Primary Tissue Culture Research Articles

Characterization of drusen formation in a primary porcine tissue culture model of dry AMD

Age-related macular degeneration (AMD) affects millions of individuals worldwide and is a leading cause of blindness in the elderly. In dry AMD, lipoproteinaceous deposits called drusen accumulate between the retinal pigment epithelium (RPE) and Bruch's membrane, leading to impairment of oxygen and nutrient trafficking to the neural retina, and degeneration of the overlying photoreceptor cells. Owing to key differences in human and animal ocular anatomy and the slowly progressing nature of the disease, AMD is not easily modeled invivo. In this study, we further characterize a "drusen-in-a-dish" primary porcine RPE model system by employing vital lipid staining to monitor sub-RPE deposition over time in monolayers of cells cultured on porous transwell membranes. We demonstrate for the first time using a semi-automated image analysis pipeline that the number and size of sub-RPE deposits increases gradually but significantly over time and confirm that sub-RPE deposits grown in culture immunostain positive for multiple known components found in human drusen. As a result, we propose that drusen-in-a-dish cell culture models represent a high-throughput and cost-scalable alternative to animal models in which to study the pathobiology of drusen accumulation and may serve as useful tools for screening novel therapeutics aimed at treating dry AMD.

HDAC inhibitors activate lipid peroxidation and ferroptosis in gastric cancer

Gastric cancer remains among the deadliest neoplasms worldwide, with limited therapeutic options. Since efficacies of targeted therapies are unsatisfactory, drugs with broader mechanisms of action rather than a single oncogene inhibition are needed. Preclinical studies have identified histone deacetylases (HDAC) as potential therapeutic targets in gastric cancer. However, the mechanism(s) of action of HDAC inhibitors (HDACi) are only partially understood. This is particularly true with regard to ferroptosis as an emerging concept of cell death.In a panel of gastric cancer cell lines with different molecular characteristics, tumor cell inhibitory effects of different HDACi were studied. Lipid peroxidation levels were measured and proteome analysis was performed for the in-depth characterization of molecular alterations upon HDAC inhibition. HDACi effects on important ferroptosis genes were validated on the mRNA and protein level.Upon HDACi treatment, lipid peroxidation was found increased in all cell lines. Class I HDACi (VK1, entinostat) showed the same toxicity profile as the pan-HDACi vorinostat. Proteome analysis revealed significant and concordant alterations in the expression of proteins related to ferroptosis induction. Key enzymes like ACSL4, POR or SLC7A11 showed distinct alterations in their expression patterns, providing an explanation for the increased lipid peroxidation. Results were also confirmed in primary human gastric cancer tissue cultures as a relevant ex vivo model.We identify the induction of ferroptosis as new mechanism of action of class I HDACi in gastric cancer. Notably, these findings were independent of the genetic background of the cell lines, thus introducing HDAC inhibition as a more general therapeutic principle.

Increasing Catecholamine Secretion Through NPY in Pheochromocytomas With False-Negative 123 I-MIBG Scintigraphy.

123 I-MIBG has been well established as a functional imaging tool, and 131 I-MIBG therapy is being considered for catecholamine-secreting tumors. Tumors with the characteristics of a noradrenergic biochemical phenotype, small, malignant, metastatic, extra-adrenal, bilateral, and hereditary, especially SDHx -related tumors, are reported to correlate with reduced MIBG uptake. However, the potential molecular mechanisms influencing MIBG uptake have been poorly studied. To identify critical genes that may enhance MIBG accumulation in pheochromocytomas (PCCs), we performed RNA-seq analyses for 16 operated patients with PCCs (6 MIBG-negative and 10 MIBG-positive) combined with RT-qPCR for 27 PCCs (5 MIBG-negative and 22 MIBG-positive) and examined primary cultures of the surgical tissues. In the present study, 6 adrenal nodules of 66 nodules surgically removed from 63 patients with PCCs (9%) were MIBG negative. MIBG, a guanethidine analog of norepinephrine, can enter chromaffin cells through active uptake via the cellular membrane, be deposited in chromaffin granules, and be released via Ca 2+ -triggered exocytosis from adrenal chromaffin cells. When we compared expression of several catecholamine biosynthesis and secretion-associated genes between MIBG-negative and MIBG-positive tumors using transcriptome analyses, we found that neuropeptide Y, which is contained in chromaffin granules, was significantly increased in MIBG-negative tumors. NPY stimulated norepinephrine secretion dose-dependently in primary cell culture derived from MIBG-positive PCC. In our study, MIBG-negative PCCs were all norepinephrine-hypersecreting tumors. These data indicate that NPY upregulation in PCCs may stimulate chromaffin granule catecholamine secretion, which is associated with false-negative 123 I-MIBG scintigraphy.

