Regular Culture Conditions Research Articles

Biological and genomic analyses of Clavispora sputumsp. nov., a novel potential fungal pathogen closely related to Clavispora lusitaniae (syn. Candida lusitaniae) and Candida auris

Several human fungal pathogens, including drug-resistant Candida auris and species of the Candida haemulonii complex, have emerged over the past two decades, posing new threats to human health. In this study, we report the isolation and identification of a novel species belonging to the genus Clavispora, herein named as Clavisporasputum, from a clinical sputum sample of a COVID-19 patient. Cl. sputum is phylogenetically closely related to fungal pathogens Clavispora lusitaniae (syn. Candida lusitaniae) and C. auris. When grown on CHROMagar Candida Plus medium, Cl. sputum exhibited a similar coloration to C. auris strain CBS12372. Cl. sputum was able to develop weak filaments on CM medium. Although Cl. sputum and Cl. lusitaniae are phylogenetically closely related, comparative genomic and synteny analyses indicated significant chromosomal rearrangements between the two species. Although Cl. sputum could not grow at 37 °C under regular culture condition, an increased fungal burden in the lung tissue of a mouse systemic infection model implies that it could be a potential opportunistic pathogenic yeast in humans.

RECK/GPR124-driven WNT signaling in pancreatic and gastric cancer cells.

RECK has been described to modulate extracellular matrix components through negative regulation of MMP activities. Recently, RECK was demonstrated to bind to an orphan G protein-coupled receptor GPR124 to mediate WNT7 signaling in nontumor contexts. Here, we attempted to clarify the role of RECK in driving WNT signaling in cancer cells. RECK and GPR124 formed a complex in 293T cells, and when both were expressed, WNT signaling was significantly enhanced in a WNT7-dependent manner. This cooperation was abolished when RECK mutants unable to bind to GPR124 were transduced. RECK stimulated the growth of KRAS-mutated pancreatic ductal adenocarcinoma (PDAC) cells with increased sensitivity to WNT inhibitor in a GPR124-dependent manner. A gastric cancer cell line SH10TC endogenously expresses both RECK and GPR124 under regular culture conditions. In this cell line, inhibited cell growth and WNT signaling as well as increased apoptosis in the GPR124 depletion was dominantly found over those in the RECK deletion. These findings suggest that RECK promotes tumor cell growth by positively modulating WNT signaling through GPR124. This study proposes that the RECK/GPR124 complex might be a good therapeutic target in PDAC and gastric cancer.

Competitive fitness and stability of ammonium-excreting Azotobacter vinelandii strains in the soil

Non-symbiotic N2-fixation would greatly increase the versatility of N-biofertilizers for sustainable agriculture. Genetic modification of diazotrophic bacteria has successfully enhanced NH4+ release. In this study, we compared the competitive fitness of A. vinelandii mutant strains, which allowed us to analyze the burden of NH4+ release under a broad dynamic range. Long-term competition assays under regular culture conditions confirmed a large burden for NH4+ release, exclusion by the wt strain, phenotypic instability, and loss of the ability to release NH4+. In contrast, co-inoculation in mild autoclaved soil showed a much longer co-existence with the wt strain and a stable NH4+ release phenotype. All genetically modified strains increased the N content and changed its chemical speciation in the soil. This study contributes one step forward towards bridging a knowledge gap between molecular biology laboratory research and the incorporation of N from the air into the soil in a molecular species suitable for plant nutrition, a crucial requirement for developing improved bacterial inoculants for economic and environmentally sustainable agriculture.Key points• Genetic engineering for NH4+ excretion imposes a fitness burden on the culture medium• Large phenotypic instability for NH4+-excreting bacteria in culture medium• Lower fitness burden and phenotypic instability for NH4+-excreting bacteria in soil

Editing efficiencies with Cas9 orthologs, Cas12a endonucleases, and temperature in rice.

