Chronic treatment with dapagliflozin protects against mitochondrial dysfunction in the liver of C57BL/6NCrl mice with high-fat diet/streptozotocin-induced diabetes mellitus.

Dapagliflozin (DAPA), a selective inhibitor of sodium/glucose cotransporter SGLT2, is currently used as a hypoglycemic agent in the treatment of diabetes mellitus. In this work, we have assessed the effect of DAPA treatment (1 mg/kg/day) on the ultrastructure and functions of the liver mitochondria of C57BL/6NCrl mice with type 2 diabetes mellitus (T2DM) induced by a high-fat diet combined with low-dose streptozotocin injections. An electron microscopy study showed that DAPA prevented the mitochondrial swelling and normalized the average mitochondrial size in hepatocytes of diabetic animals. The treatment with DAPA reversed the decline in the mtDNA copy number in the liver of diabetic mice. DAPA-treated T2DM mice showed increased expression of the Ppargc1a, Mfn2 and Drp1 in the liver tissue. The treatment of diabetic animals with DAPA normalized the mitochondrial respiratory control ratio, significantly decreased the level of lipid peroxidation products in liver mitochondria, and decreased their resistance to the opening of the mitochondrial permeability transition pore. At the same time, DAPA had no effects on the studied parameters of control animals. The paper discusses the possible mechanisms of the effect of dapagliflozin on mitochondrial dysfunction in the liver of diabetic animals.

No evidence of amplified Plasmodium falciparum plasmepsin II gene copy number in an area with artemisinin-resistant malaria along the China-Myanmar border.

BACKGROUND: The emergence and spread of artemisinin resistance in Plasmodium falciparum poses a threat to malaria eradication, including China's plan to eliminate malaria by 2020. Piperaquine (PPQ) resistance has emerged in Cambodia, compromising an important partner drug that is widely used in China in the form of dihydroartemisinin (DHA)-PPQ. Several mutations in a P. falciparum gene encoding a kelch protein on chromosome 13 (k13) are associated with artemisinin resistance and have arisen spread in the Great Mekong subregion, including the China-Myanmar border. Multiple copies of the plasmepsin II/III (pm2/3) genes, located on chromosome 14, have been shown to be associated with PPQ resistance. METHODS: The therapeutic efficacy of DHA-PPQ for the treatment of uncomplicated P. falciparum was evaluated along the China-Myanmar border from 2010 to 2014. The dry blood spots samples collected in the efficacy study prior DHA-PPQ treatment and from the local hospital by passive detection were used to amplify k13 and pm2. Polymorphisms within k13 were genotyped by capillary sequencing and pm2 copy number was quantified by relative-quantitative real-time polymerase chain reaction. Treatment outcome was evaluated with the World Health Organization protocol. A linear regression model was used to estimate the association between the day 3 positive rate and k13 mutation and the relationship of the pm2 copy number variants and k13 mutations. RESULTS: DHA-PPQ was effective for uncomplicated P. falciparum infection in Yunnan Province with cure rates > 95%. Twelve non synonymous mutations in the k13 domain were observed among the 268 samples with the prevalence of 44.0% and the predominant mutation was F446I with a prevalence of 32.8%. Only one sample was observed with multi-copies of pm2, including parasites with and without k13 mutations. The therapeutic efficacy of DHA-PPQ was > 95% along the China-Myanmar border, consistent with the lack of amplification of pm2. CONCLUSION: DHA-PPQ for uncomplicated P. falciparum infection still showed efficacy in an area with artemisinin-resistant malaria along the China-Myanmar border. There was no evidence to show PPQ resistance by clinical study and molecular markers survey. Continued monitoring of the parasite population using molecular markers will be important to track emergence and spread of resistance in this region.

An effective vaginal gel to deliver CRISPR/Cas9 system encapsulated in poly (ß-amino ester) nanoparticles for vaginal gene therapy.

