Inorganic arsenic inhibits the nucleotide excision repair pathway and reduces the expression of XPC

Chronic exposure to arsenic, most often through contaminated drinking water, has been linked to several types of cancer in humans, including skin and lung cancer. However, the mechanisms underlying its role in causing cancer are not well understood. There is evidence that exposure to arsenic can enhance the carcinogenicity of UV light in inducing skin cancers and may enhance the carcinogenicity of tobacco smoke in inducing lung cancers. The nucleotide excision repair (NER) pathway removes different types of DNA damage including those produced by UV light and components of tobacco smoke. The aim of the present study was to investigate the effect of sodium arsenite on the NER pathway in human lung fibroblasts (IMR-90 cells) and primary mouse keratinocytes. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts (6-4 PP) and cyclobutane pyrimidine dimers (CPDs). We find a concentration-dependent inhibition of the removal of 6-4 PPs and CPDs in both cell types treated with arsenite. Treatment of both cell types with arsenite resulted in a significant reduction in the abundance of XPC, a protein that is critical for DNA damage recognition in NER. The abundance of RNA expressed from several key NER genes was also significantly reduced by treatment of IMR-90 cells with arsenite. Finally, treatment of IMR-90 cells with MG-132 abrogated the reduction in XPC protein, suggesting an involvement of the proteasome in the reduction of XPC protein produced by treatment of cells with arsenic. The inhibition of NER by arsenic may reflect one mechanism underlying the role of arsenic exposure in enhancing cigarette smoke-induced lung carcinogenesis and UV light-induced skin cancer, and it may provide some insights into the emergence of arsenic trioxide as a chemotherapeutic agent.     

Interaction between APC and Fen1 during breast carcinogenesis

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER – adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) – promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.     

Interleukin-36 potently stimulates human M2 macrophages,Langerhans cells and keratinocytes to produce pro-inflammatory cytokines

Interleukin (IL)-36 cytokines belong to the IL-1 family and include three agonists, IL-36 α, β and γ and one inhibitor, IL-36 receptor antagonist (IL-36Ra). IL-36 and IL-1 (α and β) activate similar intracellular pathways via their related heterodimeric receptors, IL-36R/IL-1RAcP and IL-1R1/IL-1RAcP, respectively. However, excessive IL-36 versus IL-1 signaling induces different phenotypes in humans, which may be related to differential expression of their respective receptors.We examined the expression of IL-36R, IL-1R1 and IL-1RAcP mRNA in human peripheral blood, tonsil and skin immune cells by RT-qPCR. Monocyte-derived dendritic cells (MDDC), M0, M1 or M2-polarized macrophages, primary keratinocytes, dermal macrophages and Langerhans cells (LC) were stimulated with IL-1β or IL-36β. Cytokine production was assessed by RT-qPCR and immunoassays.The highest levels of IL-36R mRNA were found in skin-derived keratinocytes, LC, dermal macrophages and dermal CD1a⁺ DC. In the blood and in tonsils, IL-36R mRNA was predominantly found in myeloid cells. By contrast, IL-1R1 mRNA was detected in almost all cell types with higher levels in tonsil and skin compared to peripheral blood immune cells. IL-36β was as potent as IL-1β in stimulating M2 macrophages, keratinocytes and LC, less potent than IL-1β in stimulating M0 macrophages and MDDC, and exerted no effects in M1 and dermal macrophages. Levels of IL-1Ra diminished the ability of M2 macrophages to respond to IL-1.Taken together, these data are consistent with the association of excessive IL-36 signaling with an inflammatory skin phenotype and identify human LC and M2 macrophages as new IL-36 target cells.     

