose of the present study was to compare the yields and biological properties of IFP-derived MSCs and IFP-derived DFAT cells. Methods: IFPs were harvested from the knees of 8 osteoarthritis (OA) patients. DFAT cells were obtained using a ceiling culture of adipocytes isolated from the floating top layer of IFP digestion. MSCs were obtained by culturing precipitated stromal vascular fraction cells. We compared the P0 cell yields, surface antigen profile, colony formation ability, and multipotency of DFAT cells and MSCs. Results: The P0 cell yields per flask and the estimated total cell yields from 1 g of IFP were much greater for MSCs than for DFAT cells. Both MSCs and DFAT cells were positive for MSC markers. No obvious difference was observed in colony formation ability. In differentiation assays, DFAT cells produced greater amounts of lipid droplets, calcified tissue, and glycosaminoglycan than MSCs did. Adipogenic and chondrogenic gene expressions were upregulated in DFAT cells. Conclusions: IFP-derived DFAT cells showed higher adipogenic and chondrogenic potentials than IFP-derived MSCs, but they had a poor cell yield.

Toxicity of combined exposure to acrylamide and Staphylococcus aureus.

In the current study, the aim was to examine the toxicity of combined exposure to acrylamide and Staphylococcus aureus. We investigated the effect of staphylococcal enterotoxin A (SEA), a toxin produced by Staphylococcus aureus, on the oxidation induced DNA damaging potency of acrylamide in mouse spleen cells using an Formamidopyrimidine-DNA glycosylase-modified (FPG)-modified comet assay. Parameters like tail moment, tail length, and % tail DNA as indicators of DNA damage were significantly increased in the combined acrylamide and SEA treatment compared with SEA or acrylamide alone. Further, we examined the effects of acrylamide and its epoxide metabolite glycidamide on overall production of SEA, SEA mRNA gene expression, and on the formation of biofilm of S. aureus. Acrylamide significantly increased the SEA expression level and SEA production in S. aureus. Acrylamide also significantly increased biofilm formation in S. aureus without affecting its growth rate. Moreover, the addition of acrylamide significantly increased the expression of S. aureus virulence factors RNAIII and icaA in fetal bovine serum. Our results showed that combined exposure to acrylamide and S. aureus or its toxin enhanced their chemical and biological toxicities.

Acute oral toxicity of damnacanthal and its anticancer activity against colorectal tumorigenesis.

Damnacanthal is an anthraquinone, extracted, and purified from the root of Morinda citrifolia in Thailand. This study aimed to measure acute oral toxicity and to investigate the anticancer activity of damnacanthal in colorectal tumorigenesis. We found that the growth of human colorectal cancer cells was inhibited by damnacanthal in a dose- and a time-dependent manner. The growth inhibitory effect of damnacanthal was better than that of 5-FU used as a positive control in colorectal cancer cells, along with the downregulation of cell cycle protein cyclin D1. Similarly, an oral treatment of damnacanthal effectively inhibited the growth of colorectal tumor xenografts in nude mice, which was approximately 2-3-fold higher as compared to 5-FU by tumor size as well as expression of bioluminescence. Furthermore, the study of acute oral toxicity in mice exhibited a relatively low toxicity of damnacanthal with a LD50 cut-off value of 2500 mg/kg according to OECD Guideline 423. These results reveal the potential therapeutic activity of a natural damnacanthal compound as an anti-colorectal cancer drug.

Bioactivity guided isolation and identification of phenolic compounds from Citrus aurantium L. with anti-colorectal cancer cells activity by UHPLC-Q-TOF/MS.