Exosomal long non-coding RNA MALAT1: a candidate of liquid biopsy in monitoring of Wilms' tumor.

Wilms' tumor (WT) is a rare kidney cancer that primarily affects children. Exosomes are extracellular vesicles that cargo nucleic acids, proteins,etc. for cellular communication. Long non-coding RNAs (lncRNAs) have utility as biomarkers for cancer diagnosis, prognosis, and disease monitoring. We hypothesize that expression of lncRNA, metastasis-associated lung adenocarcinoma transcript-1(MALAT1), is dysregulated and possibly trafficked within exosomes to influence the tissue microenvironment for metastasis and recurrence of WT. We investigated the expression of MALAT1 in thirty WT samples by qPCR. Exosomes were isolated using a precipitated and affinity-binding-based kit, and characterized using TEM, NTA, and DLS. Mean number of exosomes was 9.01×108/mL in primary culture, 1.64×108/mL in urine, and 4.65×108/plasma:400µl. Average yield of total RNA was 1.28µg (primary-culture supernatant:1ml), 1.47µg (Urine:1ml), 1.65µg (Plasma:400 µL). We quantified MALAT1 in exosomes derived from these sources in patients of WT. Expression of MALAT1 was significantly downregulated (p=0.008) in WT samples. This is the first study that demonstrated the presence of lncRNA MALAT1 in various invasive and non-invasive samples of patients with WT(primary tissue culture, urine, and plasma samples).

Utilizing primary human airway mucociliary tissue cultures to model ramifications of chronic E-cigarette usage

Electronic cigarettes are battery powered devices that use a vape-liquid to produce a vapor that is inhaled. A consequence of the rise in e-cigarette usage was the 2019 emergence of a vaping-induced respiratory disease denoted as ‘e-cigarette or vaping use-associated lung injury’ (EVALI). One of the suspected causes of EVALI is Vitamin E Acetate (VEA), which was found to be a diluent in certain illicit vape-pens, whereas nicotine is commonly diluted in equal parts propylene glycol and vegetable glycerin (PG:VG). The prevalent use of e-cigarettes and the emergence of a novel illness has made understanding how e-cigarette vapors affect our respiratory tissues a public health concern. We have designed and produced a simple device that can operate e-cigarettes and deliver the vapor to a chamber containing a standard cell culture multi-well plate. Here we utilize our device to model the response of human airway mucociliary tissue after chronic exposure to vapors produced from either PG:VG or VEA. We note several differences between how PG:VG and VEA vapors interact with and alter airway tissue cultures and suggest potential mechanisms for how VEA-vapors can exacerbate EVALI symptoms. Our device combined with primary human airway tissue cultures make an economical and compact model system that allows for animal-free investigations into the acute and chronic consequences of e-cigarette vapors on primary respiratory cells.

Bioengineered Pancreas-Liver Crosstalk in a Microfluidic Coculture Chip Identifies Human Metabolic Response Signatures in Prediabetic Hyperglycemia.

