The purpose of this study was to assess the effects of berberine (BBR) on thermoregulation in mice exposed to hot (40°C) and cold (4°C) environmental conditions. Four groups of mice were assembled with three different dosages of BBR (0.2, 0.4, and 0.8 mg/kg) and normal saline (control). In room temperature, our largest dosage of BBR (0.8 mg/kg) can reduce rectal temperatures (Tc) of normal mice. In hot conditions, BBR can antagonize the increasing core body temperature and inhibit the expression of HSP70 and TNFα in mice; conversely, in cold conditions, BBR can antagonize the decreasing core body temperature and enhance the expression of TRPM8. This study demonstrates the dual ability of BBR in maintaining thermal balance, which is of great relevance to the regulation of HSP70, TNFα and TRPM8.
In the crystal structure of the title compound, C5H8N3
−, N—H⋯O hydrogen bonds, involving the unprotonated amino-group and the NH+ group in the pyridinium ring and dihydrogenphosphate O atoms, link the cations and anions. A long chain-like stacking of dihydrogenphosphate anions along the c-axis direction is constructed by O—H⋯O hydrogen bonds. Also along the c-axis direction, π–π stacking between inversion-related pyridinium rings [centroid–centroid distance = 3.8051 (10) Å] forms columnar stacks of cations.
Established cell transfection via nucleofection relies on nucleofection buffers with unknown and proprietary makeup due to trade secrecy, inhibiting the possibility of using this otherwise effective method for developing cell therapy. We devised a three-step method for discovering an optimal formulation for the nucleofection of any cell-line. These steps include the selection of the best nucleofection program and known buffer type, selection of the best polymer for boosting the transfection efficiency of the best buffer, and the comparison with the optimal buffer from an established commercial vendor (Amaxa). Using this 3-step selection system, competitive nucleofection formulations were discovered for multiple cell lines, which are equal to or surpass the efficiency of the Amaxa nucleofector solution in a variety of cells and cell lines, including primary adipose stem cells, muscle cells, tumor cells, and immune cells. Through the use of scanning electron microscopy, we have revealed morphological changes, which predispose for the ability of these buffers to assist in transferring plasmid DNA into the nuclear space. Our formulation may greatly reduce the cost of electroporation study in laboratory and boosts the potential of application of electroporation-based cell therapies in clinical trials.
electroporation; cell transfection; cell therapy; adipose stem cells; formulation
Molecules of the title compound, [Co(C12H8Cl2N3)3], lie on a threefold rotation axis. The tris-N,N′-chelated CoIII atom, which is located on the threefold rotation axis, shows a distorted octahedral coordination.
Adipose stem cells have a strong potential for use in cell-based therapy, but the current nucleofection technique, which relies on unknown buffers, prevents their use.
We developed an optimal nucleofection formulation for human adipose stem cells by using a three-step method that we had developed previously. This method was designed to determine the optimal formulation for nucleofection that was capable of meeting or surpassing the established commercial buffer (Amaxa), in particular for murine adipose stem cells. By using this same buffer, we determined that the same formulation yields optimal transfection efficiency in human mesenchymal stem cells.
Our findings suggest that transfection efficiency in human stem cells can be boosted with proper formulation.
Electroporation; Formulation; Stem cells; Transfection; Cell therapy
Adipose tissue contains a heterogeneous population of mature adipocytes, endothelial cells, immune cells, pericytes, and pre-adipocytic stromal/stem cells. To date, the majority of proteomic analyses have focused on intact adipose tissue or isolated adipose stromal/stem cells in vitro. In this study, human subcutaneous adipose tissue from multiple depots (arm and abdomen) obtained from female donors was separated into populations of stromal vascular fraction cells and mature adipocytes. Out of 960 features detected by 2-D gel electrophoresis, a total of 200 features displayed a 2-fold up- or down-regulation relative to each cell population. The protein identity of 136 features was determined. Immunoblot analyses comparing SVF relative to adipocytes confirmed that carbonic anhydrase II was up-regulated in both adipose depots while catalase was up-regulated in the arm only. Bioinformatic analyses of the dataset determined that cytoskeletal, glycogenic, glycolytic, lipid metabolic, and oxidative stress related pathways were highly represented as differentially regulated between the mature adipocytes and stromal vascular fraction cells. These findings extend previous reports in the literature with respect to the adipose tissue proteome and the consequences of adipogenesis. The proteins identified may have value as biomarkers for monitoring the physiology and pathology of cell populations within subcutaneous adipose depots.
DIGE; Adipose tissue; Stem cells; Adipocytes; Proteomics; LC-MS
TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)n is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)nTA(TG)m, is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.
