Evaluation of bile salt hydrolase inhibitor efficacy for modulating host bile profile and physiology using a chicken model system.

Gut microbial enzymes, bile salt hydrolases (BSHs) are the gateway enzymes for bile acid (BA) modification in the gut. This activity is a promising target for developing innovative non-antibiotic growth promoters to enhance animal production and health. Compelling evidence has shown that inhibition of BSH activity should enhance weight gain by altering the BA pool, host signalling and lipid metabolism. We recently identified a panel of promising BSH inhibitors. Here, we address the potential of them as alternative, effective, non-antibiotic feed additives, for commercial application, to promote animal growth using a chicken model. In this study, the in vivo efficacy of three BSH inhibitors (caffeic acid phenethylester, riboflavin, carnosic acid) were evaluated. 7-day old chicks (10 birds/group) were either untreated or they received one of the specific BSH inhibitors (25 mg/kg body weight) via oral gavage for 17 days. The chicks in treatment groups consistently displayed higher body weight gain than the untreated chicks. Metabolomic analysis demonstrated that BSH inhibitor treatment led to significant changes in both circulating and intestinal BA signatures in support of blunted intestinal BSH activity. Consistent with this finding, liver and intestinal tissue RNA-Seq analysis showed that carnosic acid treatment significantly altered expression of genes involved in lipid and bile acid metabolism. Taken together, this study validates microbial BSH activity inhibition as an alternative target and strategy to antibiotic treatment for animal growth promotion.

Effect of omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation to lactating sows on growth and indicators of stress in the postweaned pig1,2.

Dietary omega-3 polyunsaturated fatty acids (n-3 PUFA) are precursors for lipid metabolites that reduce inflammation. Two experiments were conducted to test the hypothesis that enriching the sow diet in n-3 PUFA during late gestation and throughout lactation reduces stress and inflammation and promotes growth in weaned pigs. A protected fish oil product (PFO; Gromega) was used to enrich the diet in n-3 PUFA. In the initial experiment, time-bred gilts were fed a gestation and lactation diet supplemented with 0% (control; n = 5), 0.25% (n = 4), 0.5% (n = 4), or 1% (n = 5) PFO from 101 ± 2 d of gestation to day 16 of lactation. Adding 1% PFO to the diet increased the n-3:n-6 PUFA ratio in colostrum and milk compared with controls (P = 0.05). A subsequent experiment was performed to determine whether supplementing the sow diet with 1% PFO improved growth and reduced circulating markers of acute inflammation and stress in the offspring. Plasma was harvested from piglets (16 per treatment group) on day 0 (d of weaning) and days 1 and 3 postweaning. Pigs from the 1% PFO treatment group weighed more (P = 0.03) on day 3 postweaning and had a greater (P ˂ 0.05) n-3:n-6 PUFA ratio in plasma on each day sampled compared with 0% PFO controls. There was an overall treatment effect on plasma total cortisol (P = 0.03) and haptoglobin (P = 0.04), with lesser concentrations in pigs on the 1% PFO diet. Plasma corticosteroid-binding globulin (CBG) concentrations were not different between treatment groups but were less (P ˂ 0.001) on days 1 and 3 when compared with day 0. The resultant free cortisol index [FCI (cortisol/CBG)] was less (P = 0.02) on days 1 and 3 for pigs from the 1% treatment group compared with the controls. An ex vivo lipopolysaccharide (LPS) challenge of whole blood collected on days 0 and 1 was used to determine whether 1% PFO attenuated release of inflammatory cytokines (IL-1ß, IL-6, and TNF-α). Blood from pigs within the 1% PFO treatment group tended (P = 0.098) to have a lesser mean concentration of TNF-α in response to LPS compared with blood from controls. These results suggest that providing a PFO supplement as 1% of the diet to sows beginning in late gestation and during lactation can increase the n-3:n-6 PUFA ratio in their offspring, which may improve growth and reduce the acute physiological stress response in the pigs postweaning.

