Normal Growth Medium Research Articles

The Potential of Bioactive Fish Collagen Oligopeptides against Hydrogen Peroxide-Induced NIH/3T3 and HUVEC Damage: The Involvement of the Mitochondria.

Extensive in vivo investigations have demonstrated the antioxidant properties of fish collagen oligopeptides (FCOPs). One of the main causes of aging and chronic non-communicable diseases is oxidative stress. Therefore, FCOPs have a broad range of applications in illness prevention and delaying aging from the standpoint of the "food is medicine" theory. However, the mechanisms that underpin the antioxidant activity of FCOPs are not completely understood. The specific objective of this essay was to investigate the antioxidant effect of FCOPs and its possible mechanism at the cellular level. Mouse embryonic fibroblasts NIH/3T3 and human vein endothelial cells (HUVECs) were exposed to 200 µM hydrogen peroxide containing different concentrations of FCOPs for 4 h and were supplemented with different concentrations of FCOPs for 24 h. Normal growth medium without FCOPs was applied for control cells. An array of assays was used to evaluate the implications of FCOPs on cellular oxidative stress status, cellular homeostasis, inflammatory levels, and mitochondrial function. We found that FCOPs exerted a protective effect by inhibiting reactive oxygen species (ROS) production, enhancing superoxide dismutase (SOD) and endothelial nitric oxide synthase (eNOS) activities and cell viability, inhibiting cell cycle arrest in the G1 phase, suppressing interleukin-1β (IL-1β), IL-6, matrix metalloproteinase-3 (MMP-3) and intercellular adhesion molecule-1(ICAM-1) secretion, downregulating nuclear factor-kappa B (NF-κB) activity, protecting mitochondrial membrane potential, and increasing ATP synthesis and NAD+ activities in both cells. FCOPs had a stronger antioxidant impact on NIH/3T3 than on HUVECs, simultaneously increasing glutathione peroxidase (GSH-Px) activity and decreasing malondialdehyde (MDA) content in NIH/3T3. These findings indicate that FCOPs have antioxidant effects on different tissue cells damaged by oxidative stress. FCOPs were therefore found to promote cellular homeostasis, inhibit inflammation, and protect mitochondria. Meanwhile, better health outcomes will be achieved by thoroughly investigating the effective dose and intervention time of FCOPs, as the absorption efficiency of FCOPs varies in different tissue cells.

Abstract 6946: Unravelling intra-tumor phenotypic heterogeneity in colorectal cancer organoids

Abstract Colorectal cancer (CRC) is one of the most diagnosed cancers worldwide. Though several treatment strategies are available today, around a million people die of this disease each year worldwide. Thus, there is still a pivotal need for new and more effective treatments. Genetic and non-genetic intra-tumor heterogeneity has emerged lately as an intrinsic feature of malignancies and as one of the factors responsible for therapy resistance or tolerance. In particular, the nature and role that phenotypically distinct, yet genetically indistinguishable, subpopulations have on tumor biology is far from being understood. Here we investigate phenotypic heterogeneity in CRC by identifying specific transcriptionally well defined cell subpopulations. We aim at deciphering the interactions between populations in terms of differentiation and plasticity, tumor growth and potentially exposed targetable vulnerabilities. To this end, we used patient-derived metastatic CRC (mCRC) organoids, including KRAS wild type (wt) and mutant (kras) models. We performed in total scRNAseq for 7 mCRC samples (4 wt; 3 kras) in different growth media: normal growth medium (supplemented with EGF) (n=1); medium without EGF (noEGF) (n=7); or treated with Cetuximab (CTX, a clinically approved monoclonal antibody against EGFR) (n=7). We found that the wt sample grown in EGF presents 2 distinct subpopulations: one with stemness characteristics (LGR5 high) and the other with a more differentiated phenotype (KRT20 high), in agreement with the current bibliography. Interestingly, in EGF deprived cultures new subpopulations appear. In particular, we were able to identify a subpopulation with distinct expression of WNT-related genes (APCDD1, WNT6, WIF1). This subpopulation is also present, and in cases increasing in fraction, when organoids are treated with CTX (in 3 out of 7 samples). Interestingly, a fraction of the WNT positive subpopulation seems to be able to proliferate under CTX treatment (Ki67 positive). We identified the negative regulator of the WNT pathway, APCDD1, as a potential marker to track this subpopulation. We took one wt and one kras sample as models to validate these findings by flow cytometry. Furthermore, sorting of APCDD1 positive and negative cells, and further RT-qPCR analysis, confirmed good correlation between antibody staining and gene expression. Moreover, we found that both types of cells are able to re-grow as organoids after re-culturing. This suggests that this WNT-associated subpopulation could be contributing, at least in part, to CTX tolerance or resistance. Our results show promising potential to help unravel basic features of intra-tumor heterogeneity both in basal conditions and upon treatment, and possibly to find new therapeutic targets. Citation Format: Sabrina J. Fletcher, Irene Catalano, Elena Grassi, Sofia Borgato, Barbara Peracino, Elena Piretto, Luca Alessandri, Raffaele Calogero, Luca Primo, Livio Trusolino, Andrea Bertotti, Alberto Puliafito. Unravelling intra-tumor phenotypic heterogeneity in colorectal cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6946.

