Delivery mode-associated gut microbiota in the first 3 months of life in a country with high obesity rates: A descriptive study.

Delivery methods during childbirth and their related gut microbiota profiles have important impacts on health later in life, they can contribute to the development of diseases such as obesity, whose highest prevalence rate is found among the Mexican child population. Coincidentally, Mexico has one of the highest global average annual rate increase in cesarean births (C-section). Since Mexico leads the world in childhood obesity, studying the relationship between childbirth delivery methods and gut microbiota profiles in this vulnerable population may be used to identify early risk factors for obesity in other developed and developing countries. The objective of this study is to determine the association between child delivery method and gut microbiota profiles in healthy Mexican newborns.Fecal samples of 57 term infants who participated in a randomized clinical trial in 2013 to study the safety of Agave fructans in newborns, were used in this study. DNA samples were extracted and used to characterize the microbiota composition using high-throughput 16S rRNA gene sequencing. The samples were further divided based on childbirth delivery method, as well as early diet. Gut microbiota profiles were determined and analyzed using cluster analysis followed by multiple correspondence analysis.An unusual high abundance of Proteobacteria was found in the gut microbiota of all Mexican infants studied, regardless of delivery method. Feces from infants born by C-section had low levels of Bacteroidetes, high levels of Firmicutes, especially Clostridium and Enterococcus, and a strikingly high ratio of Firmicutes/Bacteroidetes (F:B). Profiles enriched in Bacteroidetes and low F:B ratios, were strongly associated with vaginal delivery.The profile of gut microbiota associated with feces from Mexican infants born by C-section, may be added to the list of boosting factors for the worrying obesity epidemic in Mexico.

Expression profiling and gene ontology analysis in fathead minnow (Pimephales promelas) liver following exposure to pulp and paper mill effluents.

Many studies link pulp and paper mill effluent (PPME) exposure to adverse effects in fish populations present in the mill receiving environments. These impacts are often characteristic of endocrine disruption and may include impaired reproduction, development and survival. While these physiological endpoints are well-characterized, the molecular mechanisms causing them are not yet understood. To investigate changes in gene transcription induced by exposure to a PPME at several stages of treatment, male and female fathead minnows (FHMs) were exposed for 6 days to 25% (v/v) secondary (biologically) treated kraft effluent (TK) or 100% (v/v) combined mill outfall (CMO) from a mill producing both kraft pulp and newsprint. The gene expression changes in the livers of these fish were analyzed using a 22K oligonucleotide microarray. Exposure to TK or CMO resulted in significant changes in the expression levels of 105 and 238 targets in male FHMs and 296 and 133 targets in females, respectively. Targets were then functionally analyzed using gene ontology tools to identify the biological processes in fish hepatocytes that were affected by exposure to PPME after its secondary treatment. Proteolysis was affected in female FHMs exposed to both TK and CMO. In male FHMs, no processes were affected by TK exposure, while sterol, isoprenoid, steroid and cholesterol biosynthesis and electron transport were up-regulated by CMO exposure. The results presented in this study indicate that short-term exposure to PPMEs affects the expression of reproduction-related genes in the livers of both male and female FHMs, and that secondary treatment of PPMEs may not neutralize all of their metabolic effects in fish. Gene ontology analysis of microarray data may enable identification of biological processes altered by toxicant exposure and thus provide an additional tool for monitoring the impact of PPMEs on fish populations.

Pulp and paper mill effluents induce distinct gene expression changes linked to androgenic and estrogenic responses in the fathead minnow (Pimephales promelas).

Although effluent treatment systems within pulp and paper mills remove many toxicants and improve wastewater quality, there is a need to understand and quantify the effectiveness of the treatment process. At a combined news and kraft pulp and paper mill in northwestern Ontario, Canada, fathead minnow (FHM) reproduction and physiology were examined before, during, and after a short-term (6-d) exposure to 10% (v/v) untreated kraft mill effluent (UTK), 25% (v/v) secondary treated kraft mill effluent (TK), and 100% (v/v) combined mill outfall (CMO). Although UTK exposure significantly decreased egg production, neither TK nor CMO caused any reproductive changes. The expression of six genes responsive to endocrine-disrupting compounds, stress, or metals was then examined in livers of these fish using real-time polymerase chain reaction. In female FHMs, none of the three effluents induced significant expression changes in any genes investigated. By contrast, in males there were significant increases in the mRNA levels of androgen receptor, estrogen receptor (ER) beta, and cytochrome P4501A (CYP1A) upon UTK and TK exposure but no changes in ERalpha or vitellogenin (VTG) gene expression, whereas CMO exposure significantly increased the mRNA levels of ERalpha, VTG, and CYP1A. Together, these results suggest that kraft effluent before and after biological treatment contained compounds able to induce androgenic effects in FHMs, and that combination of kraft and newsmill effluents eliminated the androgenic compounds while inducing distinct and significant patterns of gene expression changes that were likely due to estrogenic compounds produced by the newsmill.