Agricultural intensification and urbanization negatively impact soil nematode richness and abundance: a meta-analysis.

Human activity has extensively transformed the land surface by agricultural intensification and urbanization. In soil, nematodes are the most abundant invertebrates. The effect of human interventions was assessed on overall richness, overall abundance, richness and abundance of nematodes of each trophic group and colonizer-persister (c-p) guild by comparing urban, agriculture and disturbed grassland (DGL) with natural grassland (NGL) and forest ecosystems. Meta-analyses were conducted to generate quantitative summaries from 111 published articles that met the inclusion criteria, 91 expressed data in grams and 20 expressed data in cm3. Results from data expressed per 100 g of soil indicated that overall richness was higher in forest than in NGL, DGL, urban, and agriculture ecosystems. The richness of all c-p guilds and of all trophic groups except herbivores was highest in forest ecosystems. In contrast, overall abundance was highest in DGL, agriculture and forest ecosystems. The abundance of c-p 1, c-p 2 and c-p 3 guilds and bacterivores, fungivores and herbivores was highest in disturbed ecosystems, while the abundance of c-p 4 and c-p 5 guilds and predators and omnivores was highest in relatively undisturbed ecosystems. Results from data expressed as nematodes per 100 cm3 of soil indicated that abundance followed a similar pattern, but richness often differed between the two methodologies. These meta-analyses strengthen the concept that human interventions adversely impact both richness and abundance using nematodes as soil health bioindicators.Human activity has extensively transformed the land surface by agricultural intensification and urbanization. In soil, nematodes are the most abundant invertebrates. The effect of human interventions was assessed on overall richness, overall abundance, richness and abundance of nematodes of each trophic group and colonizer-persister (c-p) guild by comparing urban, agriculture and disturbed grassland (DGL) with natural grassland (NGL) and forest ecosystems. Meta-analyses were conducted to generate quantitative summaries from 111 published articles that met the inclusion criteria, 91 expressed data in grams and 20 expressed data in cm3. Results from data expressed per 100 g of soil indicated that overall richness was higher in forest than in NGL, DGL, urban, and agriculture ecosystems. The richness of all c-p guilds and of all trophic groups except herbivores was highest in forest ecosystems. In contrast, overall abundance was highest in DGL, agriculture and forest ecosystems. The abundance of c-p 1, c-p 2 and c-p 3 guilds and bacterivores, fungivores and herbivores was highest in disturbed ecosystems, while the abundance of c-p 4 and c-p 5 guilds and predators and omnivores was highest in relatively undisturbed ecosystems. Results from data expressed as nematodes per 100 cm3 of soil indicated that abundance followed a similar pattern, but richness often differed between the two methodologies. These meta-analyses strengthen the concept that human interventions adversely impact both richness and abundance using nematodes as soil health bioindicators.

An in vivo model to assess the thermoregulatory response of lactating Holsteins to an acute heat stress event occurring after a pharmacologically-induced LH surge.

Hyperthermia occurring 10-12 h after LH surge reduces quality of maturing oocyte, thereby reducing fertility. Objective was to examine consequences of an acute heat stress and the influence of certain hormones on the thermoregulatory responses of lactating cows during this critical period. Between the months of February through May, cows were transported to a facility and maintained at a temperature-humidity index (THI) of 65.9 ±â€¯0.2 (thermoneutral) or exposed to changes in THI to simulate what may occur during an acute heat stress event (71-86 THI; heat stress); cows were rapidly cooled thereafter. Mixed model regressions with repeated measures were used to test respiration rates (RR) and rectal temperature (RT). Within 40 and 110 min of increasing THI, RR increased in a quadratic fashion (P < 0.001); RT increased by 0.04 ±â€¯0.1 °C (P < 0.001) per unit THI. Changes in RR lagged THI and preceded rises in RT. Average THI 3-days before treatment (prior THI) influenced RR (P = 0.050) and RT (P < 0.001) changes. Increased RR was more noticeable in heat-stressed cows when prior THI was in the 40 s. Rectal temperature of heat-stressed cows was 0.8 ±â€¯0.02 °C lower when prior THI was in the 40 s versus low 60 s. Levels of progesterone and luteinizing hormone before treatment were predictive of thermoregulatory response in heat-stressed cows. Rapid cooling decreased RR by 0.6 ±â€¯0.1 bpm (P < 0.001) and RT by 0.02 ±â€¯0.002 °C per min (P < 0.002). Speed and magnitude of thermoregulatory changes to an acute heat stress and after sudden cooling emphasizes importance of strategic cooling before ovulation. Efforts to do so when prior THI approaches levels expected to induce mild stress are especially important. Respiration rate is a useful indicator of the degree of hyperthermia a lactating cow is experiencing.