BACKGROUND: Gene therapy has held promises for treating specific genetic diseases. However, the key to clinical application depends on effective gene delivery. METHODS: Using a large animal model, we developed two pharmaceutical formulations for gene delivery in the pigs' vagina, which were made up of poly (ß-amino ester) (PBAE)-plasmid polyplex nanoparticles (NPs) based two gel materials, modified montmorillonite (mMMT) and hectorite (HTT). FINDINGS: By conducting flow cytometry of the cervical cells, we found that PBAE-GFP-NPs-mMMT gel was more efficient than PBAE-GFP-NPs-HTT gel in delivering exogenous DNA intravaginally. Next, we designed specific CRISPR/SpCas9 sgRNAs targeting porcine endogenous retroviruses (PERVs) and evaluated the genome editing efficacy in vivo. We discovered that PERV copy number in vaginal epithelium could be significantly reduced by the local delivery of the PBAE-SpCas9/sgRNA NPs-mMMT gel. Comparable genome editing results were also obtained by high-fidelity version of SpCas9, SpCas9-HF1 and eSpCas9, in the mMMT gel. Further, we confirmed that the expression of topically delivered SpCas9 was limited to the vagina/cervix and did not diffuse to nearby organs, which was relatively safe with low toxicity. INTERPRETATION: Our data suggested that the PBAE-NPs mMMT vaginal gel is an effective preparation for local gene therapy, yielding insights into novel therapeutic approaches to sexually transmitted disease in the genital tract. FUNDING: This work was supported by the National Science and Technology Major Project of the Ministry of science and technology of China (No. 2018ZX10301402); the National Natural Science Foundation of China (81761148025, 81871473 and 81402158); Guangzhou Science and Technology Programme (No. 201704020093); National Ten Thousand Plan-Young Top Talents of China, Fundamental Research Funds for the Central Universities (17ykzd15 and 19ykyjs07); Three Big Constructions-Supercomputing Application Cultivation Projects sponsored by National Supercomputer Center In Guangzhou; the National Research FFoundation (NRF) South Africa under BRICS Multilateral Joint Call for Proposals; grant 17-54-80078 from the Russian Foundation for Basic Research.

The distribution of functional N-cycle related genes and ammonia and nitrate nitrogen in soil profiles fertilized with mineral and organic N fertilizer.

Nitrogen transformation in soil is a complex process and the soil microbial population can regulate the potential for N mineralization, nitrification and denitrification. Here we show that agricultural soils under standard agricultural N-management are consistently characterized by a high presence of gene copies for some of the key biological activities related to the N-cycle. This led to a strong NO3- reduction (75%) passing from the soil surface (15.38 ± 11.36 g N-NO3 kg-1 on average) to the 1 m deep layer (3.92 ± 4.42 g N-NO3 kg-1 on average), and ensured low nitrate presence in the deepest layer. Under these circumstances the other soil properties play a minor role in reducing soil nitrate presence in soil. However, with excessive N fertilization, the abundance of bacterial gene copies is not sufficient to explain N leaching in soil and other factors, i.e. soil texture and rainfall, become more important in controlling these aspects.

Long INterspersed element-1 mobility as a sensor of environmental stresses.

Long INterspersed element (LINE-1, L1) retrotransposons are the most abundant transposable elements in the human genome, constituting approximately 17%. They move by a "copy-paste" mechanism, involving reverse transcription of an RNA intermediate and insertion of its cDNA copy at a new site in the genome. L1 retrotransposition (L1-RTP) can cause insertional mutations, alter gene expression, transduce exons, and induce epigenetic dysregulation. L1-RTP is generally repressed; however, a number of observations collected over about 15 years revealed that it can occur in response to environmental stresses. Moreover, emerging evidence indicates that L1-RTP can play a role in the onset of several neurological and oncological diseases in humans. In recent years, great attention has been paid to the exposome paradigm, which proposes that health effects of an environmental factor should be evaluated considering both cumulative environmental exposures and the endogenous processes resulting from the biological response. L1-RTP could be an endogenous process considered for this application. Here, we summarize the current understanding of environmental factors that can affect the retrotransposition of human L1 elements. Evidence indicates that L1-RTP alteration is triggered by numerous and various environmental stressors, such as chemical agents (heavy metals, carcinogens, oxidants, and drugs), physical agents (ionizing and non-ionizing radiations), and experiential factors (voluntary exercise, social isolation, maternal care, and environmental light/dark cycles). These data come from in vitro studies on cell lines and in vivo studies on transgenic animals: future investigations should be focused on physiologically relevant models to gain a better understanding of this topic.