Aberrant glucose homeostasis is the most common metabolic disturbance affecting one in ten adults worldwide. Prediabetic hyperglycemia due to dysfunctional interactions between different human tissues, including pancreas and liver, constitutes the largest risk factor for the development of type 2 diabetes. However, this early stage of metabolic disease has received relatively little attention. Microphysiological tissue models that emulate tissue crosstalk offer emerging opportunities to study metabolic interactions. Here, a novel modular multitissue organ-on-a-chip device is presented that allows for integrated and reciprocal communication between different 3D primary human tissue cultures. Precisely controlled heterologous perfusion of each tissue chamber is achieved through a microfluidic single "synthetic heart" pneumatic actuation unit connected to multiple tissue chambers via specific configuration of microchannel resistances. On-chip coculture experiments of organotypic primary human liver spheroids and intact primary human islets demonstrate insulin secretion and hepatic insulin response dynamics at physiological timescales upon glucose challenge. Integration of transcriptomic analyses with promoter motif activity data of 503 transcription factors reveals tissue-specific interacting molecular networks that underlie β-cell stress in prediabetic hyperglycemia. Interestingly, liver and islet cultures show surprising counter-regulation of transcriptional programs, emphasizing the power of microphysiological coculture to elucidate the systems biology of metabolic crosstalk.

Tropism of SARS-CoV-2 for human cortical astrocytes

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

Differential susceptibility to SARS‐CoV‐2 in the normal nasal mucosa and in chronic sinusitis

Human nasal mucosa is susceptible to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection and serves as a reservoir for viral replication before spreading to other organs (e.g. the lung and brain) and transmission to other individuals. Chronic rhinosinusitis (CRS) is a common respiratory tract disease and there is evidence suggesting that susceptibility to SARS‐CoV‐2 infection differs between the two known subtypes, eosinophilic CRS and non‐ECRS (NECRS). However, the mechanism of SARS‐CoV‐2 infection in the human nasal mucosa and its association with CRS has not been experimentally validated. In this study, we investigated whether the human nasal mucosa is susceptible to SARS‐CoV‐2 infection and how different endotypes of CRS impact on viral infection and progression. Primary human nasal mucosa tissue culture revealed highly efficient SARS‐CoV‐2 viral infection and production, with particularly high susceptibility in the NECRS group. The gene expression differences suggested that human nasal mucosa is highly susceptible to SARS‐CoV‐2 infection, presumably due to an increase in ACE2‐expressing cells and a deficiency in antiviral immune response, especially for NECRS. Importantly, patients with NECRS may be at a particularly high risk of viral infection and transmission, and therefore, close monitoring should be considered.

Measuring Glucose Consumption and Gluconeogenesis in 3D Human Tissue Cultures with Nanoliter Input Volumes.

Organotypic and microphysiological culture of primary human tissues and cancers has emerged as a powerful set of technologies that allow to faithfully mimic cellular metabolism and functions ex vivo. The predominant 3D culture methods include spheroids and microfluidic chips. These cultures use low cell numbers and culture volumes, which, however, poses important limitations for the available amounts of sample for downstream analyses. Here, we describe a detailed method for the measurement of glucose consumption dynamics in organotypic culture using a bienzymatic colorimetric assay that accurately quantifies glucose levels using nanoliter input volumes. As an example we utilize spheroids consisting of primary human hepatocytes. The assay has been carefully optimized and benchmarked and is compatible with both longitudinal and high-throughput screening in both static and perfused conditions. The method is straightforward and only requires a microplate reader capable of running absorbance kinetic measurements.

Organoid and Primary Epithelial Cultures of Human Prostate Show the Key Role of the Epithelial-to-Mesenchymal Transition in Generation of Tissue-Specific Stromal Cells

The prostate gland (PG) is a small organ in the male reproductive system that is currently the focus of biomedical research due to its leading position in morbidity and mortality from the tissue-specific cancer prostate cancer (PC). The PG epithelium, which undergoes a cancerous transformation, is formed and functions under the control of androgens. At the beginning of the disease, epithelial cells produce an androgen receptor (AR) and are sensitive to androgen-deprivation therapy. However, such therapy inevitably leads to the transition of the disease to the castration-resistant prostate cancer (CRPC), which manifests itself in metastasis and rapid mortality. In CRPC, the cells of the prostate epithelium change their phenotype, that may be associated with AR mutation and loss the sensitivity to specific therapy. The mechanism of PG phenotypic transformation may be hidden in the interaction and formation of the stromal and epithelial cells, which are evident during the establishment of the primary cultures. The aim of this study was to investigate the generation of human PG stromal cells in primary stromal and organoid cultures. We found that, in contrast to the rapid appearance and formation of a homogeneous population of mesenchymal cells in primary stromal cultures of most tissues, human PG cell cultures are formed initially from epithelial cells. They appear in the second week of cultivation and produce cytokeratins (CKs). A homogeneous population of mesenchymal cells producing vimentin is formed only at the end of the fourth week of cultivation. It is accompanied by the disappearance of epithelial cells. At the same time, some epithelial cells simultaneously produce CKs and vimentin. In PG organoid cultures, there is often a concomitant growth of epithelial, but not mesenchymal, cells on culture plastic. During the cultivation of epithelial cells arising from the organoid cultures, they, like the cells of the primary epithelium, exhibit the ability to spontaneous transformation into mesenchymal cells and simultaneously produce CKs and vimentin. Our data suggest that in primary and organoid PG cultures, stromal cells can be formed from epithelium due to the epithelial-to-mesenchymal transition (EMT). The tendency of PG epithelium toward spontaneous EMT may contribute to the mechanism of high sensitivity of prostate tissue to malignant transformation and metastasis. Understanding this mechanism may contribute to the development of effective antitumor therapy of prostate cancer.