Natural plants are rich sources of various bioactive compounds. Consequently, the efficiently isolation of these bioactive components has always attracted considerable attention. Our work aims to demonstrate a framework for bioactivity guided isolation of potential effective compounds from the complex food materials. We demonstrated its application for isolation of phenolic compounds with anti-proliferative activity against colorectal cancer cells (CRCs) from Citrus aurantium L. Firstly, phenolic rich fraction was successfully identified as the main effective components that could simultaneously suppress the growth of CRCs and inhibit Wnt signaling. In order to obtain the bioactive phenolic constituents, a detailed study was performed by optimizing the purification conditions. Two phenolic rich fractions (40% and 60% ethanol elution fractions) were then obtained by AB-8 macroporous resins under optimized condition. Finally, the main components (65 compounds) were tentatively identified from the 40% ethanol eluant by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) analysis. Notably, there were five of the phytochemicals (Feruloylagmatine, Haploside C, Sagittatin A, Linderagalactone C and Koparin-2'-methyl ether) which were hitherto unidentified in Citrus aurantium L. fruit. In conclusion, this study showed that under the principle of bioactivity guided strategy, phenolic constituents with potential anti-CRCs activity were isolated from Citrus aurantium L.

Canine induced pluripotent stem cells efficiently differentiate into definitive endoderm in 3D cell culture conditions using high-dose activin A.

Introduction: Endoderm-derived organs support indispensable functions in the body. Pluripotent stem cells can generate endoderm-derived cells or tissues and have excellent therapeutic potential to replace the functions of endodermal tissues. However, there is no viable method to induce endodermal precursor cells, definitive endoderm (DE), from canine induced pluripotent stem cells (ciPSCs). Methods: A ciPSC line was used in this study. In order to induce DE, ciPSCs were cultured with high dose activin A and fetal bovine serum. We considered the optimal differentiation period and starting cell density. Next, to reduce the remaining undifferentiated cells and improve the DE induction efficiency, DE was induced from 3D cell aggregates with knockout serum replacement instead of fetal bovine serum. Finally, hepatic and pancreatic induction were performed to investigate whether DE could differentiate into downstream lineages. Results: After differentiation, some cells expressed the DE markers FOXA2 and SOX17. DE induction period and starting cell density were found to be important for efficient DE induction. However, some cells remained undifferentiated even after optimization of cell density and culture period. Cell differentiation under 3D culture conditions reduced undifferentiated cells and the replacement of fetal bovine serum with knockout serum replacement improved the DE induction efficiency. After hepatic and pancreatic induction, cells expressed some early hepatic and pancreatic markers. Conclusions: A ciPSC line was successfully differentiated to DE efficiently using a high dose of activin A with knockout serum replacement under 3D cell culture conditions. We believe that this study will be fundamental to achieving the generation of canine endodermal tissues from ciPSCs.

Bridging potential of Taurine-loading PCL conduits transplanted with hEnSCs on resected sciatic nerves.

Reconstruction of nerve conduits is a promising method for functional improvement in peripheral nerve repair. Besides choosing of a suitable polymer for conduit construction, adding factors such as Taurine improve a more advantageous microenvironment for defect nerve regeneration. Showing several major biological properties of Taurine, for example, regulation of the osmotic pressure, modulation of neurogenesis, and calcium hemostasis, makes it an appropriate option for repairing of defected nerves. To this, we examined repairing effects of Taurine-loading PCL conduits cultured with human endothelial stem cells (hEnSCs) on resected sciatic nerves. PCL/Taurine/Cell conduits transplanted to a 10-mm sciatic nerve gap. Forty-two wistar rats were randomly divided to seven groups: (1) Normal group, (2) Negative control (NC), (3) Positive control (nerve Autograft group), (4) PCL conduits group (PCL), (5) Taurine loaded PCL conduits group (PCL/Taurine), (6) hEnSCs cultured on the PCL conduits (PCL/Cell), (7) hEnSCs cultured on the PCL/Taurine conduits (PCL/Taurine/Cell). Functional recovery of motor and sensory nerves, the action potential of exciting muscle and motor distal latency has seen in PCL/Taurine/Cell conduits. Histological studies showed also remarkable nerve regeneration and obvious bridging has seen in this group. In conclusion, PCL/Taurine/Cell conduits showing suitable mechanical properties and biocompatibility may improve sciatic nerve regeneration.

Development of appropriate fatty acid formulations to raise the contractility of constructed myocardial tissues.