Amyotrophic Lateral Sclerosis (Lou Gehrig's Disease); Neurodegeneration; Ribonuclear Protein (RNP); RNA-binding Protein; RNA Metabolism; FUS/TLS; Frontotemporal Dementia; MECP2; PTBP2; TDP-43
Objective: To explore the effects of insulin-like growth factor-1 (IGF-1) on migration, proliferation and differentiation of mesenchymal stem cells (MSCs). Methods: MSCs were obtained from Sprague-Dawley rats by a combination of gradient centrifugation and cell culture techniques and treated with IGF-1 at concentrations of 5–20 ng/ml. Proliferation of MSCs was determined as the mean doubling time. Expression of CXC chemokine receptor 4 (CXCR4) and migration property were determined by flow cytometry and transwell migration essay, respectively. mRNA expression of GATA-4 and collagen II was determined by reverse transcription-polymerase chain reaction (RT-PCR). Results: The mean doubling time of MSC proliferation was decreased, and the expression of CXCR4 on MSCs and migration of MSCs were increased by IGF-1, all in a dose-dependent manner, while the optimal concentration of IGF-1 on proliferation and migration was different. IGF-1 did not affect the expression of GATA-4 or collagen II mRNA. Conclusions: IGF-1 dose-dependently stimulated the proliferation of MSCs, upregulated the expression of CXCR4, and accelerated migration. There was no apparent differentiation of MSCs to cardiomyocytes or chondrocytes after culturing with IGF-1 alone.
Mesenchymal stem cells (MSCs); Proliferation; Differentiation; Insulin-like growth factor-1 (IGF-1); CXC chemokine receptor 4 (CXCR4); Migration
AIM: To investigate the anti-fibrosis effect of IκB kinase-beta inhibitor (IKK2 inhibitor IMD0354) in liver fibrosis.
METHODS: Twenty male C57BL6 mice were divided into four groups. Five high-fat fed mice were injected with lipopolysaccharide (LPS, 10 mg/kg) intraperitoneally and five high-fat fed mice were without LPS injection to build models of liver injury, and the intervention group (five mice) was injected intraperitoneally with IKK2 inhibitor (IMD 30 mg/kg for 14 d), while the remaining five mice received a normal diet as controls. Hepatic function, pathological evaluation and liver interleukin-6 (IL-6) expression were examined. Western blotting and real-time polymerase chain reaction were used to detect the expressions of nuclear factor-κB (NF-κB), alpha-smooth muscle actin (α-SMA), tumor growth factor-beta1 (TGF-β1), tumor necrosis factor-alpha (TNF-α), typeIand type III collagen proteins and mRNA.
RESULTS: A mouse model of liver injury was successfully established, and IMD decreased nuclear translocation of NF-κB p65 in liver cells. In the IMD-treated group, the levels of alanine aminotransferase (103 ± 9.77 μ/L vs 62.4 ± 7.90 μ/L, P < 0.05) and aminotransferase (295.8 ± 38.56 μ/L vs 212 ± 25.10 μ/L, P < 0.05) were significantly decreased when compared with the model groups. The histological changes were significantly ameliorated. After treatment, the expressions of IL-6 (681 ± 45.96 vs 77 ± 7.79, P < 0.05), TGF-β1 (Western blotting 5.65% ± 0.017% vs 2.73% ± 0.005%, P < 0.05), TNF-α (11.58% ± 0.0063% vs 8.86% ± 0.0050%, P < 0.05), typeIcollagen (4.49% ± 0.014% vs 1.90% ± 0.0006%, P < 0.05) and type III collagen (3.46% ± 0.008% vs 2.29% ± 0.0035%, P < 0.05) as well as α-SMA (6.19 ± 0.0036 μ/L vs 2.16 ± 0.0023 μ/L, P < 0.05) protein and mRNA were downregulated in the IMD group compared to the fibrosis control groups (P < 0.05).
CONCLUSION: IKK2 inhibitor IMD markedly improved non-alcoholic fatty liver disease in mice by lowering NF-κB activation, which could become a remedial target for liver fibrosis.
Liver fibrosis; IKK2 inhibitor; Nuclear factor-kappa B; Tumor growth factor-beta1; Interleukin-6; Alpha-smooth muscle actin; C57BL mouse
Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for ∼40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca2+) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for ∼80% of passive Ca2+ leak from the endoplasmic reticulum. Deficient Ca2+ signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca2+ leak function of presenilins is independent of their γ-secretase activity. Our data suggest a Ca2+ signaling function for presenilins and provide support for the “Ca2+ hypothesis of AD.”