HT22 cell differentiation reduces insulin receptor levels

Purpose: The brain is an insülin-sensitive organ and has widespread insulin receptor (IR) expression. IR signaling in the brain is essential for neuronal development, feeding behavior, body weight, and cognitive processes such as attention, learning, and memory. HT22 cells, which are derived from parent HT4 cells that are immortalized from primary mouse hippocampal neuronal cells are used in research related to insulin signaling. However, the role of these cells in insulin signaling is not known. In this study, we aimed to examine IR levels in cells differentiated using neurobasal medium. Material and methods: For the study, briefly, the cells were seeded in 6-well plates at 2x105 cells/well for 24 h. After the cells reached 80% confluence, the normal growth medium was replaced with a differentiation medium and the cells were incubated for 72 hours at 37 0C in 5% CO2. Western blot procedure was used to determine the expression of the IR. Result: Our results show that differentiation of HT22 cells stimulates neurite outgrowth. Furthermore, IR protein levels were significantly downregulated in differentiated HT22 cells. Conclusion: This finding may require careful consideration of the use of neurobasal medium in conditions where IR signaling is important.

Melphalan-Induced Multiple Myeloma Cells Exhibit a Senescent-like Dormant Phenotype

Multiple myeloma (MM) is an incurable plasma cell malignancy. Despite therapeutic advances that have vastly improved survival, all patients are expected to relapse. One mechanism that may contribute to MM relapse is tumor dormancy. Dormant MM cells may persist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells may then exit dormancy following treatment to drive disease relapse. Dormancy shares many features with cellular senescence, a state of stable growth arrest induced by cell stress. Chemotherapies that produce genotoxic stress have been associated with therapy-induced senescence (TIS). One such genotoxic agent, melphalan (Mel), is routinely administered to MM patients for myeloablation prior to autologous bone marrow transplant. We hypothesized that High Dose Mel (HDM) induces a senescent-like dormant phenotype in surviving MM cells. To test our hypothesis, we developed an in vitro model for HDM. We identified 10µM Mel as a concentration to induce >90% cell toxicity (HDM) in 5TGM1 mouse MM cells over 10 days. 5TGM1-GFP MM cells were cultured with HDM or vehicle (Veh) for 6 hours; cells were then cultured in normal growth medium in the presence of primary mouse (C57BL/6) bone marrow stromal cells (BMSCs). Co-cultures were imaged by fluorescent microscopy every other day to quantify cumulative population doublings (CPD) and cell morphology over the following 10 days. The effect of HDM on CPDs was also evaluated in the human RPMI-8226 MM cell line. 5TGM1-GFP cells were isolated by FACS on day 10 for evaluation of senescence histological characteristics and gene expression. All studies represent n=3 independent experiments unless otherwise indicated. HDM-5TGM1 cells exhibited a significant reduction in mean CPDs over 10 days compared to Veh (HDM 0.32±0.31, Veh 6.72±0.69, p<0.0001), indicating stable growth arrest in HDM treated cells consistent with senescence. HDM-5TGM1 cells were also significantly larger versus Veh (median size in pixels units: HDM 413.2±99.98, Veh 207±8.88, p<0.05), consistent with classical enlarged morphology in senescent cells. Preliminary assessment (n=1) of HDM-RPMI-8226 cells showed similar growth arrest (mean cumulative doublings: HDM 0.058±0.101, Veh 4.185±0.358) and enlargement (mean size in pixel units: HDM 804.1±6.048, Veh 526.9±2.115), suggesting a translatable mechanism in humans. FACS-sorted 5TGM1-GFP cells were stained for telomere associated foci (TAF), a marker of persistent DNA damage characteristic of senescent cells. HDM-5TGM1 cells showed a higher percentage of TAF+ cells (Percent TAF+ cells: HDM 39.7%±9.1%, Veh 10.6%±0.8%, n=2). Sorted HDM-5TGM1 cells also exhibited significant increases over Veh (p<0.001) in senescence markers ( Cdkn1a, Cdkn1c, Glb1), senescence cell anti-apoptosis pathway genes (SCAPs, Bcl2l1), and senescence associated secretory phenotype (SASP) genes ( Ccl5, Icam1, Mmp13). In addition, HDM-5TGM1 cells showed significant increases in myeloid markers that have previously been shown to be increased in dormant 5TGM1 ( Axl, Fcer1g, Mpeg1). Single cell RNA-sequencing was used to further evaluate senescence gene expression changes in growth arrested HDM-5TGM1 cells. Gene set enrichment analysis (GSEA) of genes differentially expressed in HDM-5TGM1 versus Veh cells was performed for custom senescence phenotyping gene sets (Senescence Up, SenUp; Senescence Growth Arrest, SenGA; Inflammatory SASP, iSASP; SCAPs; and Plasma Cell Senescence; PCSen) as well as mouse ortholog versions of a published senescence and biological aging gene set (SenMayo) and KEGG curated gene sets. GSEA of HDM-5TGM1 versus Veh cells confirmed significant (FDR<0.25) upregulation of iSASP, MayoSen, PCSen, SenUp, SCAPs, and SenGA gene sets. Concurrently, GSEA revealed significant downregulation of KEGG DNA replication, cell cycle, and nucleotide excision repair gene sets, supporting our observations of proliferative arrest and persistent DNA damage TAF. Our findings demonstrate that HDM-MM cells exhibit multiple features of senescence, suggesting a potential mechanistic role for TIS in MM dormancy and highlight a novel therapeutic target that may be present in patients post-HDM. Thus, targeting senescence may be a novel approach to eliminate dormant MM cells and prevent relapse.