Pirfenidone inhibits cryoablation induced local macrophage infiltration along with its associated TGFb1 expression and serum cytokine level in a mouse model

PurposeTo investigate the effects of pirfenidone (PFD) on post-cryoablation inflammation in a mouse model.Materials and methodsIn this IACUC-approved study, eighty Balb/c mice were randomly divided into four groups (20/group): sham + vehicle, sham + PFD, cryoablation + vehicle, and cryoablation + PFD. For cryoablation groups, a 20% freeze rate cryoablation (20 s to less than −100 °C) was used to ablate normal muscle in the right flank. For sham groups, the cryoprobe was advanced into the flank and maintained for 20 s without ablation. PFD or vehicle solution was intraperitoneally injected (5 mg/kg) at days 0, 1, 2, 3, and then every other day until day 13 after cryoablation. Mice were euthanized at days 1, 3, 7, and 14. Blood samples were used for serum IL-6, IL-10, and TGFβ1 analysis using electrochemiluminescence and ELISA assays, respectively. Immunohistochemistry-stained ablated tissues were used to analyze macrophage infiltration and local TGFβ1 expression in the border region surrounding the cryoablation-induced coagulation zone.ResultsCryoablation induced macrophage infiltration and increased TGFβ1 expression in the border of the necrotic zone, and high levels of serum IL-6, peaking at days 7 (70.5 ± 8.46/HPF), 14 (228 ± 18.36/HPF), and 7 (298.67 ± 92.63), respectively. Animals receiving PFD showed reduced macrophage infiltration (35.5 ± 16.93/HPF at day 7, p < 0.01) and cytokine levels (60.2 ± 7.6/HPF at day 14, p < 0.01). PFD also significantly reduced serum IL-6 levels (p < 0.001 vs. all non-PFD groups).ConclusionsPFD mitigates cryoablation induced muscle tissue macrophage infiltration, increased IL-6 levels, and local TGFβ1 expression in a small animal model.     

Decomposition of sugarcane bagasse with lignocellulose-derived thermotolerant and thermoresistant Penicillia and Aspergilli

To promote the decomposition of sugarcane bagasse (SCB) for conversion into value-added products and to reduce waste, the capability of fungal mixes (FMs) to degrade SCB was examined. A total of 169 isolates from SCB and non-SCB were categorized as thermotolerant and thermoresistant. Thirty-six fungal candidates were screened for the presence of polyphenol oxidase, endoglucanase (EDN) and xylanase (XLN) activities, and EDN and XLN activities were quantitated. Five identified isolates (Aspergillus flavus AG10; Aspergillus niger AG68 & NB23; and Penicillium citrinum AG93 & AG140) were selected as the best enzyme producers, and 15 moderately to highly xylolytic, cellulolytic and ligninolytic isolates were added to construct FMs. Using a Taguchi design, the top ten reducing sugar-producing FMs (no. 12 showed the maximum amount of reducing sugar, at 2.11 mg g⁻¹, followed by no. 7, 15, 2, 16, 11, 13, 6, 4, & 8) were selected as potential agents for decomposition durations of 1, 2 and 3 months. The maximum decrease in SCB materials compared with the control was generated by FM 6 (9.08% cellulose reduction); FM 13 (21.03% hemicellulose reduction); and FM 16 (9.21% lignin reduction). These results indicate the potential use of SCB as a substrate for synergistic FMs. These FMs could be applied to the large-scale composting of SCB and other related agricultural residues, thus improving the biological pretreatment of lignocellulose.     

Quantifying difference in gene expression profile between bovine blastocysts derived by in vitro fertilization and somatic cell nuclear transfer

Epigenetic reprogramming intensely occurs in somatic-cell nuclear transfer (SCNT) embryos, which highlights the importance of proper expressions of reprogramming-related genes in SCNT embryos. We here assessed gene expression profiles (GEPs) difference between bovine blastocyst groups derived by in-vitro fertilization (IVF) or SCNT; in SCNT, cumulus cells and ear skin fibroblasts were used for cSCNT and fSCNT blastocysts, respectively. We obtained GEPs of 15 reprogramming-related genes in single blastocysts using multiplex PCR and found a broad range of variations in their GEPs. Weighted root-mean-square deviation (wRMSD) analysis, which calculates the deviation of SCNT blastocysts' GEPs from IVF blastocysts' mean GEP, found a significant difference between IVF and fSCNT and between cSCNT and fSCNT blastocysts (p < 0.001) but not between IVF and cSCNT. Since the fibroblasts' GEP was more distant from the IVF blastocysts' than the cumulus cells', it might partly explain the less similarity of fSCNT blastocysts' GEPs to the IVF's mean GEP. Our wRMSD method succeeds in expressing in figures how different two comparable embryo groups of different derivations are in GEP, which would be useful to select a better embryo derivation protocol among the candidates prior to field applications.     

