Characterization of an antimicrobial peptide family from the venom gland of Heteropoda venatoria.

Spider venom boasts extensive peptide diversity, constituting a natural biochemical arsenal for defense and predation. The new family HvAMPs, including 9 homologous members, were identified from the unnormalized cDNA library of Heteropoda venatoria venom gland by Sanger sequencing. The putative mature peptide is composed of 22 aliphatic amino acid residues. The mature peptides of HvAMP1 and HvAMP5, with 3 different amino acids, were synthesized and both were shown to adopt an amphipathic α-helical structure and amphipathicity in SDS buffer by CD spectroscopy. In comparison to HvAMP1, HvAMP5 exhibits higher antibacterial activity, particularly against Gram-positive bacteria, coupled with reduced hemolytic activity and cytotoxicity. Results from SYTO 9/PI staining indicate that HvAMP5 acts by disrupting bacterial cell membranes. Analysis of the relationships between structures and functions suggests that HvAMP5 enhances antibacterial activity and reduces mammalian cell toxicity by increasing positive charge and proline substitution. The three residues variation can augment the electrostatic attraction of antibacterial peptides to the bacterial phospholipid bilayer. The present study suggests that the HvAMPs may exert lytic action against cells of different origins to increase cellular and tissue barrier permeability to facilitate spider's defense or predation. Moreover, HvAMP5 holds promise as a novel antibacterial agent for treating Gram-positive bacterial infections. Simultaneously, the numerous diverse amino acid residue substitutions within the HvAMP family offer a template for future study.

Conjugated Linoleic Acid Reduces Lipid Accumulation via Down-regulation Expression of Lipogenic Genes and Up-regulation of Apoptotic Genes in Grass Carp (Ctenopharyngodon idella) Adipocyte In Vitro.

The relationship between conjugated linoleic acid (CLA) and lipogenesis has been extensively studied in mammals and some cell lines, but it is relatively rare in fish, and the potential mechanism of action of CLA reducing fat mass remains unclear. The established primary culture model for studying lipogenesis in grass carp (Ctenopharyngodon idella) preadipocytes was used in the present study, and the objective was to explore the effects of CLA on intracellular lipid and TG content, fatty acid composition, and mRNA levels of adipogenesis transcription factors, lipase, and apoptosis genes in grass carp adipocytes in vitro. The results showed that CLA reduced the size of adipocyte and lipid droplet and decreased the content of intracellular lipid and TG, which was accompanied by a significant down-regulation of mRNA abundance in transcriptional regulators including peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer-binding protein (C/EBP) α, sterol regulatory element-binding protein (SREBP) 1c, lipase genes including fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), lipoprotein lipase (LPL). Meanwhile, it decreased the content of saturated fatty acids (SFAs) and n - 6 polyunsaturated fatty acid (n-6 PUFA) and increased the content of monounsaturated fatty acid (MUFA) and n - 3 polyunsaturated fatty acid (n-3 PUFA) in primary grass carp adipocyte. In addition, CLA induced adipocyte apoptosis through downregulated anti-apoptotic gene B-cell CLL/lymphoma 2 (Bcl-2) mRNA level and up-regulated pro-apoptotic genes tumor necrosis factor-α (TNF-α), Bcl-2-associated X protein (Bax), Caspase-3, and Caspase-9 mRNA level in a dose-dependent manner. These findings suggest that CLA can act on grass carp adipocytes through various pathways, including decreasing adipocyte size, altering fatty acid composition, inhibiting adipocyte differentiation, promoting adipocyte apoptosis, and ultimately decreasing lipid accumulation.

Cuproptosis-related prognostic signatures predict the prognosis and immunotherapy in HCC patients.