Factors in cattle affecting embryo transfer pregnancies in recipient animals.

The use of embryo transfer (ET) in cattle is important for profitability and improved genetic gains. The advent of the commercial embryo collection and transfer industry has led to advancements in multiple techniques and practices. Specific variables, however, have historically affected pregnancy rates but an understanding of the magnitude of these effects in the current industry is limited. Transfer location (cranial, middle, or caudal third of the uterine horn ipsilateral to the ovary with a CL), transfer score (range of 1-3 with 1 being excellent and 3 poor, based on difficulty of accessing the site of embryo deposition), and amount of time to complete a transfer, therefore, were recorded. These variables were collected in a setting designed to mimic commercial production practices as well as exaggerated time (due to data collection) to assess effects on pregnancy rates. Fresh and frozen in vivo-derived embryos (n = 256) from Bos taurus cows were transferred to Bos taurus recipients. There tended to be more pregnancies when embryos were deposited in the cranial part of the uterus (P = 0.08) compared to the middle and caudal third of the uterus. With a lesser degree of difficulty in transfers (score 1), there tended to be more pregnancies established (P = 0.07). When lesser time was needed for transferring embryos and collecting data, there were greater pregnancy rates (P = 0.03). Thus, these traditionally accepted variables of influence (site of embryo placement in uterus, difficulty, and time) continue to influence ET pregnancy success.

Haplotyping of Cornus florida and C. kousa chloroplasts: Insights into species-level differences and patterns of plastic DNA variation in cultivars.

Chloroplast DNA is a part of plant non-nuclear genome, and is of particular interest for lineage studies. Moreover, the non-coding regions of cpDNA display higher mutation rates than the conserved coding cpDNA, which has been employed for phylogenetic and population research. We analyzed the cpDNA of 332 gDNA samples from collections of Cornus florida and C. kousa (commercial cultivars, breeding selections, and wild kousa accessions from Asia), using the chlorotyping system developed on North America-native, wild accessions of C. florida. Our results indicated significant differences in chlorotype frequencies between the two species. Cornus florida samples were represented by all major chlorotypes previously described, whereas all C. kousa samples analyzed had only one of the chlorotype patterns shown by C. florida. The chlorotyping analytic panel was then expanded by sequencing the targeted three non-coding cpDNA regions. Results indicated a major difference in the maternally-inherited cpDNA between the two closely related Big-Bracted Cornus species. Chlorotype diversity and differences in the proportion of informative sites in the cpDNA regions of focus emphasized the importance of proper loci choice for cpDNA-based comparative studies between the closely related dogwood species. Phylogenetic analyses of the retrieved sequences for the other species of Cornus provided information on the relative utility of the cpDNA regions studied and helped delineate the groups (Big-Bracted, Cornelian Cherries, Blue/White-Fruited) within the genus. Genealogical relationships based on the cpDNA sequences and the inferred chlorotype networks indicated the need for continued analyses across further non-coding cpDNA regions to improve the phylogenetic resolution of dogwoods.

Heat stress impairs gap junction communication and cumulus function of bovine oocytes.