Osteoinductive and Osteogenic Capacity of Freeze-Dried Bovine Bone Compared to Deproteinized Bovine Bone Mineral Scaffold in Human Umbilical Cord Mesenchymal Stem Cell Culture: An In Vitro Study.

Freeze-dried bovine bone scaffold (FDBB) or decellularized FDBB (dc-FDBB) was developed as an ideal scaffold with osteoinductive properties. This research aims to compare the osteoinductive properties marked by the expression of runt-related transcription factor-2 (RUNX2) and Osterix (OSX) and the osteogenic capacity of these scaffolds imbued with human umbilical cord mesenchymal stem cells (hUCMSCs). This study was performed in five experimental groups: a negative control group (C-) of hUCMSCs with a normal growth medium, a positive control group (C + ) of hUCMSCs with an osteogenic medium, experimental group 1 (E1) with an FDBB conditioned medium (CM), and experimental group 2 (E2) with a dc-FDBB-CM, and a third experimental group (E3) consisting of a DBBM-CM. Alizarin red staining was performed to qualitatively assess osteoinductive capacity. RUNX2 and OSX expression was quantified using real-time quantification polymerase chain reaction with two replications on day six (D6) and day 12 (D12) as fold changes. This experiment revealed that hUCMSCs were positively expressed by CD73, CD90, and CD105 but were not expressed by CD34. Alizarin red staining showed that E1 had the most calcium deposition on D6 and D12, followed by E3 and then E2 The RUNX2 and OSX expression was higher in E1 but this difference was not significant. The OSX expression in E1,E2,E3 was lower on D12 and C+ of OSX had the highest expression. There was a significant difference of fold change measured between all groups (p < 0.05), and there was no significant difference between any of the groups treated with OSX and RUNX2 on D6 and D12. FDBB osteoinduction and osteogenic capacity were higher when compared with DBBM and dc-FDBB.

Extracellular Matrix Derived From Dental Pulp Stem Cells Promotes Mineralization.

