The effects of torula yeast as a protein source on apparent total tract digestibility, inflammatory markers, and fecal microbiota dysbiosis index in Labrador Retrievers with chronically poor stool quality.

This study examined the effects of varying protein sources on apparent total tract digestibility, inflammatory markers, and fecal microbiota in Labrador Retrievers with historically poor stool quality. Thirty dogs (15 male, 15 female; aged 0.93 to 11.7 yr) with stool quality scores ≤2.5 on a 5-point scale (1 representing liquid stool and 5 representing firm stool) were randomly assigned to 1 of 3 nutritionally complete diets with differing protein sources and similar macronutrient profiles: 1) chicken meal (n = 10); 2) 10% brewer's yeast (n = 10); or 3) 10% torula yeast (n = 10). Another 10 dogs (five male, five female) with normal stool quality (scores ranging from 3 to 4) received diet 1 and served as negative control (NC). All dogs were fed diet 1 for 7 days, then provided their assigned treatment diets from days 7 to 37. Daily stool scores and weekly body weights were recorded. On days 7, 21, and 36, blood serum was analyzed for c-reactive protein (CRP), and feces for calgranulin C (S100A12), α1-proteinase inhibitor (α1-PI), calprotectin, and microbiota dysbiosis index. Apparent total tract digestibility was assessed using the indicator method with 2 g titanium dioxide administered via oral capsules. Stool scores were greater in NC (P < 0.01) as designed but not affected by treatment × time interaction (P = 0.64). Body weight was greater (P = 0.01) and CRP lower (P < 0.01) in NC dogs. Dry matter and nitrogen-free extract digestibility did not differ among groups (P ≥ 0.14). Negative controls had greater fat digestibility compared to BY (94.64 ±â€…1.33% vs. 91.65 ±â€…1.25%; P = 0.02). The overall effect of treatment was significant for protein digestibility (P = 0.03), but there were no differences in individual post hoc comparisons (P ≥ 0.07). Treatment did not affect S100A12 or α1-PI (P ≥ 0.44). Calprotectin decreased at a greater rate over time in TY (P < 0.01). The dysbiosis index score for BY and TY fluctuated less over time (P = 0.01). Blautia (P = 0.03) and Clostridium hiranonis (P = 0.05) abundances were reduced in BY and TY. Dogs with chronically poor stool quality experienced reduced body weights and increased serum CRP, but TY numerically increased protein digestibility, altered the microbiome, and reduced fecal calprotectin. Torula yeast is a suitable alternative protein source in extruded canine diets, but further research is needed to understand the long-term potential for improving the plane of nutrition and modulating gut health.

Pet and human populations continue to grow and compete for nutritious, sustainable protein sources. The incorporation of alternative proteins like torula yeast can provide a solution to this problem. Torula yeast also may have additional health benefits like reducing gut inflammation. To test its effects in dogs, we fed Labrador Retrievers with chronically poor stool quality either a control diet with chicken meal, a diet with 10% brewer's yeast, or a diet with 10% torula yeast. We compared their responses to dogs with normal stool quality fed the control diet. Dogs with chronically poor stool quality had lower body weights and increased systemic inflammation compared to those with good stool quality. Calprotectin, a marker of gut inflammation, was reduced more in dogs fed torula yeast than in dogs fed chicken meal. Torula and brewer's yeast also changed the abundance of certain gut bacteria. Torula yeast may be added to dog diets with no negative effects and can alter the gut environment in Labrador Retrievers with chronically poor stool quality.

A Novel CRISPR Interference Effector Enabling Functional Gene Characterization with Synthetic Guide RNAs.

While CRISPR interference (CRISPRi) systems have been widely implemented in pooled lentiviral screening, there has been limited use with synthetic guide RNAs for the complex phenotypic readouts enabled by experiments in arrayed format. Here we describe a novel deactivated Cas9 fusion protein, dCas9-SALL1-SDS3, which produces greater target gene repression than first or second generation CRISPRi systems when used with chemically modified synthetic single guide RNAs (sgRNAs), while exhibiting high target specificity. We show that dCas9-SALL1-SDS3 interacts with key members of the histone deacetylase and Swi-independent three complexes, which are the endogenous functional effectors of SALL1 and SDS3. Synthetic sgRNAs can also be used with in vitro-transcribed dCas9-SALL1-SDS3 mRNA for short-term delivery into primary cells, including human induced pluripotent stem cells and primary T cells. Finally, we used dCas9-SALL1-SDS3 for functional gene characterization of DNA damage host factors, orthogonally to small interfering RNA, demonstrating the ability of the system to be used in arrayed-format screening.

