Alantolactone Attenuates Renal Fibrosis via Inhibition of Transforming Growth Factor ß/Smad3 Signaling Pathway.

BACKGRUOUND: Renal fibrosis is characterized by the accumulation of extracellular matrix proteins and interstitial fibrosis. Alantolactone is known to exert anticancer, anti-inflammatory, antimicrobial and antifungal effects; however, its effects on renal fibrosis remains unknown. Here, we investigated whether alantolactone attenuates renal fibrosis in mice unilateral ureteral obstruction (UUO) and evaluated the effect of alantolactone on transforming growth factor (TGF) signaling pathway in renal cells. METHODS: To evaluate the therapeutic effect of alantolactone, cell counting kit-8 (CCK-8) assay, histological staining, Western blot analysis, and real-time quantitative polymerase chain reaction were performed in UUO kidneys in vivo and in TGF-ß-treated renal cells in vitro. RESULTS: Alantolactone (0.25 to 4 µM) did not affect the viability of renal cells. Mice orally administered 5 mg/kg of alantolactone daily for 15 days did not show mortality or liver toxicity. Alantolactone decreased UUO-induced blood urea nitrogen and serum creatinine levels. In addition, it significantly alleviated renal tubulointerstitial damage and fibrosis and decreased collagen type I, fibronectin, and α-smooth muscle actin (α-SMA) expression in UUO kidneys. In NRK-49F cells, alantolactone inhibited TGF-ßstimulated expression of fibronectin, collagen type I, plasminogen activator inhibitor-1 (PAI-1), and α-SMA. In HK-2 cells, alantolactone inhibited TGF-ß-stimulated expression of collagen type I and PAI-1. Alantolactone inhibited UUO-induced phosphorylation of Smad3 in UUO kidneys. In addition, it not only decreased TGF-ß secretion but also Smad3 phosphorylation and translocation to nucleus in both kidney cell lines. CONCLUSION: Alantolactone improves renal fibrosis by inhibiting the TGF-ß/Smad3 signaling pathway in obstructive nephropathy. Thus, alantolactone is a potential therapeutic agent for chronic kidney disease.

Endoplasmic reticulum stress in pancreatic ß cells induces incretin desensitization and ß-cell dysfunction via ATF4-mediated PDE4D expression.

Pancreatic ß-cell dysfunction and eventual loss are key steps in the progression of type 2 diabetes (T2D). Endoplasmic reticulum (ER) stress responses, especially those mediated by the protein kinase RNA-like ER kinase and activating transcription factor 4 (PERK-ATF4) pathway, have been implicated in promoting these ß-cell pathologies. However, the exact molecular events surrounding the role of the PERK-ATF4 pathway in ß-cell dysfunction remain unknown. Here, we report our discovery that ATF4 promotes the expression of PDE4D, which disrupts ß-cell function via a downregulation of cAMP signaling. We found that ß-cell-specific transgenic expression of ATF4 led to early ß-cell dysfunction and loss, a phenotype that resembles accelerated T2D. Expression of ATF4, rather than C/EBP homologous protein (CHOP), promoted PDE4D expression, reduced cAMP signaling, and attenuated responses to incretins and elevated glucose. Furthermore, we found that ß-cells of leptin receptor-deficient diabetic (db/db) mice had elevated nuclear localization of ATF4 and PDE4D expression, accompanied by impaired ß-cell function. Accordingly, pharmacological inhibition of the ATF4 pathway attenuated PDE4D expression in the islets and promoted incretin-simulated glucose tolerance and insulin secretion in db/db mice. Finally, we found that inhibiting PDE4 activity with selective pharmacological inhibitors improved ß-cell function in both db/db mice and ß-cell-specific ATF4 transgenic mice. In summary, our results indicate that ER stress causes ß-cell failure via ATF4-mediated PDE4D production, suggesting the ATF4-PDE4D pathway could be a therapeutic target for protecting ß-cell function during the progression of T2D.NEW & NOTEWORTHY Endoplasmic reticulum stress has been implied to cause multiple ß-cell pathologies during the progression of type 2 diabetes (T2D). However, the precise molecular events underlying this remain unknown. Here, we discovered that elevated ATF4 activity, which was seen in T2D ß cells, attenuated ß-cell proliferation and impaired insulin secretion via PDE4D-mediated downregulation of cAMP signaling. Additionally, we demonstrated that pharmacological inhibition of the ATF4 pathway or PDE4D activity alleviated ß-cell dysfunction, suggesting its therapeutic usefulness against T2D.