Cuproptosis, an unusual type of programmed cell death mechanism of cell death, involved the disruption of specific mitochondrial metabolic enzymes in the occurrence and development of tumors. However, it was still unclear how the relationship between cuproptosis-related genes (CRGs) may contribute to hepatocellular carcinoma (HCC) potential the prognosis of HCC remained limited. Here, the landscape of 14 CRGs in HCC was evaluated using the Cancer Genome Atlas and International Cancer Genome Consortium datasets. And then, 4 CRGs (ATP7A, MTF1, GLS, and CDKN2A) were screened for the construction of risk signatures for prognosis and drug therapy. The HCC patients with CRGs high-risk showed poor prognosis than those with low risk. Moreover, the CRGs risk signature was shown to be an independent prognostic factor and associated with the immune microenvironment in HCC. Meanwhile, we constructed and verified a prognostic model based on cuproptosis-related lncRNAs (Cr-lncRNAs). We obtained 291 Cr-lncRNAs and constructed Cr-lncRNA prognosis signature based on 3 key Cr-lncRNAs (AC026356.1, NRAV, AL031985.3). The Cr-lncRNA prognosis signature was also an independent prognostic factor and associated with the immune microenvironment in HCC. Finally, the drug sensitivity database showed that 8 candidate drugs related to CRGs signature and Cr-lncRNAs signature. In summary, we evaluated and validated the CRGs and Cr-lncRNAs as potential predictive markers for prognosis, immunotherapy, and drug candidate with the personalized diagnosis and treatment of HCC.

A FRET-based ratiometric fluorescent probe for hydrogen polysulfide detection in living cells and zebrafish.

Hydrogen polysulfide (H2Sn, n > 1) is an important active sulfur molecule (RSS) in organisms, which have been considered to be involved in redox signaling and cytoprotective processes. In this work, in order to quickly and accurately detect H2Sn in biosystems, 2-fluoro-5-nitrobenzoic ester was used as the response moiety for H2Sn, and the FRET strategy was adopted to effectively connect the donor (6-hydroxy-2-naphthoic acid) and acceptor (4-substituted-1,8-naphthalimide) to construct a new ratiometric H2Sn fluorescent probe NPNA-H2Sn. NPNA-H2Sn exhibited a more than âˆ¼ 8.0-fold ratio enhancement towards H2Sn at I450/I526 and a very high sensitivity with a very low detection limit of 40.3 nM. Impressive, NPNA-H2Sn was further used for fluorescence imaging of H2Sn in living cells and zebrafish, which showed high-clear ratiometric images. Therefore, we have demonstrated that NPNA-H2Sn could be applied for ratiometric images of endogenous H2Sn in living biosystems and provide a powerful molecular tool for evaluating the physiological and pathological functions of H2Sn.

Convection-enhanced delivery of sorafenib and suppression of tumor progression in a murine model of brain melanoma through the inhibition of signal transducer and activator of transcription 3.

OBJECT Despite recent advances, metastatic melanoma remains a terminal disease, in which life-threatening brain metastasis occurs in approximately half of patients. Sorafenib is a multikinase inhibitor that induces apoptosis of melanoma cells in vitro. However, systemic administration has been ineffective because adequate tissue concentrations cannot be achieved. This study investigated if convection-enhanced delivery (CED) of sorafenib would enhance tumor control and survival via inhibition of the signal transducer and activator of transcription 3 (Stat3) pathway in a murine model of metastatic brain melanoma. METHODS Melanoma cells treated with sorafenib in vitro were examined for signaling and survival changes. The effect of sorafenib given by CED was assessed by bioluminescent imaging and animal survival. RESULTS The results showed that sorafenib induced cell death in the 4 established melanoma cell lines and in 1 primary cultured melanoma cell line. Sorafenib inhibited Stat3 phosphorylation in HTB65, WYC1, and B16 cells. Accordingly, sorafenib treatment also decreased expression of Mcl-1 mRNA in melanoma cell lines. Because sorafenib targets multiple pathways, the present study demonstrated the contribution of the Stat3 pathway by showing that mouse embryonic fibroblast (MEF) Stat3 +/+ cells were significantly more sensitive to sorafenib than MEF Stat3 -/- cells. In the murine model of melanoma brain metastasis used in this study, CED of sorafenib increased survival by 150% in the treatment group compared with animals receiving the vehicle control (p < 0.01). CED of sorafenib also significantly abrogated tumor growth. CONCLUSIONS The data from this study indicate that local delivery of sorafenib effectively controls brain melanoma. These findings validate further investigation of the use of CED to distribute molecularly targeted agents.

Role of microRNA 30a targeting insulin receptor substrate 2 in colorectal tumorigenesis.