The advent of CRISPR-Cas technology has made it the genome editing tool of choice in all kingdoms of life, including plants, which can have large, highly duplicated genomes. As a result, finding adequate target sequences that meet the specificities of a given Cas nuclease on any gene of interest remains challenging in many cases. To assess target site flexibility, we tested five different Cas9/Cas12a endonucleases (SpCas9, SaCas9, St1Cas9, Mb3Cas12a, and AsCas12a) in embryogenic rice calli from Taipei 309 at 37°C (optimal temperature for most Cas9/Cas12a proteins) and 27°C (optimal temperature for tissue culture) and measured their editing rates under regular tissue culture conditions using Illumina sequencing. StCas9 and AsCas12 were not functional as tested, regardless of the temperature used. SpCas9 was the most efficient endonuclease at either temperature, regardless of whether monoallelic or biallelic edits were considered. Mb3Cas12a at 37°C was the next most efficient endonuclease. Monoallelic edits prevailed for both SaCas9 and Mb3Cas12a at 27°C, but biallelic edits prevailed at 37°C. Overall, the use of other Cas9 orthologs, the use of Cas12a endonucleases, and the optimal temperature can expand the range of targetable sequences.

In Vitro Techniques for Shipping of Micropropagated Plant Materials

Shipping of in vitro micro-cuttings in tubes or jars is a frequently used method as the plants are more likely to quickly reproduce and comply with quarantine regulations in plant germplasm distribution. However, these containers are fragile during transportation. To diminish the risk associated with the long-distance shipping of in vitro plants, a safe and widely applicable packing and conservation technique based on microplate and slow growth was developed in this study. Potato cultivar ZHB and ginger cultivar G-2 were used to optimize the system with microplates (96 wells), vacuum-sealed packaging, and slow-growth techniques. Under regular culture conditions, packing in vacuum-sealed microplates reduced the survival of ZHB and G-2 micro-cuttings to 85.8% and 20.0%, respectively, and regeneration to 61.8% and 0%, respectively. Reducing the temperature to 10 °C maintained the survival of ZHB and G-2 micro-cuttings in the range of 83.3–100% after 60 days. Exposure to darkness decreased the survival of G-2 and inhibited regrowth. Thus, conservation in darkness at 10 °C is suggested. The effects of iron concentration and plant growth retardants were further assessed. The addition of 1/4 MS medium combined with 100 mg/L chlormequat chloride (CCC) resulted in full survival and growth inhibition of plantlets, without malformation identified. Finally, incubation with 1/4 MS medium supplemented with 100 mg/L CCC in vacuum-sealed microplates at 10 °C in the dark resulted in high survival and suppressed germination. Sweet potato HXS was incubated as well to test the broad-spectrum applications of the technique; 100% survival and 6.7% germination was gained. Morphological indices of released cuttings recovered to control levels after two cycles of subculture in MS medium. A 0.1–0.2% genetic variation was detected by SSR and ISSR, suggesting genetic stability of the conserved samples. Finally, micro-cuttings were safely transported to cities located thousands of kilometers away without package and sample damage. Our results enable easy distribution of in vitro plant germplasms.

Mammalian HEMK1 methylates glutamine residue of the GGQ motif of mitochondrial release factors

Despite limited reports on glutamine methylation, methylated glutamine is found to be highly conserved in a "GGQ" motif in both prokaryotes and eukaryotes. In bacteria, glutamine methylation of peptide chain release factors 1/2 (RF1/2) by the enzyme PrmC is essential for translational termination and transcript recycling. Two PrmC homologs, HEMK1 and HEMK2, are found in mammals. In contrast to those of HEMK2, the biochemical properties and biological significance of HEMK1 remain largely unknown. In this study, we demonstrated that HEMK1 is an active methyltransferase for the glutamine residue of the GGQ motif of all four putative mitochondrial release factors (mtRFs)—MTRF1, MTRF1L, MRPL58, and MTRFR. In HEMK1-deficient HeLa cells, GGQ motif glutamine methylation was absent in all the mtRFs. We examined cell growth and mitochondrial properties, but disruption of the HEMK1 gene had no considerable impact on the overall cell growth, mtDNA copy number, mitochondrial membrane potential, and mitochondrial protein synthesis under regular culture condition with glucose as a carbon source. Furthermore, cell growth potential of HEMK1 KO cells was still maintained in the respiratory condition with galactose medium. Our results suggest that HEMK1 mediates the GGQ methylation of all four mtRFs in human cells; however, this specific modification seems mostly dispensable in cell growth and mitochondrial protein homeostasis at least for HeLa cells under fermentative culture condition.