Perfluorooctane sulfonate (PFOS) is a persistent, bioaccumulative, and toxic chemical that has the potential for long-range transport in the environment. Its use in a wide variety of consumer products and industrial processes makes a detailed characterization of its emissions sources very challenging. These varied emissions sources all contribute to PFOS' existence within nearly all environmental media. Currently, China is the only country documented to still be producing PFOS, though there is no China PFOS emission inventory available. This study reviews the inventory methodologies for PFOS in other countries to suggest a China-specific methodology framework for a PFOS emission inventory. The suggested framework combines unknowns for PFOS-containing product penetration into the Chinese market with product lifecycle assumptions, centralizing these diverse sources into municipal sewage treatment plants. Releases from industrial sources can be quantified separately using another set of emission factors. Industrial sources likely to be relevant to the Chinese environment are identified.
Perfluorooctane sulfonate (PFOS); inventory methodology, China; Persistent Organic Pollutants; perfluorinated compounds; emissions, industrial sources; wastewater treatment plants
Lactobacillus plantarum is a widespread probiotic bacteria found in many fermented food products. In this study, the whole-cell proteins and secretory proteins of L. plantarum were separated by two-dimensional electrophoresis method. A total of 434 proteins were identified by tandem mass spectrometry, including a plasmid-encoded hypothetical protein pLP9000_05. The information of first 20 highest abundance proteins was listed for the further genetic manipulation of L. plantarum, such as construction of high-level expressions system. Furthermore, the first interaction map of L. plantarum was established by Blue-Native/SDS-PAGE technique. A heterodimeric complex composed of maltose phosphorylase Map3 and Map2, and two homodimeric complexes composed of Map3 and Map2 respectively, were identified at the same time, indicating the important roles of these proteins. These findings provided valuable information for the further proteomic researches of L. plantarum.
TDP-43, or TAR DNA-binding protein 43, is a pathological marker of a spectrum of neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. TDP-43 is an RNA/DNA-binding protein implicated in transcriptional and posttranscriptional regulation. Recent work also suggests that TDP-43 associates with cytoplasmic stress granules, which are transient structures that form in response to stress. In this study, we establish sorbitol as a novel physiological stressor that directs TDP-43 to stress granules in Hek293T cells and primary cultured glia. We quantify the association of TDP-43 with stress granules over time and show that stress granule association and size are dependent on the glycine-rich region of TDP-43, which harbors the majority of pathogenic mutations. Moreover, we establish that cells harboring wild-type and mutant TDP-43 have distinct stress responses: mutant TDP-43 forms significantly larger stress granules, and is incorporated into stress granules earlier, than wild-type TDP-43; in striking contrast, wild-type TDP-43 forms more stress granules over time, but the granule size remains relatively unchanged. We propose that mutant TDP-43 alters stress granule dynamics, which may contribute to the progression of TDP-43 proteinopathies.
AIM: To test the ability of penehyclidine hydrochloride (PHC) to attenuate intestinal injury in a rat cardiopulmonary bypass (CPB) model.
METHODS: Male Sprague-Dawley rats were randomly divided into six groups (eight each): sham-operated control; sham-operated low-dose PHC control (0.6 mg/kg); sham-operated high-dose PHC control (2.0 mg/kg); CPB vehicle control; CPB low-dose PHC (0.6 mg/kg); and CPB high-dose PHC (2.0 mg/kg). Blood samples were collected from the femoral artery 2 h after CPB for determination of plasma diamine oxidase (DAO), D-lactate and endotoxin levels. Spleen, liver, mesenteric lymph nodes and lung were removed for biochemical analyses. Intestinal tissue ultrastructure was examined by electron microscopy.
RESULTS: In the sham-operated groups, high- and low-dose-PHC had no significant impact on the levels of DAO, D-lactate and endotoxin, or the incidence of intestinal bacterial translocation (BT). Serum levels of DAO, D-lactate, endotoxin and the incidence of intestinal BT were significantly increased in the surgical groups, compared with the sham-operated groups (0.543 ± 0.061, 5.697 ± 0.272, 14.75 ± 2.46, and 0/40 vs 1.038 ± 0.252, 9.377 ± 0.769, 60.37 ± 5.63, and 30/40, respectively, all P < 0.05). PHC alleviated the biochemical and histopathological changes in a dose-dependent manner. Serum levels of DAO, D-lactate, and endotoxin and the incidence of intestinal BT in the high-dose PHC group were significantly lower than in the low-dose PHC group (0.637 ± 0.064, 6.972 ± 0.349, 29.64 ± 5.49, and 14/40 vs 0.998 ± 0.062, 7.835 ± 0.330, 38.56 ± 4.28, and 6/40, respectively, all P < 0.05).
CONCLUSION: PHC protects the structure and function of the intestinal mucosa from injury after CPB in rats.
Penehyclidine hydrochloride; Intestinal mucosa injury; Cardiopulmonary bypass
In the title compound, C14H16ClN3O2S, the dihedral angle between the 4-chlorophenyl and 1,3,4-oxadiazole rings is 67.1 (1)° and the orientation of the amide N—H and C=O bonds is anti. In the crystal, molecules are linked by N—H⋯O and N—H⋯S hydrogen bonds.