MicroRNAs (miRNAs) are dysregulated in many types of malignant diseases, including colorectal cancer. miRNA 30a (miR-30a) is a member of the miR-30 family and has been implicated in many types of cancers. In this study, we determined the expression of miR-30a in human colon cancer tissues and cell lines. miR-30a was found to be significantly downregulated in both the tissues and cell lines. Furthermore, overexpression of miR-30a inhibited, while silencing of miR-30a promoted, cell proliferation, migration, and invasion in vitro. Consistently, stable overexpression of miR-30a suppressed the growth of colon cancer cell xenografts in vivo. Moreover, bioinformatic algorithms and luciferase reporter assays revealed that insulin receptor substrate 2 (IRS2) is a direct target of miR-30a. Further functional studies suggested that repression of IRS2 by miR-30a partially mediated the tumor suppressor effect of miR-30a. In addition, miR-30a inhibited constitutive phosphorylation of Akt by targeting IRS2. Additionally, clinicopathological analysis indicated that miR-30a has an inverse correlation with the staging in patients with colon cancer. Taken together, our study provides the first evidence that miR-30a suppressed colon cancer cell growth through inhibition of IRS2. Thus, miR-30a might serve as a promising therapeutic strategy for colon cancer treatment.

MicroRNA-93 suppress colorectal cancer development via Wnt/ß-catenin pathway downregulating.

MicroRNA-93 (miR-93) is involved in several carcinoma progressions. It has been reported that miR-93 acts as a promoter or suppressor in different tumors. However, till now, the role of miR-93 in colon cancer is unclear. Herein, we have found that expression of miR-93 was lower in human colon cancer tissue and colorectal carcinoma cell lines compared with normal colon mucosa. Forced expression of miR-93 in colon cancer cells inhibits colon cancer invasion, migration, and proliferation. Furthermore, miR-93 may downregulate the Wnt/ß-catenin pathway, which was confirmed by measuring the expression level of the ß-catenin, axin, c-Myc, and cyclin-D1 in this pathway. Mothers against decapentaplegic homolog 7 (Smad7), as an essential molecular protein for nuclear accumulation of ß-catenin in the canonical Wnt signaling pathway, is predicted as a putative target gene of miR-93 by the silico method and demonstrated that it may be suppressed by targeting its 3'UTR. These findings showed that miR-93 suppresses colorectal cancer development via downregulating Wnt/ß-catenin, at least in part, by targeting Smad7. This study revealed that miR-93 is an important negative regulator in colon cancer and suggested that miR-93 may serve as a novel therapeutic agent that offers benefits for colon cancer treatment.

Penicillium marneffei infection within an osteolytic lesion in an HIV-negative patient.

Penicillium marneffei is a thermally dimorphic pathogenic fungus that causes systemic infection similar to disseminated cryptococcosis. P. marneffei is endemic in Southeast Asia, usually infecting HIV-infected individuals; infection of HIV-negative individuals is extremely rare. Here, we describe a disseminated P. marneffei infection within an osteolytic lesion in an HIV-negative patient. A 40-year-old Chinese woman presented with intermittent fever, generalized lymphadenopathy, and a skin rash. Following a sternum biopsy, the patient was diagnosed with P. marneffei infection. An emission computed tomography bone scan revealed the presence of increased radioactivity in the left clavicle and sternum, indicative of an osteolytic lesion. In addition to reporting this very rare case, we also present a brief review of the literature, highlighting the differences in clinical manifestations between HIV-positive and HIV-negative patients infected with P. marneffei as it applies to our case.

Altered expression of polycomb group genes in glioblastoma multiforme.

The Polycomb group (PcG) proteins play a critical role in histone mediated epigenetics which has been implicated in the malignant evolution of glioblastoma multiforme (GBM). By systematically interrogating The Cancer Genome Atlas (TCGA), we discovered widespread aberrant expression of the PcG members in GBM samples compared to normal brain. The most striking differences were upregulation of EZH2, PHF19, CBX8 and PHC2 and downregulation of CBX7, CBX6, EZH1 and RYBP. Interestingly, changes in EZH2, PHF19, CBX7, CBX6 and EZH1 occurred progressively as astrocytoma grade increased. We validated the aberrant expression of CBX6, CBX7, CBX8 and EZH2 in GBM cell lines by Western blotting and qRT-PCR, and further the aberrant expression of CBX6 in GBM tissue samples by immunohistochemical staining. To determine if there was functional significance to the diminished CBX6 levels in GBM, CBX6 was overexpressed in GBM cells resulting in decreased proliferative capacity. In conclusion, aberrant expression of PcG proteins in GBMs may play a role in the development or maintenance of the malignancy.