Upregulation of the ERRγ-VDAC1 axis underlies the molecular pathogenesis of pancreatitis.

Emerging evidence suggest that transcription factors play multiple roles in the development of pancreatitis, a necroinflammatory condition lacking specific therapy. Estrogen-related receptor γ (ERRγ), a pleiotropic transcription factor, has been reported to play a vital role in pancreatic acinar cell (PAC) homeostasis. However, the role of ERRγ in PAC dysfunction remains hitherto unknown. Here, we demonstrated in both mice models and human cohorts that pancreatitis is associated with an increase in ERRγ gene expression via activation of STAT3. Acinar-specific ERRγ haploinsufficiency or pharmacological inhibition of ERRγ significantly impaired the progression of pancreatitis both in vitro and in vivo. Using systematic transcriptomic analysis, we identified that voltage-dependent anion channel 1 (VDAC1) acts as a molecular mediator of ERRγ. Mechanistically, we showed that induction of ERRγ in cultured acinar cells and mouse pancreata enhanced VDAC1 expression by directly binding to specific site of the Vdac1 gene promoter and resulted in VDAC1 oligomerization. Notably, VDAC1, whose expression and oligomerization were dependent on ERRγ, modulates mitochondrial Ca2+ and ROS levels. Inhibition of the ERRγ-VDAC1 axis could alleviate mitochondrial Ca2+ accumulation, ROS formation and inhibit progression of pancreatitis. Using two different mouse models of pancreatitis, we showed that pharmacological blockade of ERRγ-VDAC1 pathway has therapeutic benefits in mitigating progression of pancreatitis. Likewise, using PRSS1R122H-Tg mice to mimic human hereditary pancreatitis, we demonstrated that ERRγ inhibitor also alleviated pancreatitis. Our findings highlight the importance of ERRγ in pancreatitis progression and suggests its therapeutic intervention for prevention and treatment of pancreatitis.

Gemigliptin exerts protective effects against doxorubicin-induced hepatotoxicity by inhibiting apoptosis via the regulation of fibroblast growth factor 21 expression.

AIM: Doxorubicin is a highly effective anticancer agent that causes hepatotoxicity and cardiotoxicity in patients. Fibroblast growth factor 21, a well-known regulator of glucose and lipid metabolism, exerts cardioprotective effects. Gemigliptin and dipeptidyl peptidase-4 inhibitors are widely used in the treatment of patients with type 2 diabetes. The protective effects of gemigliptin on hepatotoxicity via the increase in fibroblast growth factor 21 expression has not yet been elucidated. This study was designed to investigate the protective effects of gemigliptin against doxorubicin-induced hepatotoxicity via the upregulation of fibroblast growth factor 21 expression in the cultured murine hepatocyte cell line, AML12. METHODS: Murine hepatocyte AML12 cells were treated with doxorubicin, fibroblast growth factor 21 and gemigliptin in 0.5% fetal bovine serum medium for 24 h at the indicated doses. Cells were transfected with the fibroblast growth factor 21 small interfering RNA for 24 h, followed by protein isolation. RESULTS: Fibroblast growth factor 21 expression levels were increased during doxorubicin-induced hepatotoxicity in the murine hepatocyte AML12 cells. Fibroblast growth factor 21 treatment prevented doxorubicin-induced hepatotoxicity by attenuating apoptosis. Gemigliptin prevented doxorubicin-induced hepatotoxicity by upregulating fibroblast growth factor 21 expression. However, the protective effects of gemigliptin were blocked by fibroblast growth factor 21 inhibition in doxorubicin-treated AML12 cells. CONCLUSION: These results indicate that gemigliptin exhibits protective effects against doxorubicin-induced hepatotoxicity by upregulating the fibroblast growth factor 21 expression levels in the cultured murine hepatocyte AML12 cells.

Characteristics and phylogenetic analysis of the complete chloroplast genome of Paeonia japonica (Paeoniaceae).