Previous studies have demonstrated that EGF and bFGF maintain the stem cell properties of proliferating human adipose-derived stromal/stem cells (hASCs) in vitro. While the expansion and cryogenic preservation of isolated hASCs are routine, these manipulations can impact their proliferative and differentiation potential. This study examined cryogenically preserved hASCs (n = 4 donors), with respect to these functions, after culture with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) at varying concentrations (0–10 ng/ml). Relative to the control, cells supplemented with EGF and bFGF significantly increased proliferation by up to three-fold over 7–8 days. Furthermore, cryopreserved hASCs expanded in the presence of EGF and bFGF displayed increased oil red O staining following adipogenic induction. This was accompanied by significantly increased levels of several adipogenesis-related mRNAs: aP2, C/EBPα, lipoprotein lipase (LPL), PPARγ and PPARγ co-activator-1 (PGC1). Adipocytes derived from EGF- and bFGF-cultured hASCs exhibited more robust functionality based on insulin-stimulated glucose uptake and atrial natriuretic peptide (ANP)-stimulated lipolysis. These findings indicate that bFGF and EGF can be used as culture supplements to optimize the proliferative capacity of cryopreserved human ASCs and their adipogenic differentiation potential.
adipogenesis; adipose-derived stem cells; edipermal growth factor; basic fibroblast growth factor; cryopreservation; differentiation
Circadian mechanisms underlie the physiology of mammals as an adaptation to the earth’s rotation on its axis. Highly conserved core circadian regulatory proteins (CCRP) maintain an oscillatory expression profile in the central and peripheral tissues. The CCRP include both a positive and negative arm as well as downstream transcriptional regulators. Recent studies in murine models have determined that the mRNAs encoding the CCRP are present in multiple adipose tissue depots and exhibit a robust oscillatory expression profile. The current study set out to examine the expression of CCRP mRNAs in human subcutaneous adipose tissues.
Retrospective analysis of total RNA isolated from subcutaneous adipose tissue.
150 healthy female and male lean (BMI < 25), overweight (BMI between 25 and 29.99) or obese (BMI >30) subjects of varied ethnic backgrounds undergoing elective liposuction or surgical procedures.
The expression of the CCRP mRNAs displayed a significant correlation between each other and mRNAs representative of adipogenic biomarkers. Hierarchical cluster analyses of mRNAs isolated from the cohort of female Caucasian subjects (n = 116) identified three major clusters based on expression of downstream CCRP mRNAs. The mRNAs encoding D site of albumin promoter binding protein (DBP), E4 promoter binding protein 4 (E4BP4), PPARγ Co-Activator 1β (PGC-1β), and Rev-erb α were negatively correlated with BMI in a lean cluster (n = 66), positively correlated with BMI in a younger overweight/obese cluster (n = 19), and not significantly correlated with BMI in an older, overweight/obese cluster (n = 31).
These data confirm and extend findings that link the CCRP and circadian mechanisms to the risk of obesity.
Adipose tissue; BMAL1; Circadian; Clock; Cryptochrome; Double Binding Protein (DBP); E4BP4; Period; Peroxisome Proliferator Activated Receptor γ Co-Activator-1 (PGC-1); Rev-Erbα
Posterolateral spinal fusion is the standard treatment for lumbar compression fractures. Adult adipose tissue-derived stem cells (ASCs) promote osteogenesis in vivo and in vitro. The hypothesis tested in this study was that syngeneic and allogeneic ASCs on a biomaterial scaffold composed of tricalcium phosphate and collagen I will accelerate spinal fusion in a rat model. ASCs from male Fischer or ACI rats were loaded onto scaffolds (53,571 cells/mm3) and cultured in stromal media for 48 h. Male Fisher rats were assigned to 4 cohorts (n = 14/cohort) after bilateral decortication of the L4 and L5 transverse processes: (1) No treatment; (2) scaffold only; (3) scaffold + syngeneic ASCs; or (4) scaffold + allogeneic ASCs. Half of each cohort was harvested 4 or 8 weeks after surgery. Spinal fusion was evaluated with radiographs, microcomputed tomography, and light microscopy. Callus did not form in spines without scaffolds. There were no significant differences in callus formation among scaffold cohorts 4 weeks after surgery. Callus formation was more mature in both ASC cohorts versus scaffold alone 8 weeks after surgery based on microstructure as well as radiographic and microcomputed tomographic evidence of active bone formation. Inflammatory cell infiltrate was significantly lower in both ASC cohorts (syngeneic = 18.3±0.85%; allogeneic = 23.5±2.33%) versus scaffold alone (46.8±11.8%) 4 weeks after surgery. Results of this study support syngeneic and allogeneic ASC acceleration of posterior lumbar spinal fusion in a rat model.
adipose; adult stem cells; spine; fusion; rat