The intimate association of cumulus cells with one another and with the oocyte is important for regulating oocyte meiotic arrest and resumption. The objective of this study was to determine the effects of heat stress on cumulus cell communication and functions that may be related to accelerated oocyte meiosis during early maturation. Bovine cumulus-oocyte complexes underwent in vitro maturation for up to 6 h at thermoneutral control (38.5°C) or elevated (40.0, 41.0 or 42.0°C) temperatures. Gap junction communication between the cumulus cells and the oocyte was assessed using the fluorescent dye calcein after 4 h of in vitro maturation. Dye transfer was reduced in cumulus-oocyte complexes matured at 41.0°C or 42.0°C; transfer at 40.0°C was similar to control (P < 0.0001). Subsequent staining of oocytes with Hoechst revealed that oocytes matured at 41.0 or 42.0°C contained chromatin at more advanced stages of condensation. Maturation of cumulus-oocyte complexes at elevated temperatures reduced levels of active 5' adenosine monophosphate activated kinase (P = 0.03). Heat stress exposure had no effect on active extracellular-regulated kinase 1/2 in oocytes (P = 0.67), associated cumulus cells (P = 0.60) or intact cumulus-oocyte complexes (P = 0.44). Heat-induced increases in progesterone production by cumulus-oocyte complexes were detected during the first 6 h of maturation (P = 0.001). Heat-induced alterations in gap junction communication and other cumulus-cell functions likely cooperate to accelerate bovine oocyte meiotic progression.

Mitochondrial-related consequences of heat stress exposure during bovine oocyte maturation persist in early embryo development.

Hyperthermia during estrus has direct consequences on the maturing oocyte that carries over to the resultant embryo to compromise its ability to continue in development. Because early embryonic development is reliant upon maternal transcripts and other ooplasmic components, we examined impact of heat stress on bovine oocyte transcripts using microarray. Oocytes were matured at 38.5ºC for 24 h or 41.0ºC for the first 12 h of in vitro maturation; 38.5ºC thereafter. Transcriptome profile was performed on total (adenylated + deadenylated) RNA and polyadenylated mRNA populations. Heat stress exposure altered the abundance of several transcripts important for mitochondrial function. The extent to which transcript differences are coincident with functional changes was evaluated by examining reactive oxygen species, ATP content, and glutathione levels. Mitochondrial reactive oxygen species levels were increased by 6 h exposure to 41.0ºC while cytoplasmic levels were reduced compared to controls (P < 0.0001). Exposure to 41.0ºC for 12 h increased total and reduced glutathione levels in oocytes at 12 h but reduced them by 24 h (time × temperature P < 0.001). ATP content was higher in heat-stressed oocytes at 24 h (P < 0.0001). Heat-induced increases in ATP content of matured oocytes persisted in early cleavage-stage embryos (8- to 16-cell embryos; P < 0.05) but were no longer apparent in blastocysts (P > 0.05). Collectively, results indicate that direct exposure of maturing oocytes to heat stress may alter oocyte mitochondrial processes/function, which is inherited by the early embryo after fertilization.

Temporal dynamics of gut microbiota in triclocarban-exposed weaned rats.

Widely used as an antimicrobial in antibacterial bar soaps, triclocarban (3,4,4'-trichlorocarbanilide; TCC) is effective against Gram-positive bacteria but shows little efficacy against Gram-negative strains, potentially altering the composition of indigenous microflora within and on the human body. To date, the consequence of continuous or previous nonprescription antimicrobial exposure from compounds in personal care products on commensal microflora is still elusive. Previous research has shown that TCC exposure during gestation and lactation induced dysbiosis of gut microbial communities among exposed dams and neonates. However, the impact of antimicrobial exposure specifically after discontinuation of the use of TCC on the gut microbiota has not been investigated. In this study, weaned Sprague Dawley rats (postnatal day, PND 22) were provided ad lib access to TCC-supplemented diet (0.2% w/w or 0.5% w/w) for 4 weeks (phase I) followed by a 4-week washout period (phase II) to determine gut microflora changes both during continuous exposure to TCC and to determine the potential rebound following TCC withdrawal. Fecal samples were collected at baseline (PND 22) prior to TCC exposure and throughout phase I and phase II. The V4 region of 16S rDNA was sequenced from extracted total fecal DNA with the MiSeq platform. Exposure to both 0.2% w/w and 0.5% w/w TCC was sufficient to alter diversity of microbiota during phase I of treatment. This effect was further prolonged into phase II, even when TCC exposure was discontinued. Collectively, these data highlight the impact of both continuous and prior TCC exposure on gut microbial ecology and shed light onto the potential long-term health risk of daily nonprescription antimicrobial personal care product use.