Paeonia japonica, distributed throughout Asia, is a traditional medicinal herb in Korea, with many potential beneficial effects including pain-relieving, anti-inflammatory, and anti-cancer activities. Despite its high pharmacological value, the genetic information on Paeonia japonica remains limited. In this study, the chloroplast genome of P. japonica was sequenced using next-generation sequencing (NGS) technology and genome and phylogeny were analyzed using multiple tools. The chloroplast genome of P. japonica was 152,731 bp in length with an inverted repeat region of 26,656 bp, including a large single-copy region of 84,389 bp and a small single copy region of 17,030 bp. The P. japonica chloroplast genome included 113 genes comprising 80 protein-coding genes, 27 tRNA, and 5 rRNA genes. Phylogenetic analysis indicated that P. japonica and P. obovata share a close evolutionary relationship.

Lin28a ameliorates glucotoxicity-induced ß-cell dysfunction and apoptosis.

An excessive and prolonged increase in glucose levels causes ß-cell dysregulation, which is accompanied by impaired insulin synthesis and secretion, a condition known as glucotoxicity. Although it is known that both Lin28a and Lin28b regulate glucose metabolism, other molecular mechanisms that may protect against glucotoxicity are poorly understood. We investigated whether Lin28a overexpression can improve glucotoxicityinduced ß-cell dysregulation in INS-1 and primary rat islet cells. INS-1, a rat insulinoma cell line was cultured and primary rat islet cells were isolated from SD-rats. To define the effect of Lin28a in chronic high glucose-induced ß-cell dysregulation, we performed several in vitro and ex-vivo experiments. Chronic exposure to high glucose led to a downregulation of Lin28a mRNA and protein expression, followed by a decrease in insulin mRNA expression and secretion in ß-cells. The mRNA and protein expression levels of PDX-1 and BETA2, were reduced; The levels of apoptotic factors, including c-caspase3 and the Bax/Bcl-2 ratio, were increased due to glucotoxicity. Adenovirusmediated Lin28a overexpression in ß-cells reversed the glucotoxicity- induced reduction of insulin secretion and insulin mRNA expression via regulation of ß-cell-enriched transcription factors such as PDX-1 and BETA2. Adenovirus-mediated overexpression of Lin28a downregulated the glucotoxicity-induced upregulation of c-caspase3 levels and the Bax/Bcl-2 ratio, while inhibition of endogenous Lin28a by small interfering RNA resulted in their up-regulation. Lin28a counteracted glucotoxicity-induced downregulation of p-Akt and p-mTOR. Our results suggest that Lin28a protects pancreatic ß-cells from glucotoxicity through inhibition of apoptotic factors via the PI3 kinase/Akt/mTOR pathway. [BMB Reports 2021; 54(4): 215-220].

Lin28a attenuates TGF-ß-induced renal fibrosis.

Lin28a has diverse functions including regulation of cancer, reprogramming and regeneration, but whether it promotes injury or is a protective reaction to renal injury is unknown. We studied how Lin28a acts in unilateral ureteral obstruction (UUO)-induced renal fibrosis following unilateral ureteral obstruction, in a mouse model. We further defined the role of Lin28a in transforming growth factor (TGF)-signaling pathways in renal fibrosis through in vitro study using human tubular epithelium-like HK-2 cells. In the mouse unilateral ureteral obstruction model, obstruction markedly decreased the expression of Lin28a, increased the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin. In TGF-ß-stimulated HK-2 cells, the expression of Lin28a was reduced and the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin was increased. Adenovirus-mediated overexpression of Lin28a inhibited the expression of TGF-ß-stimulated type I collagen, α-SMA, vimentin and fibronectin. Lin28a inhibited TGF-ß-stimulated SMAD3 activity, via inhibition of SMAD3 phosphorylation, but not the MAPK pathway ERK, JNK or p38. Lin28a attenuates renal fibrosis in obstructive nephropathy, making its mechanism a possible therapeutic target for chronic kidney disease. [BMB Reports 2020; 53(11): 594-599].

Arg-Gly-Asp-modified elastin-like polypeptide regulates cell proliferation and cell cycle proteins via the phosphorylation of Erk and Akt in pancreatic ß-cell.