Abstract 3163: Progressive prostate cancer LNCaP subclones partially retain regressive communications with patient-derived fibroblasts

Abstract The tumor microenvironment of prostate cancer (PCa) plays an important role in disease progression, responsiveness to therapy, or inversely restraint of tumor growth. PCa cell evolution including loss of androgen receptor dependency is associated with intratumor heterogeneity, generating progressive PCa cell subclones. The tumor stroma of PCa contains multiple functionally different populations of fibroblasts that regulate PCa cell progression, e.g., cancer-promoting or cancer-restraining. Fibroblasts secret a number of growth factors, cytokines, the ECM molecules, and miRNAs in a cellular characteristic-specific manner. Thus, we hypothesize that diversity of fibroblasts may induce heterogenous PCa cell progression. To investigate the heterotypical communications between PCa cells and fibroblasts, we used 3 subclones of androgen-sensitive LNCaP cells and 4 patient-derived fibroblasts. Androgen-low-sensitive E9 and F10 cells were obtained from the parental androgen-sensitive LNCaP cell population through use of a limiting dilution method in regular culture condition. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted condition. Commercially available normal human prostate stromal cells (PrSC) was purchased from Lonza Group Ltd. Three original fibroblasts pcPrFs (-M5, -M28, -M31) were isolated from needle biopsy samples of patients with advanced PCa. To examine the effects of fibroblasts on cancer-related gene expression in PCa cells, in vitro co-culture experiment was performed using cell culture insert. In all PCa cells, LNCaP and its subclones, expressions of NFKB1, SRC, and MYC mRNA were not dramatically changed by co-culturing with 3 PCa patient-derived fibroblasts. Only in LNCaP cells, however, they were uniformly down-regulated by co-culturing with PrSC. Only in LNCaP cells, expression of CDH2 mRNA was up-regulated by co-culturing with pcPrF-M5 and pcPrF-M28. In all PCa cells, expression of NKX3-1 mRNA was uniformly up-regulated by co-culturing with pcPrF-M28 and pcPrF-M31. In LNCaP, E9, and F10 cells but not in AIDL cells, expression of GSTP1 mRNA was uniformly up-regulated by co-culturing with pcPrF-M31. Cell growth of LNCaP cells was not remarkably affected by co-culturing with pcPrF-M28 and pcPrF-M31. The heterogeneous stromal compartment in PCa tissues contains multiple functionally different populations of fibroblasts that are also associated with intratumor heterogeneity. Our data showed that up-regulations of NKX3-1 and GSTP1 mRNA expression by co-culturing with pcPrF-M28 and pcPrF-M31 were commonly observed in LNCaP subclones differing in androgen sensitivity. This result suggests that certain fibroblasts may restrain PCa cell progression by up-regulations of tumor suppressor gene expression even in progressive poorly differentiated PCa cells. Citation Format: Kenichiro Ishii, Chise Matsuda, Yasuhisa Nakagawa, Taku Shirai, Yoshifumi Hirokawa, Takeshi Sasaki, Kazuhiro Iguchi, Masatoshi Watanabe. Progressive prostate cancer LNCaP subclones partially retain regressive communications with patient-derived fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3163.