Prenatal PCBs disrupt early neuroendocrine development of the rat hypothalamus.

Neonatal exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) can interfere with hormone-sensitive developmental processes, including brain sexual differentiation. We hypothesized that disruption of these processes by gestational PCB exposure would be detectable as early as the day after birth (postnatal day (P) 1) through alterations in hypothalamic gene and protein expression. Pregnant Sprague-Dawley rats were injected twice, once each on gestational days 16 and 18, with one of the following: DMSO vehicle; the industrial PCB mixture Aroclor 1221 (A1221); a reconstituted mixture of the three most prevalent congeners found in humans, PCB138, PCB153, and PCB180; or estradiol benzoate (EB). On P1, litter composition, anogenital distance (AGD), and body weight were assessed. Pups were euthanized for immunohistochemistry of estrogen receptor α (ERα) or TUNEL labeling of apoptotic cells or quantitative PCR of 48 selected genes in the preoptic area (POA). We found that treatment with EB or A1221 had a sex-specific effect on developmental apoptosis in the neonatal anteroventral periventricular nucleus (AVPV), a sexually dimorphic hypothalamic region involved in the regulation of reproductive neuroendocrine function. In this region, exposed females had increased numbers of apoptotic nuclei, whereas there was no effect of treatment in males. For ERα, EB treatment increased immunoreactive cell numbers and density in the medial preoptic nucleus (MPN) of both males and females, while A1221 and the PCB mixture had no effect. PCR analysis of gene expression in the POA identified nine genes that were significantly altered by prenatal EDC exposure, in a manner that varied by sex and treatment. These genes included brain-derived neurotrophic factor, GABA(B) receptors-1 and -2, IGF-1, kisspeptin receptor, NMDA receptor subunits NR2b and NR2c, prodynorphin, and TGFα. Collectively, these results suggest that the disrupted sexual differentiation of the POA by prenatal EDC exposures is already evident as early as the day after birth, effects that may change the trajectory of postnatal development and compromise adult reproductive function.

Endocrine disruption of brain sexual differentiation by developmental PCB exposure.

In mammals, sexual differentiation of the hypothalamus occurs during prenatal and early postnatal development due in large part to sex differences in hormones. These early organizational processes are critically important for the attainment and maintenance of adult reproductive functions. We tested the hypothesis that perinatal exposure to polychlorinated biphenyls (PCBs) that disrupt hormonal pathways would perturb reproductive maturation and the sexually dimorphic development of neuroendocrine systems in the preoptic area (POA). Pregnant Sprague-Dawley rats were injected on gestational d 16 and 18 with vehicle (dimethylsulfoxide), Aroclor 1221 (A1221, an estrogenic PCB mix), a reconstituted PCB mixture representing those highest in human body burden (PCBs 138, 153, 180), or estradiol benzoate, an estrogenic control. Male and female pups were monitored for somatic and reproductive development. In adulthood, some rats were perfused and used for immunohistochemistry of estrogen receptor α, kisspeptin, and coexpression of Fos in GnRH neurons. Other rats were used to obtain fresh-frozen POA dissections for use in a PCR-based 48-gene expression array. Pubertal onset was advanced and estrous cyclicity irregular in endocrine-disrupted females. Furthermore, sexual differentiation of female neuroendocrine systems was masculinized/defeminized. Specifically, in the adult female anteroventral periventricular nucleus, estrogen receptor α-cell numbers and kisspeptin fiber density were significantly decreased, as was GnRH-Fos coexpression. PCR analysis identified androgen receptor, IGF-I, N-methyl-d-aspartate receptor subunit NR2b, and TGFß1 mRNAs as significantly down-regulated in endocrine-disrupted female POAs. These data suggest that developmental PCBs profoundly impair the sexual differentiation of the female hypothalamus.