Background: Extracellular matrix (ECM) plays a pivotal role in many physiological processes. ECM macromolecules and associated factors differ according to tissues, impact cell differentiation, and tissue homeostasis. Dental pulp ECM may differ from other oral tissues and impact mineralization. Thus, the present study aimed to identify the matrisome of ECM proteins derived from human dental pulp stem cells (DPSCs) and its ability to regulate mineralization even in cells which do not respond to assaults by mineralization, the human gingival fibroblasts (GF). Methods: ECM were extracted from DPSCs cultured in normal growth medium supplemented with L-ascorbic acid (N-ECM) or in osteogenic induction medium (OM-ECM). ECM decellularization (dECM) was performed using 0.5% triton X-100 in 20 mM ammonium hydroxide after 21 days. Mass spectrometry and proteomic analysis identified and quantified matrisome proteins. Results: The dECM contained ECM proteins but lacked cellular components and mineralization. Interestingly, collagens (COL6A1, COL6A2, and COL6A3) and elastic fibers (FBN1, FBLN2, FN1, and HSPG2) were significantly represented in N-ECM, while annexins (ANXA1, ANXA4, ANXA5, ANXA6, ANXA7, and ANXA11) were significantly overdetected in OM-ECM. GF were reseeded on N-dECM and OM-dECM and cultured in normal or osteogenic medium. GF were able to attach and proliferate on N-dECM and OM-dECM. Both dECM enhanced mineralization of GF at day 14 compared to tissue culture plate (TCP). In addition, OM-dECM promoted higher mineralization of GF than N-dECM although cultured in growth medium. Conclusions: ECM derived from DPSCs proved to be osteoinductive, and this knowledge supported cell-derived ECM can be further utilized for tissue engineering of mineralized tissues.

Transwell Insert-Embedded Microfluidic Devices for Time-Lapse Monitoring of Alveolar Epithelium Barrier Function under Various Stimulations.

This paper reports a transwell insert-embedded microfluidic device capable of culturing cells at an air-liquid interface (ALI), mimicking the in vivo alveolar epithelium microenvironment. Integration of a commercially available transwell insert makes the device fabrication straightforward and eliminates the tedious device assembly processes. The transwell insert can later be detached from the device for high-resolution imaging of the cells. In the experiments, the cells showing type-I pneumocyte markers are exploited to construct an in vitro alveolar epithelium model, and four culture conditions including conventional liquid/liquid culture (LLC) and air–liquid interface (ALI) cell culture in normal growth medium, and ALI cell culture with inflammatory cytokine (TNF-α) stimulation and ethanol vapor exposure are applied to investigate their effects on the alveolar epithelium barrier function. The barrier permeability is time-lapse monitored using trans-epithelial electrical resistance (TEER) measurement and immunofluorescence staining of the tight junction protein (ZO-1). The results demonstrate the functionalities of the device, and further show the applications and advantages of the constructed in vitro cell models for the lung studies.

Use of Response Surface Methodology in optimization of biomass, lipid productivity and fatty acid profiles of marine microalga Dunaliella parva for biodiesel production

Abstract In order to reduce fossil fuel consumption, renewable energy sources such as algal biomass have attracted more interest for the production of biodiesel as an energy source. In the current study, marine microalga Dunaliella parva was mainly assessed for higher biomass productivity, lipid content and productivity by the optimization of growth medium with Response Surface Methodology (RSM) for production of biodiesel. Other biochemical compounds such as carbohydrates, proteins and free amino acids content and productivity were also evaluated by RSM. Maximum biomass productivity, lipid content and productivity (48.59 mg/L/day, 39.08 % and 19.91 mg/L/day, respectively) was attained by RSM optimized medium containing 0.63 g/L nitrogen, 0.02 g/L phosphorus and 1.61 M NaCl, which are 1.2, 1.5 and 1.4 folds, respectively, higher than normal growth medium and agreed well with the predicted value (49.85 mg/L/day, 37.51 % and 19.49 mg/L/day). In addition, saturated fatty acids increased from 42.8 % to 59.7%, polyunsaturated fatty acids decreased from 54.9% to 40.3% and the biodiesel properties almost complied with international standards, ASTM D6751 and EN 14214 under optimized conditions. Thus, D. parva has potential as an alternative source for the biodiesel production of excellent quality.