Establishment and characterization of a novel treatment-related neuroendocrine prostate cancer cell line KUCaP13.

The prevalence of neuroendocrine prostate cancer (NEPC) arising from adenocarcinoma (AC) upon potent androgen receptor (AR) pathway inhibition is increasing. Deeper understanding of NEPC biology and development of novel therapeutic agents are needed. However, research is hindered by the paucity of research models, especially cell lines developed from NEPC patients. We established a novel NEPC cell line, KUCaP13, from tissue of a patient initially diagnosed with AC which later recurred as NEPC. The cell line has been maintained permanently in vitro under regular cell culture conditions and is amenable to gene engineering with lentivirus. KUCaP13 cells lack the expression of AR and overexpress NEPC‐associated genes, including SOX2, EZH2, AURKA, PEG10, POU3F2, ENO2, and FOXA2. Importantly, the cell line maintains the homozygous deletion of CHD1, which was confirmed in the primary AC of the index patient. Loss of heterozygosity of TP53 and PTEN, and an allelic loss of RB1 with a transcriptomic signature compatible with Rb pathway aberration were revealed. Knockdown of PEG10 using shRNA significantly suppressed growth in vivo. Introduction of luciferase allowed serial monitoring of cells implanted orthotopically or in the renal subcapsule. Although H3K27me was reduced by EZH2 inhibition, reversion to AC was not observed. KUCaP13 is the first patient‐derived, treatment‐related NEPC cell line with triple loss of tumor suppressors critical for NEPC development through lineage plasticity. It could be valuable in research to deepen the understanding of NEPC.

The PHO pathway regulates white-opaque switching and sexual mating in the human fungal pathogen Candida albicans.

The opportunistic fungal pathogen Candida albicans is able to switch among several morphological phenotypes in response to environmental changes. White-opaque transition is a typical phenotypic switching system involved in the regulation of pathogenesis and sexual reproduction in C. albicans. Under regular laboratory culture conditions, to undergo white-to-opaque switching, cells must first undergo homozygosis at the mating-type locus (MTLa/a or α/α) since the a1/α2 heterodimer represses the expression of the Wor1 master regulator of switching in MTLa/α heterozygous strains. In this study, we report the roles of the PHO pathway of phosphate metabolism in the regulation of white-opaque switching and sexual mating in C. albicans. We find that deletion of the PHO pathway genes PHO81, PHO80, PHO2, and PHO4 induces the opaque phenotype in MTLa/α heterozygous cells. Low concentrations of external phosphate are conducive for the opaque phenotype in both MTL homozygous and heterozygous strains. Moreover, phosphate starvation can also increase the mating efficiency in C. albicans. Consistently, the pho80/pho80 mutant mimics an artificial phosphate starvation state and mates efficiently at both lower and higher phosphate concentrations. Our study establishes a link between the PHO pathway and white-opaque epigenetic switching in C. albicans.

Alleviation of acute radiation-induced bone marrow failure in mice with human fetal placental stromal cell therapy

PurposeSelected placental mesenchymal stromal cells isolated from the fetal mesenchymal placental tissues (f-hPSCs) were tested as cell therapy of lethal acute radiation syndrome (ARS) with bone marrow regeneration and induced extramedullary hematopoiesis.Methods and materialsf-hPSCs were isolated from the chorionic plate of human placentae and further expanded in regular culture conditions. 2 × 106 f-hPSCs were injected on days 1 and 4 to 8-Gy total body irradiated (TBI) C3H mice, both intramuscularly and subcutaneously. Pre-splenectomized TBI mice were used to test the involvement of extramedullary spleen hematopoiesis in the f-hPSC-induced hematopoiesis recovery in the TBI mice. Weight and survival of the mice were followed up within the morbid period of up to 23 days following irradiation. The role of hematopoietic progenitors in the recovery of treated mice was evaluated by flow cytometry, blood cell counts, and assay of possibly relevant growth factors.Results and conclusionsThe survival rate of all groups of TBI f-hPSC-treated mice at the end of the follow-up was dramatically elevated from < 10% in untreated to ~ 80%, with a parallel regain of body weight, bone marrow (BM) recovery, and elevated circulating progenitors of blood cell lineages. Blood erythropoietin levels were elevated in all f-hPSC-treated mice. Extramedullary splenic hematopoiesis was recorded in the f-hPSC-treated mice, though splenectomized mice still had similar survival rate. Our findings suggest that the indirect f-hPSC life-saving therapy of ARS may also be applied for treating other conditions with a failure of the hematopoietic system and severe pancytopenia.