A novel approach to analyzing lung cancer mortality disparities: Using the exposome and a graph-theoretical toolchain.

OBJECTIVES: The aim is to identify exposures associated with lung cancer mortality and mortality disparities by race and gender using an exposome database coupled to a graph theoretical toolchain. METHODS: Graph theoretical algorithms were employed to extract paracliques from correlation graphs using associations between 2162 environmental exposures and lung cancer mortality rates in 2067 counties, with clique doubling applied to compute an absolute threshold of significance. Factor analysis and multiple linear regressions then were used to analyze differences in exposures associated with lung cancer mortality and mortality disparities by race and gender. RESULTS: While cigarette consumption was highly correlated with rates of lung cancer mortality for both white men and women, previously unidentified novel exposures were more closely associated with lung cancer mortality and mortality disparities for blacks, particularly black women. CONCLUSIONS: Exposures beyond smoking moderate lung cancer mortality and mortality disparities by race and gender. POLICY IMPLICATIONS: An exposome approach and database coupled with scalable combinatorial analytics provides a powerful new approach for analyzing relationships between multiple environmental exposures, pathways and health outcomes. An assessment of multiple exposures is needed to appropriately translate research findings into environmental public health practice and policy.

Congenic mice demonstrate the presence of QTLs conferring obesity and hypercholesterolemia on chromosome 1 in the TALLYHO mouse.

The TALLYHO (TH) mouse presents a metabolic syndrome of obesity, type 2 diabetes, and hyperlipidemia. Highly significant quantitative trait loci (QTLs) linked to adiposity and hypercholesterolemia were previously identified on chromosome (Chr) 1 in a genome-wide scan of F2 mice from C57BL/6J (B6) x TH. In this study, we generated congenic mouse strains that carry the Chr 1 QTLs derived from TH on a B6 background; B6.TH-Chr1-128Mb (128Mb in size) and B6.TH-Chr1-92Mb (92Mb in size, proximally overlapping). We characterized these congenic mice on chow and high fat (HF) diets. On chow, B6.TH-Chr1-128Mb congenic mice exhibited a slightly larger body fat mass compared with B6.TH-Chr1-92Mb congenic and B6 mice, while body fat mass between B6.TH-Chr1-92Mb congenic and B6 mice was comparable. Plasma total cholesterol levels were significantly higher in B6.TH-Chr1-128Mb congenics compared to B6.TH-Chr1-92Mb congenic and B6 mice. Again, there was no difference in plasma total cholesterol levels between B6.TH-Chr1-92Mb congenic and B6 mice. All animals gained more body fat and exhibited higher plasma total cholesterol levels when fed HF diets than fed chow, but these increases were greater in B6.TH-Chr1-128Mb congenics than in B6.TH-Chr1-92Mb congenic and B6 mice. These results confirmed the effect of the 128Mb TH segment from Chr 1 on body fat and plasma cholesterol values and showed that the distal segment of Chr 1 from TH is necessary to cause both phenotypes. Through bioinformatic approaches, we generated a list of potential candidate genes within the distal region of Chr 1 and tested Ifi202b and Apoa2. We conclude that Chr 1 QTLs largely confer obesity and hypercholesterolemia in TH mice and can be promising targets for identifying susceptibility genes. Congenic mouse strains will be a valuable resource for gene identification.

Behavioral and physiologic changes in Holstein steers experimentally infected with Mannheimia haemolytica.