Gene expression analysis of interferon kappa in laser capture microdissected cervical epithelium.

Optimal sample handling techniques for tissue preparation and storage, RNA extraction and quantification, and target gene detection are crucial for reliable gene expression analysis. Methods for measuring low-expressing genes, such as interferons, in human cervical samples are not described in the scientific literature. To detect interferon mRNA in human cervical samples we obtained normal and dysplastic frozen and formalin-fixed cervical biopsies from colposcopy. Histopathological diagnosis was performed by one pathologist. Cervical keratinocytes were isolated using laser capture microdissection. Immortalized keratinocytes transduced with or devoid of an HPV oncogene were used for initial method development. RNA from samples was extracted and integrity tested to compare tissue storage and extraction methods. The expression of five housekeeping genes was analyzed in cell lines and patient tissue to permit normalization between samples using quantitative real-time polymerase chain reaction. The usefulness of cDNA amplification was assessed for the detection of low-expressing interferon kappa in cervical tissue. Here we report optimal tissue storage conditions, RNA extraction, sample normalization, and transcript amplification, as well as the sensitivity of quantitative real-time polymerase chain reaction and laser capture microdissection, for interferon kappa detection in cervical tissue. Without these optimized techniques, interferon kappa detection would be unattainable in cervical samples.

A real-time PCR method for the quantification of the two isoforms of metallothionein in Lake Trout (Salvelinus namaycush).

Metallothioneins (MTs) are low-molecular-weight proteins whose physiologic roles are the regulation of essential metals Cu and Zn, sequestration of heavy metals, and free radical scavenging. Induced production of MTs in a wide variety of organisms exposed to heavy metals has made them popular exposure indicators. While it has been postulated that the three different isoforms of MT play different physiologic roles, methods to discern induction separately have not been available. The development of real-time polymerase chain reaction (real-time PCR) primers and TaqMan probes to measure the two MT isoforms found in salmonid fish are described. Assuming a high degree of homology between the isoforms and within different groups of salmonids, the sequences for MT-I and MT-II from rainbow trout were used to develop primers and probes for lake trout using the Primer3 program. Two sections of each isoform that varied by only a few nucleotides were targeted. SYBR Green validated the primer specificity, and melt curve analysis further ensured that only one product was amplified. Analysis of archived samples from fish captured in unmanipulated reference lakes or from lakes experimentally treated with cadmium or ethynylestradiol (EE2) afforded an examination of seasonal and contaminant influences on MT-I and MT-II mRNA expression.

Developmental effects of bioaccumulated selenium in eggs and larvae of two salmonid species.

Elevated concentrations of Se have been detected in cold, flowing water habitats near uranium and coal mines in Canada. Fish from these systems have concentrations of Se in their tissues that exceed toxic effect thresholds that have been established for warm-water fishes. However, the applicability of toxic effect thresholds and guidelines to cold water, lotic habitats is a matter of contention in the literature since most cases of Se toxicosis have been documented in standing, warm-water systems. To examine the possibility of impaired reproduction in wild rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinusfontinalis) near coal mining activity in the northeastern slopes region of Alberta, Canada, spawn from both species were collected from exposure and reference sites. Gametes were fertilized in the laboratory, reared to the swim-up stage, and examined for deformities. A significant relationship was observed for rainbow trout between the amount of Se in eggs and the incidence of developmental abnormalities, specifically craniofacial defects, skeletal deformities, and edema. These associations approximate exponential functions with probabilities that 15% of the population would be affected occurring between 8.8 and 10.5 microg Se per gram of wet egg weight, based on probit analysis. These relationships are similar to those described for centrarchids inhabiting a seleniferous warm-water lake. No such relationships were established for brook trout.

Waterborne ethynylestradiol induces vitellogenin and alters metallothionein expression in lake trout (Salvelinus namaycush).

Estrogenic contaminants isolated from waters receiving sewage treatment plant effluents are known to induce the egg yolk precursor vitellogenin (VTG) in male fish. Levels of the metal binding protein metallothionein (MT) have also been shown to be affected by estrogens in fish. It has been postulated that MT declines in estrogen exposed fish to facilitate transfer of the essential metal Zn to cellular components required for VTG synthesis. To examine the changes in MT and VTG concentrations in fish exposed to an estrogen contaminant, lake trout (Salvelinus namaycush) were exposed to waterborne ethynylestradiol at 0, 4, 40 or 400 ng/l(-1) for 21 days. Blood and tissues were collected after 21 days of exposure to measure circulating levels of VTG as well as MT concentrations in liver and kidney. VTG increased in male and female fish from all three exposure groups compared to control fish. MT in liver significantly decreased in males and females compared to the controls, in the two highest exposures. MT in kidney was significantly higher in both sexes of fish exposed to the two highest concentrations of ethynylestradiol. These data are supportive of a relationship between estrogen exposure and the regulation of MT. Further studies to examine the specific links between estrogen exposure, VTG induction and regulation of essential metals like Zn are required.