Folic acid attenuates hyperhomocysteinemia-induced glomerular damage in rats.

The present study investigated whether lowering plasma homocysteine (Hcy) with folic acid (FA) could attenuate hyperhomocysteinemia (HHcy)-associated glomerular damage and possible mechanisms. The HHcy animal model was established by intragastric administration with l-methionine in rats. FA was also given intragastrically. Plasma Hcy and creatinine and urinary albumin were measured. Histological and ultrastructural changes were observed by light and electron microscopes. The expression of alpha-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA) and transforming growth factor-beta1 (TGF-ß1) in the kidney was examined by immunohistochemical staining and western blot analysis. The administration of l-methionine induced HHcy in rats. The HHcy rats developed glomerulosclerosis and fibrosis. Plasma creatinine concentration and urinary albumin excretion were also significantly increased in HHcy rats. Effacement and extensively fusion of podocyte foot process was observed in HHcy rats, which was associated with decreased expression of nephrin protein in renal cortex of HHcy rats. Supplementation with FA lowered plasma Hcy significantly. Plasma creatinine concentration and urinary albumin excretion were also significantly attenuated by FA. Morphologically, HHcy-associated glomerulosclerosis, fibrosis, podocyte foot process effacement and loss of podocyte nephrin, were significantly improved by FA. The expressions of α-SMA, PCNA and TGF-ß1 were increased in renal cortex of HHcy rats, and which were also partially reversed by FA. These data suggest that elevated plasma Hcy is an important pathogenic factor for glomerular damage. Lowering plasma Hcy by FA can inhibit TGF-ß1 expression and attenuate HHcy-induced glomerular damage.

Cytotoxic T lymphocyte trafficking and survival in an augmented fibrin matrix carrier.

Cell-based therapies have intriguing potential for the treatment of a variety of neurological disorders. One such example is genetically engineered cytotoxic T lymphocytes (CTLs) that are being investigated in brain tumor clinical trials. The development of methods for CTL delivery is critical to their use in the laboratory and clinical setting. In our study, we determined whether CTLs can migrate through fibrin matrices and if their migration, survival, and function could be modulated by adding chemokines to the matrix. Our results indicated that CTLs can freely migrate through fibrin matrices. As expected, the addition of the monocyte chemotactic protein-1 (MCP-1), also known as chemokine C-C motif ligand 2 (CCL2), to the surrounding media increased egress of the CTLs out of the fibrin clot. Interleukin (IL) -2 and/or IL-15 embedded in the matrix enhanced T cell survival and further promoted T cell migration. The interleukin-13 receptor alpha 2 specific (IL-13R alpha2) T cells that traveled out of the fibrin clot retained the capacity to kill U251 glioma cells. In summary, CTLs can survive and migrate robustly in fibrin matrices. These processes can be influenced by modification of matrix constituents. We conclude that fibrin matrices may be suitable T cell carriers and can be used to facilitate understanding of T cell interaction with the surrounding microenvironment.

[Effects of erythromycin on hydrogen peroxide-induced interleukin-8 synthesis and regulation of glutathione in human bronchial epithelial cells].

OBJECTIVE: To study the effects of erythromycin on Hydrogen peroxide (H2O2)-induced interleukin-8 synthesis and regulation of glutathione in human bronchial epithelial cells. METHODS: Human bronchial epithelial (16HBE) growth curve was recorded by MTT, cells were divided into three groups (1) control (incubation for 24, 36, 48) (2) H2O2 (Pre-incubation for 24, 36, 48 h before adding H2O2 (3) H2O2 + EM (Pre-incubation EM for 24, 36, 48 h before adding H2O2). IL-8 levels were measured in culture supernatants by ELISA, activation of transcription factor NF-kappaB and AP-1 in HBE was evaluated by Electrophoretic mobility shift assay (EMSA). Intracellular GSH and Gamma-GCS concentrations were measured by spectrophotometric assay, gamma-GCS-HS protein were determined by Western blot. RESULTS: Erythromycin (1 microg/ml, 10 microg/ml) and H2O2 (0.01 mM, 0.1 mM) have no effects on cell growth, Preincubation with EM (5 microg/ml) for 36 h and 48 h significantly inhibit H2O2 (0.01 mmol/L) induced increase of IL-8 levels in HBE supernatants, in the mean time decrease the expression of NF-kB and AP-1. Preincubation with EM (5 microg/ml) for 48 h significantly inhibit H2O2 (0.01 mmol/L) induced increase of gamma-GCS levels, gamma-GCS-HS protein expression and AP-1 binding of gamma-GCS-HS promoter in HBE. However GSH and gamma-GCS-HS protein expression in EM + H2O2 group significantly higher than those in control group. CONCLUSION: Erythromycin inhibits oxidant-mediated IL-8 levels through down-regulation of NF-kB and AP-1 binding in HBE, which can further influence the synthesis of GSH and expression gamma-GCS in HBE.