OBJECTIVE: Enhancement of ß-cell proliferation plays an important role in maintaining ß-cell mass and function, and in improving pancreatic ß-cell survival before transplantation. Extracellular matrix (ECM) components increase the adhesion and proliferation of ß-cells, and the RGD-modified elastin-like polypeptide (RGD-ELP, REP) has been described as a bioactive matrix. In this study, we investigated whether REP could enhance ß-cell adhesion and proliferation and elucidated the signaling pathways involved. METHODS: We investigated the effect of REP on cell adhesion, proliferation and insulin secretion via assays using Rin-m and rat islets. Crystal violet, CCK-8, and BrdU assay, FACS, western blot, real time q-PCR analyses and insulin ELISA were examined. To explain the associated mechanisms, phosphorylation of Akt and extracellular signal-regulated kinase (Erk) were measured. RESULTS: REP more increased the adhesion, proliferation and survival of Rin-m cells compared to elastin-like poly peptide (ELP) without RGD-motif. The enhancement of ß-cell proliferation by REP was associated with increased cyclin D1, cyclin D2 and cdk6, and decreased p27 levels. When ß-cells were cultured on REP, Erk and the phosphatidylinositol 3-kinase (PI3-kinase) downstream effector, Akt was stimulated. Treatment with the Erk pathway inhibitor and PI3-kinase inhibitor decreased REP-induced ß-cell adhesion and proliferation, and regulated REP-induced cell cycle proteins. Additionally, REP increased the mRNA and protein levels of insulin and its transcription factor, PDX-1, and insulin secretion. CONCLUSIONS: Our results demonstrate that the up-regulation of the PI3K/Akt and Erk signaling pathways and the regulation of cell cycle proteins by REP could serve as effective strategies for improving pancreatic ß-cell adhesion and proliferation.

RGD-containing elastin-like polypeptide improves islet transplantation outcomes in diabetic mice.

Successful islet transplantation critically depends on the isolation of healthy islets. However, the islet isolation procedure itself contributes to islet death due to the destruction of intra- and peri-islet extracellular matrices (ECMs) during digestion. We investigated whether an RGD-containing elastin-like polypeptide (REP) could function as a self-assembling matrix to replenish ECMs and protects islets from cell death. Immediately following isolation, islets were coated with REP coacervate particles via isothermal adsorption of an REP solution followed by thermal gelation. REP-coated islets displayed increased viability and insulin secretory capacity in pretransplant culture compared to untreated islets. Co-transplantation of REP-treated islets and REP beneath the renal sub-capsule in streptozotocin-induced diabetic mice restored normoglycemia and serum insulin levels. Mice that received co-transplants maintained normoglycemia for a longer period of time than those receiving untreated islets without REP. Moreover, co-transplantation sites exhibited enhanced ß-cell proliferation and vascularization. Thus, the REP-based coacervation strategy improve the survival, function and therapeutic potential of transplanted islets. STATEMENT OF SIGNIFICANCE: 1). An artificial matrix polypeptide comprised of thermoresponsive elastin-like peptides and integrin-stimulatory RGD ligands (REP) to reconstitute damaged or lost matrices. 2). Through body temperature-induced coacervation, REP reconstitutes intra-islet environment and enhances islet viability and insulin secretion by activating the pro-survival and insulin signaling pathways. 3). REP-coated islets were transplanted together with the matrix polypeptide under the kidney sub-capsule of mice, it develops a new peri-insular environment, which protects the islet grafts from immune rejection thus extending islet longevity. 4). Our data suggest that in situ self-assembly of biomimetic islet environments become a new platform allowing for improved islet transplantation at extrahepatic sites.

Glutamate decarboxylase 67 contributes to compensatory insulin secretion in aged pancreatic islets.

Pancreatic islets play an essential role in regulating blood glucose levels. Age-dependent development of glucose intolerance and insulin resistance results in hyperglycemia, which in turn stimulates insulin synthesis and secretion from aged islets, to fulfill the increased demand for insulin. However, the mechanism underlying enhanced insulin secretion remains unknown. Glutamic acid decarboxylase 67 (GAD67) catalyzes the conversion of glutamate into γ-aminobutyric acid (GABA) and CO2. Both glutamate and GABA can affect islet function. Here, we investigated the role of GAD67 in insulin secretion in young (3 month old) and aged (24 month old) C57BL/6J male mice. Unlike young mice, aged mice displayed glucose-intolerance and insulin-resistance. However, aged mice secreted more insulin and showed lower fed blood glucose levels than young mice. GAD67 levels in primary islets increased with aging and in response to high glucose levels. Inhibition of GAD67 activity using a potent inhibitor of GAD, 3-mercaptopropionic acid, abrogated glucose-stimulated insulin secretion from a pancreatic ß-cell line and from young and aged islets. Collectively, our results suggest that blood glucose levels regulate GAD67 expression, which contributes to ß-cell responses to impaired glucose homeostasis caused by advanced aging.