Cardiovascular Effects of Polychlorinated Biphenyls and Their Major Metabolites.

Background:Xenobiotic metabolism is complex, and accounting for bioactivation and detoxification processes of chemicals remains among the most challenging aspects for decision making with in vitro new approach methods data.Objectives:Considering the physiological relevance of human organotypic culture models and their utility for high-throughput screening, we hypothesized that multidimensional chemical-biological profiling of chemicals and their major metabolites is a sensible alternative for the toxicological characterization of parent molecules vs. metabolites in vitro.Methods:In this study, we tested 25 polychlorinated biphenyls (PCBs) [PCB 3, 11, 52, 126, 136, and 153 and their relevant metabolites (hydroxylated, methoxylated, sulfated, and quinone)] in concentration–response () for effects in human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) and endothelial cells (ECs) (iPSC-derived and HUVECs). Functional phenotypic end points included effects on beating parameters and intracellular flux in CMs and inhibition of tubulogenesis in ECs. High-content imaging was used to evaluate cytotoxicity, mitochondrial integrity, and oxidative stress.Results:Data integration of a total of 19 physicochemical descriptors and 36 in vitro phenotypes revealed that chlorination status and metabolite class are strong predictors of the in vitro cardiovascular effects of PCBs. Oxidation of PCBs, especially to di-hydroxylated and quinone metabolites, was associated with the most pronounced effects, whereas sulfation and methoxylation of PCBs resulted in diminished bioactivity.Discussion:Risk characterization analysis showed that although in vitro derived effective concentrations exceeded the levels measured in the general population, risks cannot be ruled out due to the potential for population variability in susceptibility and the need to fill data gaps using read-across approaches. This study demonstrated a strategy for how in vitro data can be used to characterize human health risks from PCBs and their metabolites. https://doi.org/10.1289/EHP7030

Recognition of synthetic polyanionic ligands underlies "spontaneous" reactivity of Vγ1 γδTCRs.

Although γδTCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several Vγ1‐containing TCRs, including the Vγ1Vδ6 TCR of γδNKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self‐ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by Vγ1Vδ6 and related TCRs, but not by TCRs from several other γδ or innate‐like αβ T cells, and demonstrated that both γ and δ chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo Vγ1 γδ T cells. Taken together, these results explain long‐standing observations on the spontaneous reactivity of Vγ1Vδ6 TCR and demonstrate an unexpected antigen presentation‐independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.

Electromagnetic field treatment increases purinergic receptor P2X7 expression and activates its downstream Akt/GSK3\u03b2/\u03b2-catenin axis in mesenchymal stem cells under osteogenic induction