OBJECTIVE To evaluate changes in behavior and surfactant protein (SP) A and D concentrations in serum and bronchoalveolar lavage fluid (BALF) samples of calves experimentally infected with Mannheimia haemolytica. ANIMALS Twelve 4- to 5-month-old Holstein steers. PROCEDURES Calves were divided into 2 treatment groups and instrumented with a data logger to collect behavioral data. After 10 days of acclimation, calves were experimentally inoculated with 3 × 109 CFUs to 5 × 109 CFUs of M haemolytica suspended in approximately 5 mL of PBS solution (infected calves; n = 6) or 5 mL of PBS solution without M haemolytica (control calves; 6) through a catheter into the right accessory lung lobe. Calves were clinically evaluated twice daily for 7 days after inoculation. Blood and BALF samples were collected from all calves at predetermined times for determination of serum and BALF SP-A and SP-D concentrations. Serum and BALF concentrations of SP-A and SP-D and behavioral data were evaluated over time and between treatment groups. RESULTS Compared with control calves, infected calves spent more time lying in general and more time lying on the right side during the 24 hours and 6 days after inoculation, respectively. Mean rectal temperature for infected calves (41.3°C) was significantly greater than that for control calves (39.2°C) 12 hours after inoculation. Mean respiratory rate for infected calves (52.5 breaths/min) was significantly greater than that for control calves (45.4 breaths/min) throughout the observation period. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated continuous behavioral monitoring may improve detection of calves with respiratory tract disease.

Highly Dynamic Anion-Quadrupole Networks in Proteins.

The dynamics of anion-quadrupole (or anion-π) interactions formed between negatively charged (Asp/Glu) and aromatic (Phe) side chains are for the first time computationally characterized in RmlC (Protein Data Bank entry 1EP0 ), a homodimeric epimerase. Empirical force field-based molecular dynamics simulations predict anion-quadrupole pairs and triplets (anion-anion-π and anion-π-π) are formed by the protein during the simulated trajectory, which suggests that the anion-quadrupole interactions may provide a significant contribution to the overall stability of the protein, with an average of -1.6 kcal/mol per pair. Some anion-π interactions are predicted to form during the trajectory, extending the number of anion-quadrupole interactions beyond those predicted from crystal structure analysis. At the same time, some anion-π pairs observed in the crystal structure exhibit marginal stability. Overall, most anion-π interactions alternate between an "on" state, with significantly stabilizing energies, and an "off" state, with marginal or null stabilizing energies. The way proteins possibly compensate for transient loss of anion-quadrupole interactions is characterized in the RmlC aspartate 84-phenylalanine 112 anion-quadrupole pair observed in the crystal structure. A double-mutant cycle analysis of the thermal stability suggests a possible loss of anion-π interactions compensated by variations of hydration of the residues and formation of compensating electrostatic interactions. These results suggest that near-planar anion-quadrupole pairs can exist, sometimes transiently, which may play a role in maintaining the structural stability and function of the protein, in an otherwise very dynamic interplay of a nonbonded interaction network as well as solvent effects.

Temporal Development of Gut Microbiota in Triclocarban Exposed Pregnant and Neonatal Rats.

Alteration of gut microbial colonization process may influence susceptibility of the newborn/infant to infectious and chronic disease. Infectious disease risk leads to widespread use of non-prescription antimicrobials in household products such as Triclocarban (TCC), an antimicrobial compound in personal care products. TCC concentrates in and is transferred through the milk to suckling offspring. TCC exposure during gestation and lactation significantly reduced phylogenetic diversity (PD) among exposed dams and neonates. Among dams using weighted UniFrac distances, TCC induced significant dysbiosis of gut microbiota by gestational day (GD) 18, a trend that continued after delivery. Similarly, an overall restructuring of gut microbiota occurred in neonates. By postnatal day (PND) 12, communities separated based on exposure status and became significantly different at PND 16. The ability of TCC to drive microbial dysbiosis warrants future investigation to evaluate the safety of non-prescription antimicrobial use, including TCC, during critical exposure windows.