The first case of hand infection caused by Dermabacter jinjuensis in a symmetrical peripheral gangrene patient.

INTRODUCTION: Strains of the genus Dermabacter is a recently established species, recognized as relatively rare opportunistic human pathogen, and is infrequently isolated from clinical specimens, including blood cultures, abscesses, wounds, bone, eye, and skin. PRESENTATION OF CASE: We present a 78-year old female with chronic symmetrical peripheral gangrene and hand infection. The patient underwent surgical debridement with amputation on gangrene with infection of both fingers. At 2 weeks postoperatively, pus discharge was newly observed and the patient underwent reoperation. In the subsequent reinfection, unknown organism has been repeatedly identified, may be the most likely causative agent. On the basis of phenotypic and genotypic distinctness and DNA-DNA hybridization results, new strain should be placed in the genus Dermabacter as representing a novel species, for which the name Dermabacter jinjuensis sp. nov. is proposed. DISCUSSION: We judged the novel species as the causative bacteria. Because of, a novel species called D. jinjuensis was repeatedly identified more than common bacteria. It can be considered as a postoperative nosocomial infection or opportunistic infection. It is not clear how the infection of D. jinjensis occurred. CONCLUSION: This is the first reported case of a human D. jinjuensis infection. We were able to treat patients without any complications by operative treatment and administering appropriate antimicrobial agents according to antibiotics susceptibility test.

Enhanced Therapeutic Treatment of Colorectal Cancer Using Surface-Modified Nanoporous Acupuncture Needles.

Acupuncture originated within the auspices of Oriental medicine, and today is used as an alternative method for treating various diseases and symptoms. The physiological mechanisms of acupuncture appear to involve the release of endogenous opiates and neurotransmitters, with the signals mediating through electrical stimulation of the central nervous system (CNS). Earlier we reported a nanoporous stainless steel acupuncture needle with enhanced therapeutic properties, evaluated by electrophysiological and behavioral responses in Sprague-Dawley (SD) rats. Herein, we investigate molecular changes in colorectal cancer (CRC) rats by acupuncture treatment using the nanoporous needles. Treatment at acupoint HT7 is found most effective at reducing average tumor size, ß-catenin expression levels, and the number of aberrant crypt foci in the colon endothelium. Surface modification of acupuncture needles further enhances the therapeutic effects of acupuncture treatment in CRC rats.

Reverse Expression of Aging-Associated Molecules through Transfection of miRNAs to Aged Mice.

Molecular changes during aging have been studied to understand the mechanism of aging progress. Herein, changes in microRNA (miRNA) expression in the whole blood of mice were studied to systemically reverse aging and propose them as non-invasive biomarkers. Through next-generation sequencing analysis, we selected 27 differentially expressed miRNAs during aging. The most recognized function involved was liver steatosis, a type of non-alcoholic fatty liver disease (NAFLD). Among 27 miRNAs, six were predicted to be involved in NAFLD, miR-16-5p, miR-17-5p, miR-21a-5p, miR-30c-5p, miR-103-3p, and miR-130a-3p; alterations in their blood and liver levels were confirmed by real-time qPCR. The expression of the genes associated in the network of these miRNAs, Bcl2, Ppara, E2f1, E2f2, Akt, Ccnd1, and Smad2/3, also was altered in the liver of aged mice. Following transfection of these miRNAs into 18-month-old mice, levels of miR-21a-5p, miR-103-3p, and miR-30c-5p increased, and their related genes exhibited a reversed expression in the liver. Expression of Mre11a, p16INK4a, and Mtor, reported to be aging-associated molecules, also was reversed in the livers of miRNA-transfected mice. These miRNAs could be non-invasive biomarkers for aging, and they might induce a reverse regulation of aging-associated pathways. This study provides preliminary data on reverse aging, which could be applied further for treatments of adult diseases.

Integrin-binding elastin-like polypeptide as an in situ gelling delivery matrix enhances the therapeutic efficacy of adipose stem cells in healing full-thickness cutaneous wounds.