Evaluation of the antiviral activity of orlistat (tetrahydrolipstatin) against dengue virus, Japanese encephalitis virus, Zika virus and chikungunya virus.

Many mosquito transmitted viruses of the genera Alphavirus and Flavivirus are human pathogens of significant concern, and there is currently no specific antiviral for any member of these two genera. This study sought to investigate the broad utility of orlistat (tetrahydrolipstatin) in reducing virus infection for several mosquito borne viruses including flaviviruses (dengue virus (DENV; nine isolates analyzed), Japanese encephalitis virus (JEV; one isolate analyzed) and Zika virus (ZIKV; 2 isolates analyzed)) as well as an alphavirus (chikungunya virus; CHIKV; 2 isolates analyzed). Three different treatment regimens were evaluated, namely pre-treatment (only), post-treatment (only) and pre- and post-treatment, and three factors were evaluated, namely level of infection, virus titer and genome copy number. Results showed that all three treatment modalities were able to significantly reduce virus titer for all viruses investigated, with the exception of three isolates of DENV in the pre-treatment only regimen. Pre- and post-treatment was more effective in reducing the level of infection and genome copy number of all viruses investigated than either pre-treatment or post-treatment alone. Collectively, these results suggest orlistat has potential as a broad-spectrum agent against multiple mosquito transmitted viruses.

Impact of Wuyiencin Application on the Soil Microbial Community and Fate of Typical Antibiotic Resistance Genes.

Antibiotic resistance genes (ARGs) have raised numerous concerns in recent years as emerging environmental contaminants. At present, research on environmental contamination by antibiotics focuses on medical, animal husbandry, and aquaculture fields, with few studies on environmental contamination by agricultural antibiotics in the field of plant protection. Wuyiencin is a low toxicity, high efficiency, and broad-spectrum agricultural antibiotic. It has been widely used in agricultural production and it effectively controls crop fungal diseases. In the present study, pot experiments with four soil treatments (A, B, C and D) were set up in a greenhouse to investigate the effect of the application of wuyiencin on the fate of typical ARGs and microbial community. Eight typical ARGs were detected by real-time PCR and the microbial communities were analyzed using high-throughput sequencing. The results showed that wuyiencin neither significantly influenced ARG abundance and absolute gene copy numbers, nor significantly varied microbial community among treatments. Since it only was short-term results, and the detection number of ARGs was limited, whether wuyiencin is safe or not to ecological environment when using for long-term will need further deep research.

Resistance of Leishmania infantum to allopurinol is associated with chromosome and gene copy number variations including decrease in the S-adenosylmethionine synthetase (METK) gene copy number.

Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL), a widespread, life-threatening disease. This parasite is responsible for the majority of human VL cases in Brazil, the Middle East, China, Central Asia and the Mediterranean basin. Its main reservoir are domestic dogs which, similar to human patients, may develop severe visceral disease and die if not treated. The drug allopurinol is used for the long-term maintenance of dogs with canine leishmaniasis. Following our report of allopurinol resistance in treated relapsed dogs, we investigated the mechanisms and markers of resistance to this drug. Whole genome sequencing (WGS) of clinical resistant and susceptible strains, and laboratory induced resistant parasites, was carried out in order to detect genetic changes associated with resistance. Significant gene copy number variation (CNV) was found between resistant and susceptible isolates at several loci, including a locus on chromosome 30 containing the genes LinJ.30.3550 through LinJ.30.3580. A reduction in copy number for LinJ.30.3560, encoding the S-adenosylmethionine synthetase (METK) gene, was found in two resistant clinical isolates and four induced resistant clonal strains. Using quantitative real time PCR, this reduction in METK copy number was also found in three additional resistant clinical isolates. Furthermore, inhibition of S-adenosylmethionine synthetase encoded by the METK gene in allopurinol susceptible strains resulted in increased allopurinol resistance, confirming its role in resistance to allopurinol. In conclusion, this study identified genetic changes associated with L. infantum resistance to allopurinol and the reduction in METK copy number identified may serve as a marker for resistance in dogs, and reduced protein activity correlated with increased allopurinol resistance.