Dynamic Physiological Culture of Ex Vivo Human Tissue: A Systematic Review.

Simple SummaryWithin cancer research, a strong emphasis is placed on the development of models that accurately reproduce the conditions in which tumours develop and grow. A limitation of several models is that they fail to replicate the tumour’s blood supply. Our aim was to evaluate the concurrent literature regarding dynamic physiological culture techniques that have been used to successfully culture human tissue. We conducted a systematic review of the literature and identified 22 articles that described the use of different dynamic culture techniques in order to create a system that was physiologically representative. The most common method described was the use of perfusion culture. This article serves as a detailed reference of novel technologies that can be implemented within cancer research in order to improve the physiological conditions of current culture techniques. Realistic cancer models will translate into a greater understanding of the disease which will directly impact on patient outcomes.Conventional static culture fails to replicate the physiological conditions that exist in vivo. Recent advances in biomedical engineering have resulted in the creation of novel dynamic culturing systems that permit the recapitulation of normal physiological processes ex vivo. Whilst the physiological benefit for its use in the culture of two-dimensional cellular monolayer has been validated, its role in the context of primary human tissue culture has yet to be determined. This systematic review identified 22 articles that combined dynamic physiological culture techniques with primary human tissue culture. The most frequent method described (55%) utilised dynamic perfusion culture. A diverse range of primary human tissue was successfully cultured. The median duration of successful ex vivo culture of primary human tissue for all articles was eight days; however, a wide range was noted (5 h–60 days). Six articles (27%) reported successful culture of primary human tissue for greater than 20 days. This review illustrates the physiological benefit of combining dynamic culture with primary human tissue culture in both long-term culture success rates and preservation of native functionality of the tissue ex vivo. Further research efforts should focus on developing precise biochemical sensors that would allow for real-time monitoring and automated self-regulation of the culture system in order to maintain homeostasis. Combining these techniques allows the creation of an accurate system that can be used to gain a greater understanding of human physiology.

Effect of a Topical Formulation on Infective Viral Load in Lambs Naturally Infected with Orf Virus.

IntroductionOrf is a highly contagious eruptive viral disease of the skin and mucosa of sheep and goats. Although vaccination with live or attenuated orf virus is the preferred option for disease control, the vaccine is unavailable in many countries. Treatment of orf lesions involves standard hygiene and in numerous cases, management of presumptive secondary infections with antibiotics, increasing risks of antimicrobial resistance (AMR). The wound dressing formulation Tri-Solfen® containing two local anaesthetics (lignocaine and bupivacaine), adrenaline and an antiseptic (cetrimide) in a gel formulation was developed for pain relief in sheep undergoing surgical husbandry procedures in Australia. Recently, TS therapy was found to reduce suffering and enhance recovery in cattle and buffalo with oral and skin lesions due to foot-and-mouth disease (FMD) virus infection. It was noted that TS has a low pH and is potentially viricidal, potentially aiding disease control.MethodsOne-month-old lambs (n=14), naturally infected with orf, were recruited from a farm during a natural outbreak of the disease. The animals were selected at the early stages of the infection and randomly divided into two cohorts: Group A (n=11) treated with the topical wound gel formulation (TS); and Group B (n=3) an untreated control group. Swabs were obtained before treatment (T0) and on days one (T1), 3 (T2) and 5 (T3) post-treatment, then submitted to direct DNA extraction with real-time PCR quantification, plus incubation with primary tissue cultures from ovine skin fibroblasts (OSF) and T-immortalized goat embryonic fibroblasts (TIGEF).ResultsAlthough no significant differences were found in the clinical progression of the lesions and PCR quantification (p=0.722) between these small cohorts, there was a significant difference (p<0.05) in reduction in infective viral load between the groups when assessed in OSF cell cultures between T0 and T3.ConclusionThese preliminary findings suggest that treatment of early stage lesions with this TS may reduce the infective viral load present in orf lesions.

Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress.

Chronic hypoxic stress induces epigenetic modifications mainly DNA methylation in cardiac fibroblasts, inactivating tumor suppressor genes (RASSF1A) and activating kinases (ERK1/2) leading to fibroblast proliferation and cardiac fibrosis. The Ras/ERK signaling pathway is an intracellular signal transduction critically involved in fibroblast proliferation. RASSF1A functions through its effect on downstream ERK1/2. The antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), decreases oxidative stress from chronic hypoxia, but its effects on these epigenetic changes have not been fully explored. To test our hypothesis, we used an in-vitro model: wild-type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG). The studied animals were housed in hypoxia (10% O2) for 21 days. The right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western blot analyses. Primary C57BL6 mouse cardiac fibroblast tissue culture was used to study the in-vitro model, the downstream effects of RASSF-1 expression and methylation, and its relation to ERK1/2. Our findings showed a significant increase in cardiac fibrosis markers: Collagen 1, alpha smooth muscle actin (ASMA), and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p < 0.05). The expression of DNA methylation enzymes (DNMT 1&3b) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p < 0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p < 0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p < 0.05). Methylation of the RASSF1A promoter region was significantly reduced in the TG hypoxic group compared to the WT hypoxic group (0.59 vs. 0.75, respectively). Based on our findings, we can speculate that EC-SOD significantly attenuates RASSF1A gene methylation and can alleviate cardiac fibrosis induced by hypoxia.

Modeling human peri-implantation placental development and function†.

It is very difficult to gain a better understanding of the events in human pregnancy that occur during and just after implantation because such pregnancies are not yet clinically detectable. Animal models of human placentation are inadequate. In vitro models that utilize immortalized cell lines and cells derived from trophoblast cancers have multiple limitations. Primary cell and tissue cultures often have limited lifespans and cannot be obtained from the peri-implantation period. We present here two contemporary models of human peri-implantation placental development: extended blastocyst culture and stem-cell derived trophoblast culture. We discuss current research efforts that employ these models and how such models might be used in the future to study the "black box" stage of human pregnancy.

Targeting the glucocorticoid receptor signature gene Mono Amine Oxidase-A enhances the efficacy of chemo- and anti-androgen therapy in advanced prostate cancer

Despite increasing options for treatment of castration-resistant prostate cancer, development of drug resistance is inevitable. The glucocorticoid receptor (GR) is a prime suspect for acquired therapy resistance, as prostate cancer (PCa) cells are able to increase GR signaling during anti-androgen therapy and thereby circumvent androgen receptor (AR)-blockade and cell death. As standard AR-directed therapies fail to block the GR and GR inhibitors might result in intolerable side effects, the identification of GR signature genes, which are better suited for a targeted approach, is of clinical importance. Therefore, the specific epithelial and stromal GR signature was determined in cancer-associated fibroblasts as well as in abiraterone and enzalutamide-resistant cells after glucocorticoid (GC) treatment. Microarray and ChIP analysis identified MAO-A as a directly up-regulated mutual epithelial and stromal GR target, which is induced after GC treatment and during PCa progression. Elevated MAO-A levels were confirmed in in vitro cell models, in primary tissue cultures after GC treatment, and in patients after neoadjuvant chemotherapy with GCs. MAO-A expression correlates with GR/AR activity as well as with a reduced progression-free survival. Pharmacological MAO-A inhibition combined with 2nd generation AR signaling inhibitors or chemotherapeutics results in impaired growth of androgen-dependent, androgen-independent, and long-term anti-androgen-treated cells. In summary, these findings demonstrate that targeting MAO-A represents an innovative therapeutic strategy to synergistically block GR and AR dependent PCa cell growth and thereby overcome therapy resistance.