Rejuvenated ageing mesenchymal stem cells by stepwise preconditioning ameliorates surgery-induced osteoarthritis in rabbits

AimsAgeing-related incompetence becomes a major hurdle for the clinical translation of adult stem cells in the treatment of osteoarthritis (OA). This study aims to investigate the effect of stepwise preconditioning on cellular behaviours in human mesenchymal stem cells (hMSCs) from ageing patients, and to verify their therapeutic effect in an OA animal model.MethodsMesenchymal stem cells (MSCs) were isolated from ageing patients and preconditioned with chondrogenic differentiation medium, followed by normal growth medium. Cellular assays including Bromodeoxyuridine / 5-bromo-2'-deoxyuridine (BrdU), quantitative polymerase chain reaction (q-PCR), β-Gal, Rosette forming, and histological staining were compared in the manipulated human mesenchymal stem cells (hM-MSCs) and their controls. The anterior cruciate ligament transection (ACLT) rabbit models were locally injected with two millions, four millions, or eight millions of hM-MSCs or phosphate-buffered saline (PBS). Osteoarthritis Research Society International (OARSI) scoring was performed to measure the pathological changes in the affected joints after staining. Micro-CT analysis was conducted to determine the microstructural changes in subchondral bone.ResultsStepwise preconditioning approach significantly enhanced the proliferation and chondrogenic potential of ageing hMSCs at early passage. Interestingly, remarkably lower immunogenicity and senescence was also found in hM-MSCs. Data from animal studies showed cartilage damage was retarded and subchondral bone remodelling was prevented by the treatment of preconditioned MSCs. The therapeutic effect depended on the number of cells applied to animals, with the best effect observed when treated with eight millions of hM-MSCs.ConclusionThis study demonstrated a reliable and feasible stepwise preconditioning strategy to improve the safety and efficacy of ageing MSCs for the prevention of OA development.Cite this article: Bone Joint Res 2021;10(1):10–21.

Functional differentiation of nitrate-reducing isolates regulated by long-term fertilization in a rice paddy soil

Fertilization can cause obvious shifts in nitrate-reducing community composition in agricultural ecosystems; however, little is known about the behaviors and functional characters of isolated nitrate reducers adapted to a specific environment. In this study, 849 nitrate-reducing bacteria were isolated from various fertilization treatments in a long-term paddy field experiment; the isolates were further characterized in functions with both culture-dependent and independent methodologies. The results showed that CK (no fertilizer) treatment had four genera with even relative abundance, whereas the other three treatments had their own predominant genera with Chromobacterium in nitrogen (N) fertilizer, Serratia in NPK (nitrogen, phosphorus, and potassium fertilizer) and Enterobacter in NPKC treatment (NPK plus rice straw). The isolates of Serratia and Enterobacter grew faster and produced significantly more nitrites than those of Chromobacterium and Burkholderia in the normal growth medium, suggesting that the dominant isolates from nutrient-rich environment, such as NPK and NPKC treatments, are better adapted to high nutritional conditions. On the contrary, the strains of Chromobacterium and Burkholderia possessed stronger nitrite production ability in comparison with the isolates of Serratia and Enterobacter in the diluted growth medium, indicating that the selected isolates from CK and N treatments have the capability to develop under nutrient-limiting conditions. Our results indicated that the behaviors and functions of nitrate reducers appears to be important in adapting to their dwelling habitats.

Hypothermic preservation of endothelialized gas-exchange membranes.

Endothelialization of the blood contacting surfaces of blood-contacting medical devices, such as cardiovascular prostheses or biohybrid oxygenators, represents a plausible strategy for increasing their hemocompatibility. Nevertheless, isolation and expansion of autologous endothelial cells (ECs) usually requires multiple processing steps and time to obtain sufficient cell numbers. This excludes endothelialization from application in acute situations. Off-the-shelf availability of cell-seeded biohybrid devices could be potentially facilitated by hypothermic storage. In this study, the survival of cord-blood-derived endothelial colony forming cells (ECFCs) that were seeded onto polymethylpentene (PMP) gas-exchange membranes and stored for up to 2 weeks in different commercially available and commonly used preservation media was measured. While storage at 4°C in normal growth medium (EGM-2) for 3 days resulted in massive disruption of the ECFC monolayer and a significant decline in viability, ECFC monolayers preserved in Chillprotec could recover after up to 14 days with negligible effects on their integrity and viability. ECFC monolayers preserved in Celsior, HTS-FRS, or Rokepie medium showed a significant decrease in viability after 7 days or longer periods. These results demonstrated the feasibility of hypothermic preservation of ECFC monolayers on gas-exchange membranes for up to 2 weeks, with potential application on the preservation of pre-endothelialized oxygenators and further biohybrid cardiovascular devices.

RNA Sequencing Reveals Cancer-Associated Changes in Laryngeal Cells Exposed to Non-Acid Pepsin.