One crucial issue in stem cell therapy used for tissue repair is often the lack of selective carriers to deliver stem cells to the site of injury where the native extracellular matrix is pathologically damaged or lost. Therefore, it is necessary to develop a biomaterial that is permissive to stem cells and is suitable to replace injured or missing matrix. The major aim of this study is to investigate the potential of an RGD-containing elastin-like polypeptide (REP) with the structure TGPG[VGRGD(VGVPG)6]20WPC to engraft adipose stem cells (ASC) to full-thickness excisional wounds in mice. We implanted REP into the wound defects via body temperature-induced in situ aggregation. Engrafted REP exhibited a half-life of 2.6days in the wounds and did not elicit any pathological immune responses. REP itself significantly accelerated wound closure and reepithelialization and upregulated the expression of dermal tissue components. A combined administration of REP and ASC formed a hydrogel-like ASC/REP composite, which provided better neovascularization than the use of ASCs alone and increased the viability of transplanted ASC, improving overall wound healing. In vitro and in vivo mechanistic investigations suggested that REP enhances ASC survival at least in part via the Fak/Src adhesion-induced upregulation of Mek/Erk and PI3K/Akt survival pathways. We conclude that REP is a promising therapeutic agent for the improvement of stem cell-based therapy for enhanced tissue regeneration and repair.

Dermabacter jinjuensis sp. nov., a novel species of the genus Dermabacter isolated from a clinical specimen.

A Gram-stain-positive, catalase-positive, facultatively anaerobic, non-motile, coryneform bacterium, designated strain 32T, was isolated from a closed pus sample from a patient having finger necrosis in Korea. Strain 32T was considered as representing a novel species according to its initial identification by matrix-assisted laser desorption/ionization-time-of-flight MS. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 32T belonged to the genus Dermabacter and was closely related to Dermabacter hominis DSM 7083T (=ATCC 49369T) (98.34 % similarity). Optimal growth was observed at 30-40 °C and pH 7. Growth occurred in the presence of 0-6 % (w/v) NaCl. Menaquinones MK-8, MK-7 and MK-9 were the major respiratory quinones. The major polar lipids were phosphatidylethanolamine, phosphatidylcholine, glycolipid and two unknown lipids. The major cellular fatty acids were anteiso-C17 : 0, anteiso-C15 : 0, iso-C16 : 0 and iso-C15 : 0. The DNA G+C content of strain 32T was 62.58 mol%, and the mean level of DNA-DNA relatedness between strain 32T and D. hominis ATCC 49369T was 49±1.6 %. Based on the phenotypic and genotypic characteristics, strain 32T is confirmed to represent a novel species of the genus Dermabacter, for which the name Dermabacter jinjuensis sp. nov. is proposed. The type strain is 32T (=NCCP 16133T=DSM 101003T).

Blood levels of cadmium and lead in residents near abandoned metal mine areas in Korea.

We analyzed national data on blood lead levels (BLL) and blood cadmium levels (BCL) in residents living near 38 abandoned metal mining areas (n = 5,682, 18-96 years old) in Korea that were collected by the first Health Effect Surveillance for Residents in Abandoned Metal mines (HESRAM) from 2008 to 2011. The geometric mean BCL and BLL were 1.60 µg/L (95 % CI = 1.57-1.62 µg/L) and 2.87 µg/dL (95 % CI = 2.84-2.90 µg/dL), respectively, notably higher than levels in the general population in Korea and other countries. We found significantly higher BLL and BCL levels in people living within 2 km of an abandoned metal mine (n = 3,165, BCL = 1.87 µg/L, BLL = 2.91 µg/dL) compared to people living more than 2 km away (n = 2,517, BCL = 1.31 µg/L, BLL = 2.82 µg/dL; P < 0.0001) and to the general population values reported in the literature.

Cytotoxicity and inhibition of intercellular interaction in N2a neurospheroids by perfluorooctanoic acid and perfluorooctanesulfonic acid.

Effects of perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) on the neuronal lineage marker expression, cell-cell interaction, caspase-3 mRNA transcription and reactive oxygen species production by N2a neuronal cells were assesses in 3-dimensional (3D) spheroid cultures, and the cytotoxicity were thoroughly compared with those of a conventional 2D monolayer-based toxicity assay. Increasing concentrations of PFOA or PFOS resulted in an increase in cell death. The half maximal inhibitory concentrations measured with spheroids were approximately one and a half times greater than the respective values for monolayer cells. Necrosis was prevalent in spheroids regardless of the dose, whereas the major injury mechanism in monolayers was dependent on compound concentration. Both PFOA and PFOS inhibited neuronal, astrocyte and oligodendrocyte marker gene expression by monolayers and spheroids grown under undifferentiated and all-trans-retinoic acid-induced differentiating conditions. In the presence of PFOA or PFOS, expression levels of E-cadherin and connexin-43 mRNAs were significantly downregulated, and spheroids were dissociated into single cell populations, indicating that the compounds affect the synthesis of E-cadherin and connexin-43 at the transcriptional level. Results from 3D cultures may provide an insight into potential inhibitory mode of action on gap junctional intercellular communication.