Sulfonated cryogel scaffolds for focal delivery in ex-vivo brain tissue cultures

The human brain has unique features that are difficult to study in animal models, including the mechanisms underlying neurodevelopmental and psychiatric disorders. Despite recent advances in human primary brain tissue culture systems, the use of these models to elucidate cellular disease mechanisms remains limited. A major reason for this is the lack of tools available to precisely manipulate a specific area of the tissue in a reproducible manner. Here we report an easy-to-use tool for site-specific manipulation of human brain tissue in culture. We show that line-shaped cryogel scaffolds synthesized with precise microscale dimensions allow the targeted delivery of a reagent to a specific region of human brain tissue in culture. 3-sulfopropyl acrylate (SPA) was incorporated into the cryogel network to yield a negative surface charge for the reversible binding of molecular cargo. The fluorescent dyes BODIPY and DiI were used as model cargos to show that placement of dye loaded scaffolds onto brain tissue in culture resulted in controlled delivery without a burst release, and labelling of specific regions without tissue damage. We further show that cryogels can deliver tetrodotoxin to tissue, inhibiting neuronal function in a reversible manner. The robust nature and precise dimensions of the cryogel resulted in a user-friendly and reproducible tool to manipulate primary human tissue cultures. These easy-to-use cryogels offer an innovate approach for more complex manipulations of ex-vivo tissue.

The isobaric tags for relative and absolute quantification-based quantitative proteomics of fresh tissue-derived secretome in hepatocellular carcinoma

IntroductionProteomics technology platforms offer an extremely useful tool for the discovery of new cancer biomarkers. Secreted proteins play important roles in signal transduction, cellular growth, proliferation, differentiation, and apoptosis. This study aimed to investigate the molecular signatures of the hepatocellular carcinoma (HCC) by quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ) with liquid chromatography-tandem mass spectrometry (LC-MS/MS).Material and methodsIn this study, we used an iTRAQ-based quantitative proteomic approach to analyse the secretome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from the primary cultures of cancerous tissues, the surrounding noncancerous tissues, and distal noncancerous tissues.ResultsA proteomic analysis of the CM proteins allowed for a total of 5214 identified proteins, of which 190 and 44 proteins were dysregulated in the HCC tissues/distal noncancerous tissues (HCC/DN group) and the adjacent noncancerous tissues/distal noncancerous tissues (AN/DN group) compared with the distal noncancerous tissues. The dysregulated proteins in the HCC/DN group were concentrated in mitogen-activated protein kinase (MAPK) signalling and Janus kinase-signal transducer and activator of the transcription (JAK-STAT) signalling, but the dysregulated proteins in the AN/DN group were more concentrated in the basal material metabolism.ConclusionsThe secretome profile alternations and signalling pathways were associated with HCC incidence and development. The dysregulated proteins in the HCC/DN group were concentrated in the MAPK signalling and JAK-STAT signalling, but the dysregulated proteins in the AN/DN group were more concentrated in the basal material metabolism.

Repeated electromagnetic field stimulation lowers amyloid-\u03b2 peptide levels in primary human mixed brain tissue cultures

Late Onset Alzheimer’s Disease is the most common cause of dementia, characterized by extracellular deposition of plaques primarily of amyloid-β (Aβ) peptide and tangles primarily of hyperphosphorylated tau protein. We present data to suggest a noninvasive strategy to decrease potentially toxic Aβ levels, using repeated electromagnetic field stimulation (REMFS) in primary human brain (PHB) cultures. We examined effects of REMFS on Aβ levels (Aβ40 and Aβ42, that are 40 or 42 amino acid residues in length, respectively) in PHB cultures at different frequencies, powers, and specific absorption rates (SAR). PHB cultures at day in vitro 7 (DIV7) treated with 64 MHz, and 1 hour daily for 14 days (DIV 21) had significantly reduced levels of secreted Aβ40 (p = 001) and Aβ42 (p = 0.029) peptides, compared to untreated cultures. PHB cultures (DIV7) treated at 64 MHz, for 1 or 2 hour during 14 days also produced significantly lower Aβ levels. PHB cultures (DIV28) treated with 64 MHz 1 hour/day during 4 or 8 days produced a similar significant reduction in Aβ40 levels. 0.4 W/kg was the minimum SAR required to produce a biological effect. Exposure did not result in cellular toxicity nor significant changes in secreted Aβ precursor protein-α (sAPPα) levels, suggesting the decrease in Aβ did not likely result from redirection toward the α-secretase pathway. EMF frequency and power used in our work is utilized in human magnetic resonance imaging (MRI, thus suggesting REMFS can be further developed in clinical settings to modulate Aβ deposition.