Nimotuzumab combined with chemotherapy as first-line treatment for advanced lung squamous cell carcinoma.

BACKGROUND: This study was conducted to evaluate the efficacy and safety of nimotuzumab combined with chemotherapy as first-line therapy in advanced lung squamous cell carcinoma (LSCC), and to explore predictive biomarkers of the efficacy of nimotuzumab. METHODS: A retrospective study was conducted of patients with advanced LSCC administered nimotuzumab combined with chemotherapy as first-line therapy from June 2012 to December 2016 at the Department of Respiratory Medicine, Peking Union Medical College Hospital. The associations between EGFR expression, EGFR gene copy numbers, and clinical efficacy were detected by immunohistochemistry and fluorescence in situ hybridization (FISH). RESULTS: Twenty-six patients were enrolled, including 22 men and 4 women. The objective response rate was 50% and the disease control rate was 100%. The median progression-free survival (PFS) and overall survival were 6.7 and 16.3 months, respectively. Patients whose samples were tested via FISH and showed positive EGFR expression had a trend of longer median PFS (10.0 months; P = 0.10). Adverse effects included 15 cases (57.7%) of bone marrow suppression, 15 (57.7%) of sensory neuropathy, 14 (53.8%) of alopecia, nine (34.6%) of nausea/vomiting and one case (3.8%) of elevated creatinine level. All adverse effects were attributed to chemotherapy. CONCLUSION: Nimotuzumab combined with chemotherapy might be a possible option as first-line therapy in patients with advanced LSCC. EGFR gene copy number examined by FISH might be a possible predictive biomarker.

Chronic toxicity of graphene and graphene oxide in sequencing batch bioreactors: A comparative investigation.

The present study investigates the chronic toxicity of graphene (G) and graphene oxide (GO) in activated sludge. Sequencing batch bioreactors were fed with influents containing 0, 1 and 5mgL-1 of GO or G (12h cycles) for ten days. Reduction in performance of the bioreactors in relation to chemical oxygen demand, ammonia and phosphate removals was observed after three days in the bioreactors fed with 5mgL-1 of nanomaterials. After about eight days, these reactors reached a steady state nutrient removal, which corresponded to recovery of certain groups of ammonia oxidizing bacteria and phosphate accumulating bacteria despite the increasing accumulation of nanomaterials in the sludge. These results suggested that biological treatment can be affected transiently by initial exposure to the nanomaterials, but certain groups of microorganisms, less sensitive to these nanomaterials, can potentially strive in the presence of these nanomaterials. Results of 16S rRNA gene deep sequencing showed that G and GO affected differently the microbial communities in the activated sludge. Between the two nanomaterials investigated, GO presented the highest impact in nutrient removal, gene abundance and changes in microbial population structures.

IncX3 plasmid mediated occurrence of blaNDM-4 within Escherichia coli ST448 from India.

This study was designed to investigate blaNDM-4 encoded within IncX3 type plasmid and their copy number alteration under carbapenem pressure within clinical isolates of Escherichia coli. NDM-4 producing E. coli isolates were collected from an Indian hospital and transferability as well as plasmid incompatibility typing was determined. Genetic environment and antibiogram profiling was carried out. Quantitative Real Time PCR was done to determine the change in plasmid copy number under concentration gradient carbapenem stress. Multilocus sequence typing and pulsed field gel electrophoresis was performed for typing of isolates. Four multidrug resistant isolates were found to harbour transconjugable blaNDM-4 carrying within IncX3 type plasmid. The blaNDM-4 was flanked by insertion sequences ISAba125 and IS5 in the upstream region whereas bleMBL was present in the downstream area. Copy number results indicated that the blaNDM-4 gene was maintained high in plasmid under exposure of ertapenem. All the strains belonged to ST448 and PFGE analysis revealed three different pulsotypes. This is the first report of blaNDM-4 encoded IncX3 type plasmid in E. coli of ST448 and needs a systematic screening policy to rapid detection of NDM-4 poducing strains to prevent dissemination of this resistant determinant in future.