BackgroundImbalance in bone formation and resorption is a crucial component of the pathological process leading to osteoporosis. Electromagnetic fields (EMFs) have been reported to be beneficial to osteogenesis, although the exact mechanism has not been fully clarified. Purinergic receptor P2X7 is expressed in osteoblasts and is reported to participate in the regulation of bone metabolism.ObjectivesTo elucidate the link between EMFs and P2X7 expression and investigate its potential as a novel therapeutic target in osteoporosis.MethodWe investigated the effect of EMFs on P2X7 expression and downstream signaling in human bone marrow mesenchymal stem cells (h-MSCs). We also established an ovariectomized (OVX) osteoporosis rat model to evaluate the therapeutic efficacy of combining EMFs with P2X7 agonists.ResultsEMF treatment increased P2X7 expression in h-MSCs under conditions of osteogenic induction but not under regular culture conditions. P2X7 or PI3K/Akt inhibition partially inhibited the pro-osteogenic effect of EMF and lowered the EMF-stimulated activity of the Akt/GSK3β/β-catenin axis. No additive effect of this suppression was observed following simultaneous inhibition of P2X7 and PI3K/Akt. EMF treatment in the presence of a P2X7 agonist had a greater effect in increasing osteogenic marker expression than that of EMF treatment alone. In the OVX osteoporosis model, the therapeutic efficacy of combining EMFs with P2X7 agonists was superior to that of EMF treatment alone.ConclusionsEMF treatment increases P2X7 expression by h-MSCs during osteogenic differentiation, leading to activation of the Akt/GSK3β/β-catenin axis, which promotes the osteogenesis. Our findings also indicate that combined EMF and P2X7 agonist treatment may be an effective novel strategy for osteoporosis therapy.

Abstract 2026: Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer

Abstract Well-differentiated prostate cancer (PCa) cells are generally androgen/androgen receptor (AR)-dependent, i.e. AR signaling regulates cell cycle and differentiation. Loss of AR signaling after androgen deprivation therapy (ADT) triggers AR-independent outgrowth, generating poorly differentiated uncontrollable PCa cells. To prevent development and progression of castration-resistant prostate cancer (CRPC), we hypothesize that preservation of AR signaling after ADT is an important target. Tumor stroma surrounding PCa cells is enriched in fibroblasts secreting AR-activating factors, e.g. EGF, IGF1, and IL-6. Thus, we investigated the role of fibroblasts in AR activation of human prostate cancer LNCaP sublines differing in androgen sensitivity under androgen deprivation. Androgen-low-sensitive E9 and F10 cells were obtained from the parental androgen-sensitive LNCaP cell population through use of a limiting dilution method in regular culture condition. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted condition. Original fibroblasts pcPrFs (-M5, -M6, -M7) were isolated from needle biopsy samples of patients with PCa. The PCa cells alone or PCa cells plus pcPrF-M5 were grafted beneath the renal capsule of male athymic nude mice, and then mice were castrated on 14 days post transplantation. Tumor volume of E9 cells + pcPrF-M5 became diminished post castration but that of F10 and AIDL cells + pcPrF-M5 was gradually increased even post castration. In E9 cells + pcPrF-M5 tumors, serum PSA became detected on day 21 post castration. Interestingly, serum PSA was gradually increased even in F10 cells inoculated alone. In AIDL tumors with or without pcPrF-M5, serum PSA was not detected because of mutated AR in AIDL cells. Cell growth of E9 and AIDL cells was directly increased by treating with EGF, whereas that in F10 cells was not affected. Phosphorylation of AKT and ERK1/2 in E9 and AIDL cells was also significantly increased by treating with EGF, whereas that in F10 cells was not affected. In the condition of co-cultures with fibroblasts, PSA production was directly increased in E9 cells but not in F10 and AIDL cells. Protein expression of AR splice variant 7 (AR-V7) was only detected in F10 cells. The reduced AR-dependency of PCa cells is an important clinical development because of its association with the cell’ progression to CRPC. Here our results showed that fibroblasts secreting AR-activating factors preserved AR signaling in E9 cells after ADT, indicating that this type of PCa cells can be controlled by ADT. In contrast, we suggest that loss of fibroblasts-dependent AR activation in F10 and AIDL cells may be responsible for development and progression of CRPC. Citation Format: Kenichiro Ishii, Izumi Matsuoka, Takeshi Sasaki, Manabu Kato, Kohei Nishikawa, Hideki Kanda, Yoshifumi Hirokawa, Kazuhiro Iguchi, Kiminobu Arima, Masatoshi Watanabe, Yoshiki Sugimura. Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2026.