Rab8 attenuates Wnt signaling and is required for mesenchymal differentiation into adipocytes

Differentiation of mesenchymal stem cells into adipocyte requires coordination of external stimuli and depends upon the functionality of the primary cilium. The Rab8 small GTPases are regulators of intracellular transport of membrane-bound structural and signaling cargo. However, the physiological contribution of the intrinsic trafficking network controlled by Rab8 to mesenchymal tissue differentiation has not been fully defined in vivo and in primary tissue cultures. Here, we show that mouse embryonic fibroblasts (MEFs) lacking Rab8 have severely impaired adipocyte differentiation in vivo and ex vivo. Immunofluorescent localization and biochemical analyses of Rab8a-deficient, Rab8b-deficient, and Rab8a and Rab8b double-deficient MEFs revealed that Rab8 controls the Lrp6 vesicular compartment, clearance of basal signalosome, traffic of frizzled two receptor, and thereby a proper attenuation of Wnt signaling in differentiating MEFs. Upon induction of adipogenesis program, Rab8a- and Rab8b-deficient MEFs exhibited severely defective lipid-droplet formation and abnormal cilia morphology, despite overall intact cilia growth and ciliary cargo transport. Our results suggest that intracellular Rab8 traffic regulates induction of adipogenesis via proper positioning of Wnt receptors for signaling control in mesenchymal cells.

Gut sensing of dietary amino acids, peptides and proteins, and feed-intake regulation in pigs

Amino acids (AA) play key metabolic roles in the regulation of feed intake, growth, and immunity among other physiological functions. Dietary AA supplements (i.e. non-protein-bound synthetic AA) and proteins (through peptides and free AA coming from digestion) regulate appetite by stimulating the release of gut hormones from enteroendocrine cells located throughout the gastrointestinal tract, via activation of transmembrane receptors of the G-protein coupled transmembrane receptor (GPCR) family, including calcium sensing receptor, GPCR of the class C Group 6 Member A (GPRC6A), T1R1–T1R3 of taste receptor family 1 and lysophosphatidic acid receptor 5 (GPR92). In addition, growing evidence supports the role of AA transporters in mediating AA sensing in the gut. While most of the current literature available on the topic relates to research undertaken in laboratory rodents, there has been a growing demand on the confirmation of these physiological mechanisms in pigs. In pigs, limiting essential AA L-tryptophan, and branched-chain AA L-leucine and L-isoleucine were shown to stimulate anorexigenic hormones cholecystokinin (CCK) and glucagon-like peptide 1 mediated by calcium sensing receptor and T1R1–T1R3, using primary tissue cultures. Tryptophan was shown to stimulate the orexigenic hormone ghrelin in pigs. In addition, L-lysine and L-methionine have also been shown to affect feed intake. Other free AA affecting CCK and glucagon-like peptide 1 levels in pigs are L-phenylalanine and L-arginine. Similar functions have been reported for some dietary proteins such as casein, lectin, or whey and for protein hydrolysates. Overall, this review uncovers recent evidence that dietary non-protein-bound AA are efficient modulators of appetite on the basis of their strong effects on orexigenic and anorexigenic hormones in pigs. However, little has been explored to assess the relevance of essential versus non-essential dietary AA impact on feed intake. Further understanding of the role of AA and proteins on appetite modulation and the mechanisms behind gut hormone release could prove to be a relevant tool to improve swine nutrition and performance.