Role of mitochondrial function in the invasiveness of human colon cancer cells.

We investigated the role of mitochondrial function in the invasiveness of human colorectal cancer (CRC) cell lines, using paired primary SW480 and metastatic SW620 cells, and appraised the clinical relevance of the alteration of mtDNA copy number in 33 pairs of CRC specimens after surgical resection. Suppression of mitochondrial function was achieved by the exposure of cells to oligomycin A (OA) or by knockdown of mitochondrial transcriptional factor A (TFAM) to evaluate their effects on energy metabolism, reactive oxygen species, protein expression levels of epithelial-mesenchymal transition (EMT) markers and invasive activity of CRC cells. We found that SW620 cells expressed higher levels of TFAM and mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) and cytochrome c oxidase subunit II (COX-II) and nuclear DNA-encoded NADH ubiquinone oxidoreductase subunit A9 (NDUFA9), iron-sulfur protein subunit B of succinate dehydrogenase (SDHB), ubiquinol­cytochrome c reductase core protein I/II (UQCRC1/2) and cytochrome c oxidase subunit IV (COX-IV) when compared with the SW480 cells. The mtDNA copy number, ADP-triggered oxygen consumption rate (OCR) and respiratory control ratio (RCR) of succinate-supported respiration in the SW620 cells were higher than those noted in the SW480 cells. The intracellular levels of H2O2 and O2-• in the SW620 cells were lower than levels noted in the SW480 cells. Moreover, SW620 cells displayed lower protein levels of hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI) and lactate dehydrogenase (LDH), and lower lactate production rate, and expressed higher levels of EMT markers N-cadherin, vimentin and Snail, and showed higher Transwell migration and invasion activities as compared with the SW480 cells. After OA treatment, SW620 cells exhibited a decrease in OCR and RCR of succinate-supported respiration, an increase in lactate production rate and intracellular levels of H2O2 and O2-•. Moreover, the level of vimentin and Transwell migration activity of the SW620 cells were decreased. After TFAM knockdown, the protein levels of TFAM, ND6 and COX-II, and mtDNA copy number, OCR and RCR of succinate-supported respiration in the SW620-KD#4 and SW620-KD#5 cells were all lower than those noted in the SW620­Control cells. By contrast, the protein level of HK-II, lactate production rate, the intracellular levels of H2O2 and O2-• in the SW620-KD#4 and SW620-KD#5 cells were all higher than those noted in the SW620-Control cells. Subsequently, both SW620-KD#4 and SW620-KD#5 cells had lower Transwell invasion activity than did the SW620-Control cells. Furthermore, we found that deeper invasion (P=0.025) and longer tumor length (P=0.069) were associated with higher mtDNA copy ratios in the 33 pairs of CRC specimens obtained from surgical resection. Taken together, we conclude that higher mtDNA copy number and mitochondrial function may confer an invasive advantage to CRCs.

Not only P-glycoprotein: Amplification of the ABCB1-containing chromosome region 7q21 confers multidrug resistance upon cancer cells by coordinated overexpression of an assortment of resistance-related proteins.