Local cryostimulation acutely preserves maximum isometric handgrip strength following fatigue in young women

Several types of cryostimulation have been recently proposed to rapidly lower skin temperature therefore gaining a possible neuro/muscular recovery after strenuous exercise or, more generally, in sports. Local cryostimulation may be a viable and relatively portable tool to obtain physiological benefits in previously-efforted muscular districts. However, cohesive and standardized cryo-exposure protocols are lacking as well as the righteous procedure to efficaciously combine duration, treatments and temperature in relation to desirable effects on muscular strength. In this randomized-controlled study, fifty young women were tested for maximum isometric handgrip strength, before and after exhausting contractions.Following the fatiguing protocol, the intervention group (cryo, n = 25, 24.7 ± 2.5 years, BMI 21.7 ± 1.8 kg/m²) underwent a 6-min local cryostimulation (−160 °C) on the extensor-flexor muscles of the dominant arm, while control-matched peers sat rested in a thermo-neutral room (22 ± 0.5 °C). Handgrip tests were repeated at baseline (T0), after cryostimulation (T1), and 15 min after T1 (T2). Throughout the protocol, the AUC of the strength performance was significantly higher in the cryo- compared to control group (P = 0.006). In particular, following fatigue and cryostimulation, the cryo group preserved higher strength at T1 with respect to controls (26.8 ± 2.8 vs 23.9 ± 2.8 kg, Bonferroni's post-hoc, P < 0.01). Likewise, ventral and dorsal temperature, recorded with a thermal camera, were lower in cryo- than control group (P < 0.0001).In conclusion, a brief session of local cryostimulation may acutely preserve maximal isometric force in young women following a fatiguing protocol. These findings may have implications in orchestrating strategies of district muscular recovery.     

Konjac ceramide (kCer) regulates keratinocyte migration by Sema3A-like repulsion mechanism

Previously, we proposed the following mechanism for konjac ceramide (kCer)-mediated neurite outgrowth inhibition: kCer binds to Nrp as a Sema3A agonist, resulting in Nrp1/PlexA complex formation and activation of the Sema3A signaling pathway to induce phosphorylation of CRMP2 and microtubule depolymerization. The Sema3A/Nrp1 signaling pathway is known to be also expressed in normal human keratinocytes. To determine whether kCer can function in human keratinocytes as it does in neurites, that is, if it can bind to Nrp1 in place of Sema3A, we studied the effect of kCer on HaCaT cell migration activity. Using a trans-well chamber assay, we compared the effects of Sema3A and kCer on serum-derived cell migration activity. kCer showed Sema3A-like suppression of cell migration activity and induction of cellular Cofilin phosphorylation. In addition, kCer and Sema3A inhibited histamine (His)-enhanced migration of immature HaCaT cells. We have demonstrated that kCer does not interact with histaime receptors H1R or H4R directly, but we speculate that kCer may transduce a signal downstream of the His signaling pathway.     