Laryngopharyngeal reflux (LPR) is a common affliction that contributes to laryngeal inflammation, symptoms that impact quality of life, and life-threatening illnesses such as cancer. Effective treatment strategies for LPR are lacking. Pepsin is a proinflammatory and carcinogenic element of refluxate. Investigation of molecular pathways involved in pepsin-mediated damage may lead to identification of novel biomarkers and therapeutic targets for LPR. In this study, RNA sequencing was used to examine changes in human laryngeal epithelial cells following brief pepsin insult. Cells were immortalized to generate a model to aid future study of laryngeal injury and therapeutics. In vitro translational. Laryngeal epithelial cells were cultured from a patient without signs or symptoms of LPR or laryngeal cancer. Cells were treated with 0.1 mg/ml pepsin for 1 hour or normal growth media (control) prior to RNA sequencing. Cells were immortalized via HPV E6/7 and characterized by microscopy, immunohistochemistry, G-banding, and soft agar assay. Three hundred ninety-seven genes exhibited differences in expression with pepsin treatment (P < .05). Pathway analysis revealed association with cancer and related signaling processes including dysregulation of cancer-associated molecules, Metastasis-Associated Lung Adenocarcinoma Transcript 1 and KRT82, and the long-noncoding RNA, lipoprotein receptor-related protein 1 (LRP1)-AS, which regulates the putative pepsin receptor LRP1. A single, brief exposure to pepsin activated cancer-associated signaling pathways in laryngeal cells in vitro, revealing novel mechanisms by which chronic reflux may contribute to carcinogenesis. The cell line developed herein represents a novel tool in which to investigate pepsin-dysregulated pathways identified by RNA sequencing and disparities of tumor proneness of laryngeal subsites. N/A Laryngoscope, 131:121-129, 2021.

Adipose-Derived Mesenchymal Stem Cells Enhance Ovarian Cancer Growth and Metastasis by Increasing Thymosin Beta 4X-Linked Expression.

As shown in our previous studies, growth and metastasis of ovarian cancer can be regulated by adipose-derived mesenchymal stem cells (ADSCs). However, the underlying mechanism has not yet been revealed. In this study, a proteomics analysis was performed to compare protein expression treated with and without ADSCs in ovarian cancer cells. Protein levels were altered in ovarian cancer cells due to the treatment of ADSCs. Thymosin beta 4 X-linked (TMSB4X) levels changed dramatically, and this protein was identified as one of the most important candidate molecules contributing to the tumour-promoting effects of ADSCs. Compared with the cells that are cultured in the normal growth medium, the TMSB4X levels cultured in ADSC-conditioned medium increased significantly in ovarian cancer cells. Furthermore, the growth and invasion of cancer cells were decreased, even in the ADSC-conditioned medium treatment group (P < 0.05), by the inhibition of TMSB4X. As shown in the bioluminescence images captured in vivo, increased ovarian cancer's growth and metastasis, along with elevated TMSB4X expression, were observed in the group of ADSC-conditioned medium, and the tumour-promoting effect of ADSCs was attenuated by the inhibition of TMSB4X. Based on our findings, increased TMSB4X expression may play a role in accelerating the ADSC-mediated proliferation, invasion, and migration of ovarian cancers.

The Parkinson's disease gene PINK1 activates Akt via PINK1 kinase-dependent regulation of the phospholipid PI(3,4,5)P3.

Akt signalling is central to cell survival, metabolism, protein and lipid homeostasis, and is impaired in Parkinson's disease (PD). Akt activation is reduced in the brain in PD, and by many PD-causing genes, including PINK1 This study investigated the mechanisms by which PINK1 regulates Akt signalling. Our results reveal for the first time that PINK1 constitutively activates Akt in a PINK1-kinase dependent manner in the absence of growth factors, and enhances Akt activation in normal growth medium. In PINK1-modified MEFs, agonist-induced Akt signalling failed in the absence of PINK1, due to PINK1 kinase-dependent increases in PI(3,4,5)P3 at both plasma membrane and Golgi being significantly impaired. In the absence of PINK1, PI(3,4,5)P3 levels did not increase in the Golgi, and there was significant Golgi fragmentation, a recognised characteristic of PD neuropathology. PINK1 kinase activity protected the Golgi from fragmentation in an Akt-dependent fashion. This study demonstrates a new role for PINK1 as a primary upstream activator of Akt via PINK1 kinase-dependent regulation of its primary activator PI(3,4,5)P3, providing novel mechanistic information on how loss of PINK1 impairs Akt signalling in PD.This article has an associated First Person interview with the first author of the paper.

HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida

Edwardsiella piscicida is a severe fish pathogen. Haem utilization systems play an important role in bacterial adversity adaptation and pathogenicity. In this study, a speculative haem utilization protein, HutZEp, was characterized in E. piscicida. hutZEp is encoded with two other genes, hutW and hutX, in an operon that is similar to the haem utilization operon hutWXZ identified in V. cholerae. However, protein activity analysis showed that HutZEp is probably not related to hemin utilization. To explore the biological role of HutZEp, a markerless hutZEp in-frame mutant strain, TX01ΔhutZ, was constructed. Deletion of hutZEp did not significantly affect bacterial growth in normal medium, in iron-deficient conditions, or in the presence of haem but significantly retarded bacterial biofilm growth. The expression of known genes related to biofilm growth was not affected by hutZEp deletion, which indicated that HutZEp was probably a novel factor promoting biofilm formation in E. piscicida. Compared to the wild-type TX01, TX01ΔhutZ exhibited markedly compromised tolerance to acid stress and host serum stress. Pathogenicity analysis showed that inactivation of hutZEp significantly impaired the ability of E. piscicida to invade and reproduce in host cells and to infect host tissue. In contrast to TX01, TX01ΔhutZ was defective in blocking host macrophage activation. The expression of hutZEp was directly regulated by the ferric uptake regulator Fur. This study is the first functional characterization of HutZ in a fish pathogen, and these findings suggested that HutZEp is essential for E. piscicida biofilm formation and contributes to host infection.

Microfluidic Collective Cell Migration Assay for Study of Endothelial Cell Proliferation and Migration under Combinations of Oxygen Gradients, Tensions, and Drug Treatments

Proliferation and migration of endothelial cells play an important role in many biological activities, and they can be regulated by various microenvironmental factors. In this paper, a novel microfluidic collective cell migration assay is developed to study endothelial cell migration and proliferation under combinations of three oxygen conditions: normoxia, oxygen gradient, and hypoxia and three medium compositions: normal growth medium, the medium with cytochalasin-D for actin polymerization inhibition, and with YC-1 for hypoxia-inducible factor (HIF) inhibition. The microfluidic device designed in the paper allows cell patterns formed with consistent dimensions using laminar flow patterning. In addition, stable oxygen gradients can be generated within the device by a spatially confined chemical reaction method. The device can be operated in conventional cell incubators with minimal chemical reagents and instrumentation for practical applications. The results show directional collective cell migration of the endothelial cells under the oxygen gradients for all the medium compositions. The directional behavior has never been discussed before, and indicates critical roles of oxygen gradients in guiding endothelial cell migration during various biological activities. The developed assay provides a practical yet powerful tool for further in vitro study of endothelial cell behaviors under various physiological microenvironments.

Nfat5 is involved in the hyperosmotic regulation of Tmem184b: a putative modulator of ibuprofen transport in renal MDCK I cells.

Nuclear factor of activated T cells 5 (NFAT5) is a transcription factor involved in the regulation of several genes involved in the response to extracellular hyperosmolality. Recently, the uptake of ibuprofen by an as yet unknown carrier was suggested in Madin‐Darby canine kidney (MDCK) I cells exposed to hyperosmolality. We therefore speculated that Nfat5 could be involved in the regulation of this ibuprofen carrier. Reverse transfection with siRNA against Nfat5 was used to knock down Nfat5 in MDCK I cells. The uptake of both radiolabelled taurine and ibuprofen was measured in MDCK I cells, first treated with siRNA against Nfat5 and afterwards cultivated with raffinose‐supplemented normal growth medium (500 mOsm) for 24 h. The siRNA transfection resulted in knockdown of Nfat5, and uptake of both taurine and ibuprofen was significantly decreased in transfected MDCK I cells. The decrease in ibuprofen uptake indicates that Nfat5 is involved in upregulation of the ibuprofen carrier. A transcriptome analysis of MDCK I cells treated with siRNA against Nfat5 revealed 989 genes upregulated by Nfat5 during hyperosmotic exposure. From these genes, the gene product transmembrane protein 184b was found to be regulated by Nfat5, and Tmem184b was the only potential gene product involved in the uptake of ibuprofen in MDCK I cells.DatasetThe RNA sequencing dataset is available from the NCBI Gene Expression 452 Omnibus (https://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE122074.