The development of drug resistance continues to be a dominant hindrance toward curative cancer treatment. Overexpression of a wide-spectrum of ATP-dependent efflux pumps, and in particular of ABCB1 (P-glycoprotein or MDR1) is a well-known resistance mechanism for a plethora of cancer chemotherapeutics including for example taxenes, anthracyclines, Vinca alkaloids, and epipodopyllotoxins, demonstrated by a large array of published papers, both in tumor cell lines and in a variety of tumors, including various solid tumors and hematological malignancies. Upon repeated or even single dose treatment of cultured tumor cells or tumors in vivo with anti-tumor agents such as paclitaxel and doxorubicin, increased ABCB1 copy number has been demonstrated, resulting from chromosomal amplification events at 7q11.2-21 locus, leading to marked P-glycoprotein overexpression, and multidrug resistance (MDR). Clearly however, additional mechanisms such as single nucleotide polymorphisms (SNPs) and epigenetic modifications have shown a role in the overexpression of ABCB1 and of other MDR efflux pumps. However, notwithstanding the design of 4 generations of ABCB1 inhibitors and the wealth of information on the biochemistry and substrate specificity of ABC transporters, translation of this vast knowledge from the bench to the bedside has proven to be unexpectedly difficult. Many studies show that upon repeated treatment schedules of cell cultures or tumors with taxenes and anthracyclines as well as other chemotherapeutic drugs, amplification, and/or overexpression of a series of genes genomically surrounding the ABCB1 locus, is observed. Consequently, altered levels of other proteins may contribute to the establishment of the MDR phenotype, and lead to poor clinical outcome. Thus, the genes contained in this ABCB1 amplicon including ABCB4, SRI, DBF4, TMEM243, and RUNDC3B are overexpressed in many cancers, and especially in MDR tumors, while TP53TG1 and DMTF1 are bona fide tumor suppressors. This review describes the role of these genes in cancer and especially in the acquisition of MDR, elucidates possible connections in transcriptional regulation (co-amplification/repression) of genes belonging to the same ABCB1 amplicon region, and delineates their novel emerging contributions to tumor biology and possible strategies to overcome cancer MDR.

Ephedrine-induced mitophagy via oxidative stress in human hepatic stellate cells.

The herb Ephedra sinica (also known as Chinese ephedra or Ma Huang), used in traditional Chinese medicine, contains alkaloids identical to ephedrine and pseudoephedrine as its principal active constituents. Recent studies have reported that ephedrine has various side effects in the cardiovascular and nervous systems. In addition, herbal Ephedra, a plant containing many pharmacologically active alkaloids, principally ephedrine, has been reported to cause acute hepatitis. Many studies reported clinical cases, however, the cellular mechanism of liver toxicity by ephedrine remains unknown. In this study, we investigated hepatotoxicity and key regulation of mitophagy in ephedrine-treated LX-2 cells. Ephedrine triggered mitochondrial oxidative stress and depolarization. Mitochondrial swelling and autolysosome were observed in ephedrine-treated cells. Ephedrine also inhibited mitochondrial biogenesis, and the mitochondrial copy number was decreased. Parkin siRNA recovered the ephedrine-induced mitochondrial damage. Excessive mitophagy lead to cell death through imbalance of autophagic flux. Moreover, antioxidants and reducing Parkin level could serve as therapeutic targets for ephedrine-induced hepatotoxicity.

Tetramethylpyrazine blocks TFAM degradation and up-regulates mitochondrial DNA copy number by interacting with TFAM.

The natural small molecule compound: 2,3,5,6-tetramethylpyrazine (TMP), is a major component of the Chinese medicine Chuanxiong, which has wide clinical applications in dilating blood vessels, inhibiting platelet aggregation and treating thrombosis. Recent work suggests that TMP is also an antitumour agent. Despite its chemotherapeutic potential, the mechanism(s) underlying TMP action are unknown. Herein, we demonstrate that TMP binds to mitochondrial transcription factor A (TFAM) and blocks its degradation by the mitochondrial Lon protease. TFAM is a key regulator of mtDNA replication, transcription and transmission. Our previous work showed that when TFAM is not bound to DNA, it is rapidly degraded by the ATP-dependent Lon protease, which is essential for mitochondrial proteostasis. In cultured cells, TMP specifically blocks Lon-mediated degradation of TFAM, leading to TFAM accumulation and subsequent up-regulation of mtDNA content in cells with substantially low levels of mtDNA. In vitro protease assays show that TMP does not directly inhibit mitochondrial Lon, rather interacts with TFAM and blocks degradation. Pull-down assays show that biotinylated TMP interacts with TFAM. These findings suggest a novel mechanism whereby TMP stabilizes TFAM and confers resistance to Lon-mediated degradation, thereby promoting mtDNA up-regulation in cells with low mtDNA content.

Obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy.

Mitochondria are the most prominent organelle in the oocyte. Somatic cells maintain a healthy population of mitochondria by degrading damaged mitochondria via mitophagy, a specialized autophagy pathway. However, evidence from previous work investigating the more general macroautophagy pathway in oocytes suggests that mitophagy may not be active in the oocyte. This would leave the vast numbers of mitochondria - poised to be inherited by the offspring - vulnerable to damage. Here we test the hypothesis that inactive mitophagy in the oocyte underlies maternal transmission of dysfunctional mitochondria. To determine whether oocytes can complete mitophagy, we used either CCCP or AntimycinA to depolarize mitochondria and trigger mitophagy. After depolarization, we did not detect co-localization of mitochondria with autophagosomes and mitochondrial DNA copy number remained unchanged, indicating the non-functional mitochondrial population was not removed. To investigate the impact of an absence of mitophagy in oocytes with damaged mitochondria on offspring mitochondrial function, we utilized in vitro fertilization of high fat high sugar (HF/HS)-exposed oocytes, which have lower mitochondrial membrane potential and damaged mitochondria. Here, we demonstrate that blastocysts generated from HF/HS oocytes have decreased mitochondrial membrane potential, lower metabolites involved in ATP generation, and accumulation of PINK1, a mitophagy marker protein. This mitochondrial phenotype in the blastocyst mirrors the phenotype we show in HF/HS exposed oocytes. Taken together, these data suggest that the mechanisms governing oocyte mitophagy are fundamentally distinct from those governing somatic cell mitophagy and that the absence of mitophagy in the setting of HF/HS exposure contributes to the oocyte-to-blastocyst transmission of dysfunctional mitochondria.

Multiple sclerosis-associated retrovirus, Epstein-Barr virus, and vitamin D status in patients with relapsing remitting multiple sclerosis.

The relationship between infections and autoimmune diseases is complex and there are several reports highlighting the role of human endogenous retroviruses (HERVs) in these patients. The levels of multiple sclerosis-associated retrovirus (MSRV)-type DNA of Env gene was measured in peripheral blood mononuclear cells from 52 patients with relapsing-remitting multiple sclerosis (RRMS) and 40 healthy controls using specific quantitative PCR (qPCR) analysis. Furthermore, we analyzed the status of HERV-W/MSRV in these patients with regards to both EBV (DNA load and anti-EBNA1 IgG antibody) and vitamin D concentration. MSRV DNA copy number were significantly higher in RRMS patients than healthy controls (P < 0.0001). Interestingly, an inverse correlation was found between MSRV DNA copy number and serum vitamin D concentration (P < 0.01), but not for EBV load or anti-EBNA-1 IgG antibody.

Atrazine exposure elicits copy number alterations in the zebrafish genome.

Atrazine is an agricultural herbicide used throughout the Midwestern United States that frequently contaminates potable water supplies resulting in human exposure. Using the zebrafish model system, an embryonic atrazine exposure was previously reported to decrease spawning rates with an increase in progesterone and ovarian follicular atresia in adult females. In addition, alterations in genes associated with distinct molecular pathways of the endocrine system were observed in brain and gonad tissue of the adult females and males. Current hypotheses for mechanistic changes in the developmental origins of health and disease include genetic (e.g., copy number alterations) or epigenetic (e.g., DNA methylation) mechanisms. As such, in the current study we investigated whether an atrazine exposure would generate copy number alterations (CNAs) in the zebrafish genome. A zebrafish fibroblast cell line was used to limit detection to CNAs caused by the chemical exposure. First, cells were exposed to a range of atrazine concentrations and a crystal violet assay was completed, showing confluency decreased by ~60% at 46.3µM. Cells were then exposed to 0, 0.463, 4.63, or 46.3µM atrazine and array comparative genomic hybridization completed. Results showed 34, 21, and 44 CNAs in the 0.463, 4.63, and 46.3µM treatments, respectively. Furthermore, CNAs were associated with previously reported gene expression alterations in adult male and female zebrafish. This study demonstrates that atrazine exposure can generate CNAs that are linked to gene expression alterations observed in adult zebrafish exposed to atrazine during embryogenesis providing a mechanism of the developmental origins of atrazine endocrine disruption.