RASSF6-TRIM16 axis promotes cell proliferation,migration and invasion in esophageal squamous cell carcinoma

Ras-association(RA) domain family number 6(RASSF6) is a member of the Ras-association domain protein family.It is epigenetically inactive and negatively regulates the malignant progression of some tumors.However,its precise role in esophageal squamous cell carcinoma(ESCC) has not been reported.In this study,we performed immunohistochemistry(IHC) assay.The results show that RASSF6 is upregulated in ESCC and that the elevated expression level of RASSF6 is associated with lymph node metastasis and poor survival of ESCC patients.Consistent with the clinical obse rvations,the upregulation of RASSF6 greatly promotes ESCC cell proliferation,migration and invasion as well as the cell cycle transition to Gl/S phase in vitro.According to models in vivo,the downregulation of RASSF6 considerably inhibits ESCC tumor growth and lung metastasis.Mechanistically,RASSF6 negatively regulates the tumor suppressor tripartite-motif-containing protein 16(TRIM16) by promoting its ubiquitination-dependent degradation and eventually activates pathways associated with the cell cycle and epithelialmesenchymal transition(EMT).Together,these results indicate that the RASSF6-TRIM16 axis is a key effector in ESCC progression and that RASSF6 serves as a potential target for the treatment of ESCC.     

Identification of critical regions within the TIR domain of IL-1 receptor type I

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC′ helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.     

Development of an anti-bear podoplanin monoclonal antibody PMab-247 for immunohistochemical analysis

Sensitive and specific monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues because PDPN is known as a lymphatic endothelial cell maker in pathology. Recently, we established anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, horse, goat, tiger, alpaca, and Tasmanian devil. However, anti-bear PDPN (bPDPN) has not been established yet. In this study, we immunized mice with bPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/bPDPN) cells, and screened mAbs against bPDPN using flow cytometry. One of the mAbs, PMab-247 (IgG₁, kappa), specifically detected CHO/bPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-247 for the functional analyses of bPDPN.     

Immunological response induced by cryoablation against murine H22 hepatoma cell line in vivo

ObjectivesTo describe immunological consequences induced by cryoablation against H22 cells in vivo.MethodsAdult BALB/c mice underwent subcutaneous implantation of H22 cells. All of them were assigned into three groups randomly: group A (false surgery), group B (cryoablation) and group C (cryoablation plus Freund's adjuvant). Animals were sacrificed 1, 2 and 3 weeks after treatment. Serum IFN-γ and IL-4, Th1/Th2 in spleens and cytotoxicity were detected.ResultsCompared with that of group A, (1) INF-γ of group B was higher, but IL-4 was lower; cryoablation plus Freund's adjuvant enhanced these effects. (2) Th1/Th2 rose significantly in both group B and group C. (3) Strong cytolytic activity against H22 cells of group B and group C was found on day 7, 14 and 21.ConclusionsOur study showed a marked shift toward Th1 and IFN-γ expression after cryoablation, with an immuno-stimulatory effect against murine H22 hepatoma Cell.     

Effects of different sucrose concentrations on vitrified porcine preantral follicles: Qualitative and quantitative analysis

The aim of the present study was to perform a qualitative and quantitative analysis of the effect of different sucrose concentrations combined with ethylene glycol in the preservation of vitrified porcine preantral follicles. Fragments of ovarian cortex were vitrified in cryotubes containing 200 μl of the vitrification solution (30% Ethylene Glycol; 20% Fetal Bovine Serum; 0 M–0.25 M – 0.75 M or 1 M sucrose) and stored in liquid nitrogen for a week. Histological analysis showed that after vitrification the number of normal follicles decreased compared to the fresh tissue (control). The percentage of normal primordial follicles was sucrose dose dependent. The percentage of normal primary follicles was similar in 0 M or 0.25 M sucrose, while higher concentrations (0.75 M and 1 M) increased significantly the percentage of abnormal follicles (p < 0.05). Morphometric analysis showed a statistically significant reduction in the total area of primordial follicles with 0.75 M sucrose and a significant increase in the cytoplasmic area of primordial follicles with 0 M sucrose (p < 0.05). The qualitative and the quantitative analysis appear to be a complementary tool when choosing a vitrification protocol. For our cryopreservation system - vitrification of ovarian cortex slices in cryotubes-the best vitrification medium was TCM 199-Hepes with 30% de ethylene glycol, 20% of Fetal Bovine Serum and 0 or 0.25 M sucrose. The present study shows that the use of high sucrose concentrations in the vitrification solution has a deleterious effect on the preservation of porcine preantral follicles contained in ovarian tissue. Consequently, its use at 0.75 M or 1 M wouldn't be recommended.     