Mechanically Biomimetic Gelatin-Gellan Gum Hydrogels for 3D Culture of Beating Human Cardiomyocytes.

To promote the transition of cell cultures from 2D to 3D, hydrogels are needed to biomimic the extracellular matrix (ECM). One potential material for this purpose is gellan gum (GG), a biocompatible and mechanically tunable hydrogel. However, GG alone does not provide attachment sites for cells to thrive in 3D. One option for biofunctionalization is the introduction of gelatin, a derivative of the abundant ECM protein collagen. Unfortunately, gelatin lacks cross-linking moieties, making the production of self-standing hydrogels difficult under physiological conditions. Here, we explore the functionalization of GG with gelatin at biologically relevant concentrations using semiorthogonal, cytocompatible, and facile chemistry based on hydrazone reaction. These hydrogels exhibit mechanical behavior, especially elasticity, which resembles the cardiac tissue. The use of optical projection tomography for 3D cell microscopy demonstrates good cytocompatibility and elongation of human fibroblasts (WI-38). In addition, human-induced pluripotent stem cell-derived cardiomyocytes attach to the hydrogels and recover their spontaneous beating in 24 h culture. Beating is studied using in-house-built phase contrast video analysis software, and it is comparable with the beating of control cardiomyocytes under regular culture conditions. These hydrogels provide a promising platform to transition cardiac tissue engineering and disease modeling from 2D to 3D.

In vitro study of the effect of human antigen R on lysosomal acidification during autophagy in mouse cardiomyocytes

In vitro study of the effect of human antigen R on lysosomal acidification during autophagy in mouse cardiomyocytes

Role of hexokinase Ⅱ in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro

Role of hexokinase Ⅱ in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro

Enhanced Biofilm Formation and Membrane Vesicle Release by Escherichia coli Expressing a Commonly Occurring Plasmid Gene, kil.

Escherichia coli is one of the most prevalent microorganisms forming biofilms on indwelling medical devices, as well as a representative model to study the biology and ecology of biofilms. Here, we report that a small plasmid gene, kil, enhances biofilm formation of E. coli. The kil gene is widely conserved among naturally occurring colicinogenic plasmids such as ColE1 plasmid, and is also present in some plasmid derivatives used as cloning vectors. First, we found that overexpression of the kil gene product dramatically increased biofilm mass enriched with extracellular DNA in the outer membrane-compromised strain RN102, a deep rough LPS mutant E. coli K-12 derivative. We also found that the kil-enhanced biofilm formation was further promoted by addition of physiologically relevant concentrations of Mg2+, not only in the case of RN102, but also with the parental strain BW25113, which retains intact core-oligosaccharide LPS. Biofilm formation by kil-expressing BW25113 strain (BW25113 kil+) was significantly inhibited by protease but not DNase I. In addition, a large amount of proteinous materials were released from the BW25113 kil+ cells. These materials contained soluble cytoplasmic and periplasmic proteins, and insoluble membrane vesicles (MVs). The kil-induced MVs were composed of not only outer membrane/periplasmic proteins, but also inner membrane/cytoplasmic proteins, indicating that MVs from both of the outer and inner membranes could be released into the extracellular milieu. Subcellular fractionation analysis revealed that the Kil proteins translocated to both the outer and inner membranes in whole cells of BW25113 kil+. Furthermore, the BW25113 kil+ showed not only reduced viability in the stationary growth phase, but also increased susceptibility to killing by predator bacteria, Vibrio cholerae expressing the type VI secretion system, despite no obvious change in morphology and physiology of the bacterial membrane under regular culture conditions. Taken together, our findings suggest that there is risk of increasing biofilm formation and spreading of numerous MVs releasing various cellular components due to kil gene expression. From another point of view, our findings could also offer efficient MV production strategies using a conditional kil vector in biotechnological applications.