Toxic effects of ionic liquid 1-octyl-3-methylimidazolium tetrafluoroborate on soil enzyme activity and soil microbial community diversity.

Ionic liquids (ILs) were considered as "green" solvents and have been used widely because of their excellent properties. But ILs are not as "green" as has been suggested, and the toxic effects of ILs on organisms have been shown in recent years. In the present study, the toxic effects of the IL 1-octyl-3-methylimidazolium tetrafluoroborate ([Omim]BF4) on soil enzyme activity and soil microbial communities at three different concentrations (1.0, 5.0 and 10.0mg/kg) and a control treatment over 40 days of incubation time (sampled on days 10, 20, 30 and 40) were examined under laboratory conditions. The concentrations of [Omim]BF4 in soils were detected by high performance liquid chromatography (HPLC) and the results indicated that [Omim]BF4 were maintained stable in the soil during the exposure period. However, the enzyme activity results showed that urease activity was stimulated on day 20 and then decreased after 30 days of incubation. The activity of ß-glucosidase was stimulated after 20 days of incubation in both treatment groups. Moreover, both dehydrogenase and acid phosphatase were inhibited at a high level (10.0mg/kg) only on day 20. The analysis of terminal restriction fragment length polymorphism (T-RFLP) revealed that the soil microbial community structures were altered by [Omim]BF4 and that the soil microbial diversity and evenness of high levels (5.0mg/kg and 10.0mg/kg) treatments were decreased. Moreover, the dominant structure of the microbial communities was not changed by [Omim]BF4. Furthermore, the abundance of the ammonia monooxygenase (amoA) genes of both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) was examined using real time polymerase chain reaction (RT-PCR). The results revealed that the copy numbers of the amoA-gene were decreased by [Omim]BF4 with the 5.0 and 10.0mg/kg treatments. Based on the experiment, we concluded that high levels (5.0 and 10.0mg/kg) of [Omim]BF4 could have significantly toxic effects on soil enzyme activities and the diversity of the microbial communities.

Anterior Gradient 2 (AGR2) Induced Epidermal Growth Factor Receptor (EGFR) Signaling Is Essential for Murine Pancreatitis-Associated Tissue Regeneration.

A recently published study identified Anterior Gradient 2 (AGR2) as a regulator of EGFR signaling by promoting receptor presentation from the endoplasmic reticulum to the cell surface. AGR2 also promotes tissue regeneration in amphibians and fish. Whether AGR2-induced EGFR signaling is essential for tissue regeneration in higher vertebrates was evaluated using a well-characterized murine model for pancreatitis. The impact of AGR2 expression and EGFR signaling on tissue regeneration was evaluated using the caerulein-induced pancreatitis mouse model. EGFR signaling and cell proliferation were examined in the context of the AGR2-/- null mouse or with the EGFR-specific tyrosine kinase inhibitor, AG1478. In addition, the Hippo signaling coactivator YAP1 was evaluated in the context of AGR2 expression during pancreatitis. Pancreatitis-induced AGR2 expression enabled EGFR translocation to the plasma membrane, the initiation of cell signaling, and cell proliferation. EGFR signaling and tissue regeneration were partially inhibited by the tyrosine kinase inhibitor AG1478, but absent in the AGR2-/- null mouse. AG1478-treated and AGR2-/- null mice with pancreatitis died whereas all wild-type controls recovered. YAP1 activation was also dependent on pancreatitis-induced AGR2 expression. AGR2-induced EGFR signaling was essential for tissue regeneration and recovery from pancreatitis. The results establish tissue regeneration as a major function of AGR2-induced EGFR signaling in adult higher vertebrates. Enhanced AGR2 expression and EGFR signaling are also universally present in human pancreatic cancer, which support a linkage between tissue injury, regeneration, and cancer pathogenesis.