Cell death mechanisms in GT1-7 GnRH cells exposed to polychlorinated biphenyls PCB74, PCB118, and PCB153.

Exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) causes functional deficits in neuroendocrine systems. We used an immortalized hypothalamic GT1-7 cell line, which synthesizes the neuroendocrine peptide gonadotropin-releasing hormone (GnRH), to examine the neurotoxic and endocrine disrupting effects of PCBs and their mechanisms of action. Cells were treated for 1, 4, 8, or 24 h with a range of doses of a representative PCB from each of three classes: coplanar (2,4,4',5-tetrachlorobiphenyl: PCB74), dioxin-like coplanar (2',3,4,4',5' pentachlorobiphenyl: PCB118), non-coplanar (2,2',4,4',5,5'-hexachlorobiphenyl: PCB153), or their combination. GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation were quantified. In general, GnRH peptide levels were suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter timepoints. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs reduced viability and increased both apoptotic and necrotic cell death. Although the effects for the three classes of PCBs were often similar, subtle differences in responses, together with evidence that the combination of PCBs acted slightly different from individual PCBs, suggest that the three tested PCB compounds may act via slightly different or more than one mechanism. These results provide evidence that PCB congeners have endocrine disrupting and/or neurotoxic effects on the hypothalamic GnRH cell line, a finding that has implications for environmental endocrine disruption in animals.

The recreational drug ecstasy disrupts the hypothalamic-pituitary-gonadal reproductive axis in adult male rats.

Reproductive function involves an interaction of three regulatory levels: hypothalamus, pituitary, and gonad. The primary drive upon this system comes from hypothalamic gonadotropin-releasing hormone (GnRH) neurosecretory cells, which receive afferent inputs from other neurotransmitter systems in the central nervous system to result in the proper coordination of reproduction and the environment. Here, we hypothesized that the recreational drug (+/-)-3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy'), which acts through several of the neurotransmitter systems that affect GnRH neurons, suppresses the hypothalamic-pituitary-gonadal reproductive axis of male rats. Adult male Sprague-Dawley rats self-administered saline or MDMA either once (acute) or for 20 days (chronic) and were euthanized 7 days following the last administration. We quantified hypothalamic GnRH mRNA, serum luteinizing hormone concentrations, and serum testosterone levels as indices of hypothalamic, pituitary, and gonadal functions, respectively. The results indicate that the hypothalamic and gonadal levels of the hypothalamic-pituitary-gonadal axis are significantly altered by MDMA, with GnRH mRNA and serum testosterone levels suppressed in rats administered MDMA compared to saline. Furthermore, our finding that hypothalamic GnRH mRNA levels are suppressed in the context of low testosterone concentrations suggests that the central GnRH neurosecretory system may be a primary target of inhibitory regulation by MDMA usage.

Estrogenic environmental endocrine-disrupting chemical effects on reproductive neuroendocrine function and dysfunction across the life cycle.

Endocrine disrupting chemicals (EDCs) are natural or synthetic compounds that interfere with the normal function of an organism's endocrine system. Many EDCs are resistant to biodegradation, due to their structural stability, and persist in the environment. The focus of this review is on natural and artificial EDCs that act through estrogenic mechanisms to affect reproductive neuroendocrine systems. This endocrine axis comprises the hypothalamic gonadotropin-releasing hormone (GnRH), pituitary gonadotropins, and gonadal steroid hormones, including estrogens. Although it is not surprising that EDCs that mimic or antagonize estrogen receptors may exert actions upon reproductive targets, the mechanisms for these effects are complex and involve all three levels of the hypothalamic-pituitary-gonadal (HPG) system. Nevertheless, considerable evidence links exposure to estrogenic environmental EDCs with neuroendocrine reproductive deficits in wildlife and in humans. The effects of an EDC are variable across the life cycle of an animal, and are particularly potent when exposure occurs during fetal and early postnatal development. As a consequence, abnormal sexual differentiation, disrupted reproductive function, or inappropriate sexual behavior may be detected later in life. This review will cover the effects of two representative classes of estrogenic EDCs, phytoestrogens and polychlorinated biphenyls (PCBs), on neuroendocrine reproductive function, from molecules to behavior, across the vertebrate life cycle. Finally, we identify the gaps of knowledge in this field and suggest future directions for study.