Morphogenetic and neuronal characterization of human neuroblastoma multicellular spheroids cultured under undifferentiated and all-trans-retinoic acid-differentiated conditions.

In this study, we aimed to compare the morphogenetic and neuronal characteristics between monolayer cells and spheroids. For this purpose, we established spheroid formation by growing SH-SY5Y cells on the hydrophobic surfaces of thermally-collapsed elastin-like polypeptide. After 4 days of culture, the relative proliferation of the cells within spheroids was approximately 92% of the values for monolayer cultures. As measured by quantitative assays for mRNA and protein expressions, the production of synaptophysin and neuronspecific enolase (NSE) as well as the contents of cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins are much higher in spheroids than in monolayer cells. Under the all-trans-retinoic acid (RA)-induced differentiation condition, spheroids extended neurites and further up-regulated the expression of synaptophysin, NSE, CAMs, and ECM proteins. Our data indicate that RA-differentiated SH-SY5Y neurospheroids are functionally matured neuronal architectures.

Improved neural progenitor cell proliferation and differentiation on poly(lactide-co-glycolide) scaffolds coated with elastin-like polypeptide.

Poly(lactide-co-glycolide) (PLGA) and elastin-like polypeptide (ELP) have been widely used as a biodegradable scaffold and thermoresponsive matrix, respectively. However, little attention has focused on the combinatorial use of these biomaterials for tissue engineering applications. An ELP matrix TGPG[VGRGD(VGVPG)6]20WPC (referred to as REP) contains multiple Arg-Gly-Asp motifs. This study fabricated porous PLGA scaffolds coated with various concentration of matrix via thermally induced phase transition to improve adhesion-mediated proliferation and differentiation of neural progenitor cells. Matrix-coated scaffolds were characterized by FTIR, SEM, and hematoxylin and eosin staining with respect to coating efficiency, porosity, and pore size and shape. On the matrix-coated scaffolds, cells grew as a single cell or associated each other to form a multicellular layer or cluster. In biological evaluations, cell adhesion and proliferation were significantly promoted in a matrix concentration-dependent manner. More importantly, in combination with retinoic acid, the differentiation of progenitor cells into neuronal and astroglial lineages was highly stimulated in the cells cultured on matrix-coated scaffolds than on untreated controls. Taken together, our results indicated that the REP matrix-functionalized PLGA scaffolds are suitable for improving neuronal cell functions, and thus applicable for neural tissue engineering.

Assessment of the levels of airborne bacteria, Gram-negative bacteria, and fungi in hospital lobbies.

AIMS: We assessed the levels of airborne bacteria, Gram-negative bacteria (GNB), and fungi in six hospital lobbies, and investigated the environmental and hospital characteristics that affected the airborne microorganism levels. METHODS: An Andersen single-stage sampler equipped with appropriate nutrition plate agar was used to collect the samples. The three types of microorganisms were repeatedly collected at a fixed location in each hospital (assumed to be representative of the entire hospital lobby) from 08:00 through 24:00, with a sampling time of less than 5 min. Temperature and relative humidity were simultaneously monitored. RESULTS: Multiple regression analysis was used to identify the major factors affecting microorganism levels. The average levels of bacteria (7.2 × 10(2) CFU/m(3)), GNB (1.7 × 10 CFU/m(3)), and fungi (7.7 × 10 CFU/m(3)) indicated that all hospital lobbies were generally contaminated. Season was the only factor that significantly affected the levels of all microorganisms (p < 0.0001), where contamination was the highest during the summer, significantly higher than during the winter. Other significant factors varied by microorganism, as follows: airborne bacteria (number of people in the lobby, sampling time), GNB (scale of hospital), and fungi (humidity and air temperature). CONCLUSIONS: Hospital lobby air was generally contaminated with microorganisms, including bacteria, GNB, and fungi. Environmental factors that may significantly influence the airborne concentrations of these agents should be managed to minimize airborne levels.