[Cigarette smoking coacervate up-regulates the expression of gamma-glutamylcysteine synthetase in alveolar epithelial cells mediated by activator protein-1].

OBJECTIVE: To investigate the effects of cigarette smoking coacervate (CSC) on the expression and activation of gamma-glutamylcysteine synthetase (GCS), a rate-limitating enzyme in the synthesis of glutathione (reduced form). METHODS: Rat alveolar epithelial cells of the line CCL149 were cultured and exposed to CSC of the concentrations of 10, 1, and 0.1 microg/ml for 1, 4, 8, 12, 24, and 48 hours respectively. RT-PCR was used to detect the mRNA expression of gamma-GCS, and Western blotting was used to detect the protein expression of gamma-GCS. CCL149 cells were transfected with pGL3/gamma-GCS or blank pGL3 plasmid. The luciferase activity was examined Gel retardation assay was used to detect the binding level of activator protein (AP)-1 with the region of the GCLC promoter in CCL-149 cell. RESULTS: The gamma-GCS mRNA expression levels of the CCL149 cells exposed to CSC > 1 microg/ml for 12, 24, and 48 hours were significantly higher than that of the control group (all P < 0.05). The gamma-GCS protein expression levels of the CCL149 cells exposed to CSC > 1 microg/ml for 12, 24, and 48 hours were significantly higher than that of the control group (all P < 0.05). The gamma-GCS protein activity of the CCL149 cells treated with CSC of the concentrations of 10 microg/ml and 1 microg/ml decreased 1, 4, and 8 hours after and then increased in comparison with the control group (all P < 0.05). The gamma-GCS protein activity levels of the CCL149 cells treated with CSC of the concentration of 0.1 microg/ml for less than 48 hours was not significantly different from those of the control group (all P > 0.05), and the gamma-GCS protein activity level of the CCL149 cells treated with CSC of the concentration of 0.1 microg/ml for 48 hours was significantly higher than that of the control group (P < 0.05). The activity of the luciferase with the plasmids containing 5-flanking regulatory region of rat GCLC gene and the activity of gamma-GCS in the CCL149 cells significantly increased after stimulation of CSC for 12, 24 and 48 hours (all P < 0.05). The binding levels of AP-1 with the region of the GCLC promoter in the CCL149 cells treated with CSC for 12, 24, and 48 hours were significantly increased. CONCLUSION: CSC up-regulates the expression of gamma-GCS by activation of the redox-sensitive transcription factor AP-1.

[Changes of antioxidative capacity and endothelial function before and after treatment among patients with high altitude pulmonary edema].

OBJECTIVE: To investigate the changes of antioxidative capacity and endothelial function among patients with high altitude pulmonary edema (HAPE). METHODS: The serum levels of SOD, MDA, GSH, NO, NOS and ET-1 were measured before and after treatment among 34 cases of patients with HAPE, and 20 local healthy volunteers served as control. RESULTS: The serum levels of SOD, GSH, NO and NOS were lower in patient-group before treatment than after treatment and those in control-group significantly (P < 0.01), while the concentration of MDA and ET-1 were higher in patient-group before treatment than after treatment and those in the control-group significantly (P < 0.01). The serum levels of SOD, MDA, GSH, NO, NOS and ET-1 were not different between patient-group after treatment and the control-group (P > 0.05). CONCLUSION: The results indicated that changes of SOD, MDA, GSH, NO, NOS and ET-1 may participate in the course of HAPE.