α-Glucosidase Inhibitory Effect of Fermented Fruit Juice of Morinda Citrifolia L and Combination Effect with Acarbose

Fermented fruit juice of M.citrifoliais supposed to be the future nutraceutical beverage due to its antidiabetic and antioxidant activities. The purposes of this study were to characterize the fermented juice microbiologically and chemically and to evaluate itsα-glucosidase inhibition and radical scavenging activities in vitro. The fruit of M.citrifolia was fermented and the fruit juice was obtained and evaluated for its radical scavenging activity based on a DPPH assay. It's in vitroanti diabetic activity on α-glucosidase inhibition was investigated, including its combined effect with acarbose by a Chou-Talalay method. The inhibition mode was evaluated by Line weaver-Burk plots. The juice was identified for its microbiome with 16S sequencing method and pictured with SEM. The bioactive compounds were analysed with LC-MS. The main microbiome was yeast and tentatively identified as Candida. The yeast was not able to grow in the normal growth medium for yeast, such as sabouroud agar. The TPC of the juice was 1,193 μg GAE/ml. The main compounds identified by an LC-MS were short-chain fatty acids (α-ketoglutaric acid and malic acid). The fermented fruit juice showed good α-glucosidase inhibitory and antioxidant activities with IC50 of 28.99 and 14.09μgGAE/ml, respectively. The kinetic study showed a non-competitive inhibition on α-glucosidase. The combination of the juice with acarbose at higher concentrations produced an additive effect on α-glucosidase. However, at lower concentrations, an antagonistic effect was observed. The fermented fruit juice of M.citrifoliais a good beverage with strong antidiabetic and antioxidant effects.

Effect of Fluoride Doping in Laponite Nanoplatelets on Osteogenic Differentiation of Human Dental Follicle Stem Cells (hDFSCs)

Bioactive nanosilicates are emerging prominent next generation biomaterials due to their intrinsic functional properties such as advanced biochemical and biophysical cues. Recent studies show interesting dose-dependent effect of fluoride ions on the stem cells. Despite of interesting properties of fluoride ions as well as nanosilicate, there is no reported literature on the effect of fluoride-doped nanosilicates on stem cells. We have systematically evaluated the interaction of fluoride nanosilicate platelets (NS + F) with human dental follicle stem cells (hDFSCs) to probe the cytotoxicity, cellular transport (internalization) and osteogenic differentiation capabilities in comparison with already reported nanosilicate platelets without fluoride (NS − F). To understand the osteoinductive and osteoconductive properties of the nanosilicate system, nanosilicate treated hDFSCs are cultured in three different medium namely normal growth medium, osteoconductive medium, and osteoinductive medium up to 21 d. NS + F treated stem cells show higher ALP activity, osteopontin levels and significant alizarin red staining compared to NS − F treated cells. This study highlights that the particles having fluoride additives (NS + F) aid in enhancing the osteogenic differentiation capabilities of hDFSCs thus potential nanobiomaterial for periodontal bone tissue regeneration.

Mid1 affects ion transport, cell wall integrity, and host penetration of the entomopathogenic fungus Metarhizium acridum.

Calcium signaling plays important roles in stress tolerance and virulence in fungi. Mid1, an accessory protein of Cch1 calcium channel, has been discussed in baker's yeast and some filamentous fungi. However, functions of the Mid1 gene in entomopathogenic fungi are not clear. In this study, the Mid1 gene was functionally characterized by deleting it in the entomopathogenic fungus Metarhizium acridum. The growth of the ΔMaMid1 mutant was similar as the wild type on normal growth medium, but inhibited by exogenous Ca2+, Fe2+, Mg2+, Mn2+, Li+, and calcium chelator ethylene glycol tetraacetic acid (EGTA). Cation transportation-related genes were upregulated and intracellular calcium concentration was decreased in ΔMaMid1. Deletion of the MaMid1 gene impaired the tolerance to cell wall-disrupting agents but had no impact on heat or ultraviolet irradiation tolerance compared with the wild type. Bioassays showed that ΔMaMid1 had decreased virulence, with defects in the ability to penetrate the host cuticle. Compared with the wild type, appressorium formation on locust wings and fungal growth in the insect hemocoel were significantly decreased in the ΔMaMid1 mutant in a bioassay through topical inoculation. The phenotypes of ΔMaMid1 were fully restored in a complementation strain. Taken together, our study demonstrates that the MaMid1 affects intracellular ion homeostasis and contributes to virulence by affecting the initial penetration process in M. acridum.