Introduction: Heart disease is a major cause of mortality worldwide, and the annual number of deaths due to heart disease has increased in recent years. Although heart failure is usually managed with medicines, the ultimate treatment for end-stage disease is heart transplantation or an artificial heart. However, the use of these surgical strategies is limited by issues such as thrombosis, rejection and donor shortages. Regenerative therapies, such as the transplantation of cultured cells and tissues constructed using tissue engineering techniques, are receiving great attention as possible alternative treatments for heart failure. Research is ongoing into the potential clinical use of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). However, the energy-producing capacity of cardiomyocytes maintained under previous culture conditions is lower than that of adult primary cardiomyocytes due to immaturity and a reliance on glucose metabolism. Therefore, the aims of this study were to compare the types of fatty acids metabolized between cardiomyocytes in culture and heart cells in vivo and investigate whether the addition of fatty acids to the culture medium affected energy production by cardiomyocytes. Methods: A fatty acid-containing medium was developed based on an analysis of fatty acid consumption by rat primary cardiomyocytes (rat-CMs), and the effects of this medium on adenosine triphosphate (ATP) production were investigated through bioluminescence imaging of luciferase-expressing rat-CMs. Next, the fatty acid content of the medium was further adjusted based on analyses of fatty acid utilization by porcine hearts and hiPSC-CMs. Oxygen consumption analyses were performed to explore whether the fatty acid-containing medium induced hiPSC-CMs to switch from anaerobic metabolism to aerobic metabolism. Furthermore, the effects of the medium on contractile force generated by hiPSC-CM-derived tissue were evaluated. Results: Rat serum, human serum and porcine plasma contained similar types of fatty acid (oleic acid, stearic acid, linoleic acid, palmitic acid and arachidonic acid). The types of fatty acid consumed were also similar between rat-CMs, hiPSC-CMs and porcine heart. The addition of fatty acids to the culture medium increased the bioluminescence of luciferase-expressing rat-CMs (an indirect measure of ATP level), oxygen consumption by hiPSC-CMs, and contractile force generated by cardiac tissues constructed from hiPSC-CMs. Conclusions: hiPSC-CMs metabolize similar types of fatty acid to those consumed by rat-CMs and porcine hearts. Furthermore, the addition of these fatty acids to the culture medium increased energy production by rat-CMs and hiPSC-CMs and enhanced the contractility of myocardial tissue generated from hiPSC-CMs. These findings suggest that the addition of fatty acids to the culture medium stimulates aerobic energy production by cardiomyocytes through ß-oxidation. Since cardiomyocytes cultured in standard media rely primarily on anaerobic glucose metabolism and remain in an immature state, further research is merited to establish whether the addition of fatty acids to the culture medium would improve the energy-producing capacity and maturity of hiPSC-CMs and cardiac tissue constructed from these cells. It is possible that optimizing the metabolism of cultured cardiomyocytes, which require high energy production to sustain their contractile function, will improve the properties of hiPSC-CM-derived tissue, allowing it to be better utilized for disease modeling, drug screening and regenerative therapies for heart failure.

Carbon dots conjugated to SN38 for improved colorectal anticancer therapy.

Irinotecan (CTP-11) is one of the standard therapies for colorectal cancer (CRC). CTP-11 is enzymatically converted to the hydrophobic 7-ethyl-10-hydroxycamptothecin (SN38), a one hundred-fold more active metabolite. Conjugation of hydrophobic anticancer drugs to nanomaterials is a strategy to improve their solubility, efficacy, and selectivity. Carbon dots (CDs) have garnered interest for their small sizes (<10 â€‹nm), low toxicity, high water solubility, and bright fluorescence. This paper describes the use of CDs to improve drug vehiculation, stability, and chemotherapeutic efficiency of SN38 through a direct intracellular uptake in CRC. The covalent conjugation of SN38 to CDs via a carbamate bond provides a CD-SN38 hybrid material for slow, sustained, and pH-responsive drug release. CD-SN38 successfully penetrates the CRC cells with a release in the nucleus affecting first the cell cycle and then the cytoskeleton. Moreover, CD-SN38 leads to a deregulation of the extracellular matrix (ECM), one of the major components of the cancer niche considered a possible target therapy for reducing the cancer progression. This work shows the combined therapeutic and imaging potential of CD-based hybrid materials for the treatment of CRC. Future efforts for targeted therapy of chronic diseases characterized by altered ECM deposition, such as chronic kidney disease and chronic allograft nephropathy in kidney transplant patients are envisaged.