A novel human cell culture model to study visceral smooth muscle phenotypic modulation in health and disease.

Adaptation of the smooth muscle cell (SMC) phenotype is essential for homeostasis and is often involved in pathologies of visceral organs (e.g., uterus, bladder, gastrointestinal tract). In vitro studies of the behavior of visceral SMCs under (patho)-physiological conditions are hampered by a spontaneous, uncontrolled phenotypic modulation of visceral SMCs under regular tissue culture conditions. We aimed to develop a new visceral SMC culture model that allows controlled phenotypic modulation. Human uterine SMCs [ULTR and telomerase-immortalized human myometrial cells (hTERT-HM)] were grown to confluency and kept for up to 6 days on regular tissue culture surfaces or basement membrane (BM) matrix-coated surfaces in the presence of 0-10% serum. mRNA and protein expression and localization of SMC-specific phenotype markers and their transcriptional regulators were investigated by quantitative PCR, Western blotting, and immunofluorescence. Maintaining visceral SMCs confluent for 6 days increased α-smooth muscle actin (1.9-fold) and smooth muscle protein 22-α (3.1-fold), whereas smooth muscle myosin heavy chain was only slightly upregulated (1.3-fold). Culturing on a BM matrix-coated surface further increased these proteins and also markedly promoted mRNA expression of γ-smooth muscle actin (15.0-fold), smoothelin (3.5-fold), h-caldesmon (5.2-fold), serum response factor (7.6-fold), and myocardin (8.1-fold). Whereas additional serum deprivation only minimally affected contractile markers, platelet-derived growth factor-BB and transforming growth factor β1 consistently reduced versus increased their expression. In conclusion, we present a simple and reproducible visceral SMC culture system that allows controlled phenotypic modulation toward both the synthetic and the contractile phenotype. This may greatly facilitate the identification of factors that drive visceral SMC phenotypic changes in health and disease.

Interleukin-6 induces VEGF secretion from prostate cancer cells in a manner independent of androgen receptor activation.

The reduced androgen-sensitivity of prostate cancer (PCa) cells is an important clinical development because of its association with the cells' progression to castration-resistant prostate cancer (CRPC). During androgen deprivation therapy (ADT), stroma-derived growth factors and cytokines can activate the androgen receptor (AR). For example, IL-6 is a multifunctional cytokine that is involved in the malignancy of PCa cells through AR activation. In the present study, we used an androgen-sensitive human PCa cell line (LNCaP) and its sublines to investigate the relationship between the responsiveness of PCa cells to IL-6 treatment and the cellular AR signaling pathway. The androgen-low-sensitive F10 and E9 cells were obtained from LNCaP cells by limiting dilution method in regular culture condition. In contrast, the androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging in hormone-depleted condition. Original carcinoma-associated fibroblasts (CAFs) PCaSC-8 and PCaSC-9 cells were isolated from needle biopsy samples of PCa patients. In fibroblasts derived from PCa patients, IL-6 secretion was generally higher than that observed with normal fibroblasts. In contrast, IL-6 secretion was not detected in LNCaP and its sublines. The soluble IL-6 receptor was detected in PCa cells but not in fibroblasts. IL-6 treatment suppressed cell growth of LNCaP, F10, and E9 cells but not AIDL cells and it was accompanied with neuroendocrine-like differentiation. Induction of PSA secretion was observed in IL-6-treated LNCaP and F10 cells. VEGF secretion was strongly induced in IL-6-treated LNCaP and AIDL cells. IL-6-induced VEGF secretion was significantly suppressed by a PI3K inhibitor (LY294002) and it was accompanied by inhibited phosphorylation of Akt. Our results suggest that IL-6 might induce VEGF secretion from PCa cells in a manner independent of AR activation. To prevent IL-6-induced VEGF secretion, inhibition of the PI3K/AKT signaling pathway could be an important pharmacological goal regardless of ADT.