Cat preantral follicle survival after prolonged cooled storage followed by vitrification

The aim of this study was to investigate the impact of prolonged storage at 4 °C on survival of cat preantral follicles (PAFs) pre- and post-vitrification. Ovaries were obtained from 12 queens and transported at 4 ºC within 2–6 h. Parts of the ovaries were stored for an additional 24 h or 72 h. The ovarian cortex was dissected, analyzed for viability (neutral red - NR) and morphology (histology - HE and ultrastructural analysis by TEM) and vitrified. We used 2 mm biopsy punches to obtain equal size pieces as the experimental units. After NR assessment, each sample was fixed and embedded in paraffin for HE staining to determine the number of morphologically intact follicles. Another 2 mm piece of ovary was subjected to TEM. NR viability assessment and HE results showed a similar tendency with PAF survival postvitrification even after prolonged cooling at 24 h and 72 h. With TEM, integrity of mitochondria, plasma and basal membranes as well as the presence of pre-granulose cells of PAFs were documented postvitrification for the control group and 24 h prolonged storage group, but not after 72 h storage. Our results showed that cat PAFs can survive prolonged storage followed by vitrification. The described set of techniques are applicable towards creating a gamete bank for endangered feline species.     

SitA contributes to the virulence of Klebsiella pneumoniae in a mouse infection model

Klebsiella pneumoniae is an opportunistic pathogen, which causes a wide range of nosocomial infections. Recently, antibiotic resistance makes K. pneumoniae infection difficult to deal with. Investigation on virulence determinants of K. pneumoniae can provide more information about pathogenesis and unveil new targets for treatment or vaccine development. In this study, SitA, a Fur-regulated divalent cation transporter, was found significantly increased when K. pneumoniae was cultured in a nutrient-limited condition. A sitA-deletion strain (ΔsitA) was created to characterize the importance of SitA in virulence. ΔsitA showed higher sensitivity toward hydroperoxide than its parental strain. In a mouse intraperitoneal infection model, the survival rate of mice infected with ΔsitA strain increased greatly when compared with that of mice infected with the parental strain, suggesting that sitA deletion attenuates the bacterial virulence in vivo. To test whether ΔsitA strain is a potential vaccine candidate, mice were immunized with inactivated bacteria and then challenged with the wild-type strain. The results showed that using ΔsitA mutant protected mice better than using the wild-type strain or the capsule-negative congenic bacteria. In summary, SitA was found being important for the growth of K. pneumoniae in vivo and deleting sitA might be a potential approach to generate vaccines against K. pneumoniae.     

PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG_2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.     

Optimization of a protective medium for enhancing the viability of freeze-dried Bacillus amyloliquefaciens B1408 based on response surface methodology

Response surface methodology (RSM) is a commonly used system to optimize cryoprotectants of biocontrol strains when they are subjected to preparations. Various kinds of cryoprotectants and centrifugal conditions were tested to improve the survival of biocontrol agents after freeze-drying. To determine the optimum levels of incorporation of three cryoprotectants (glucose, trehalose and xylitol) in the freeze-drying process of strain Bacillus amyloliquefaciens B1408, a range of experiments based on Box-Behnken Design (BBD) were conducted. The results indicated that the suitable centrifugation conditions were 5000 r/min,10 min and the optimum concentrations of cryoprotectants were glucose 1.00%, trehalose 4.74% and xylitol 1.45%. The proven survival rate of cells after freeze-drying was 91.24%. These results convincingly demonstrated that freeze-drying could be used to preparation of biocontrol strain B1408. This study provides a theoretical basis for commercial possibilities and formulation development.