Mycorrhizal Stimulation of Leaf Gas Exchange in Relation to Root Colonization, Shoot Size, Leaf Phosphorus and Nitrogen: A Quantitative Analysis of the Literature Using Meta-Regression.

Arbuscular mycorrhizal (AM) symbiosis often stimulates gas exchange rates of the host plant. This may relate to mycorrhizal effects on host nutrition and growth rate, or the influence may occur independently of these. Using meta-regression, we tested the strength of the relationship between AM-induced increases in gas exchange, and AM size and leaf mineral effects across the literature. With only a few exceptions, AM stimulation of carbon exchange rate (CER), stomatal conductance (g s), and transpiration rate (E) has been significantly associated with mycorrhizal stimulation of shoot dry weight, leaf phosphorus, leaf nitrogen:phosphorus ratio, and percent root colonization. The sizeable mycorrhizal stimulation of CER, by 49% over all studies, has been about twice as large as the mycorrhizal stimulation of g s and E (28 and 26%, respectively). CER has been over twice as sensitive as g s and four times as sensitive as E to mycorrhizal colonization rates. The AM-induced stimulation of CER increased by 19% with each AM-induced doubling of shoot size; the AM effect was about half as large for g s and E. The ratio of leaf N to leaf P has been more closely associated with mycorrhizal influence on leaf gas exchange than leaf P alone. The mycorrhizal influence on CER has declined markedly over the 35 years of published investigations.

Developmental consequences of supplementing with matrix metallopeptidase-9 during in vitro maturation of heat-stressed bovine oocytes.

Because latent form of matrix metallopeptidase-9 (proMMP9) levels are positively related to blastocyst development, it was hypothesized that addition during maturation may improve development of heat-stressed oocytes. To test hypothesis, 0, 30 or 300 ng/ml human proMMP9 (hMMP9) was added at 18 h of in vitro maturation (hIVM) to cumulus-oocyte complexes matured at 38.5 or 41.0ºC (first 12 h only). Heat stress decreased 24 hIVM proMMP9 levels only in 0 and 30 ng/ml groups and increased progesterone in 0 and 300 ng/ml hMMP9 groups. Heat stress decreased cleavage and blastocyst development. Independent of maturation temperature, hMMP9 at 18 hIVM decreased blastocyst development. In a second study, cumulus-oocyte complexes were matured for 24 h at 38.5 or 41.0ºC (HS first 12 h only) with 0 or 300 ng/ml hMMP9 added at 12 hIVM. Without hMMP9, heat stress decreased 24 hIVM proMMP9 levels and increased progesterone production. Addition of 300 ng/ml of hMMP9 produced equivalent levels of proMMP9 at 24 hIVM (271 vs. 279 ± 77 for 38.5ºC and 41.0ºC treated oocytes, respectively). Heat stress did not affect ability of oocytes to cleave but reduced blastocyst development. Independent of temperature, hMMP9 decreased cleavage and blastocyst development. In summary, hMMP9 supplementation during IVM did not improve development of heat-stressed oocytes even when it was added for the entire maturation period. At doses tested, hMMP9 appeared detrimental to development when supplemented during the last 12 or 6 h of oocyte maturation.

Influence of Total Knee Arthroplasty on Gait Mechanics of the Replaced and Non-Replaced Limb During Stair Negotiation.

This study compared biomechanics during stair ascent in replaced and non-replaced limbs of total knee arthroplasty (TKA) patients with control limbs of healthy participants. Thirteen TKA patients and fifteen controls performed stair ascent. Replaced and non-replaced knees of TKA patients were less flexed at contact compared to controls. The loading response peak knee extension moment was greater in control and non-replaced knees compared with replaced. The push-off peak knee abduction moment was elevated in replaced limbs compared to controls. Loading and push-off peak hip abduction moments were greater in replaced limbs compared to controls. The push-off peak hip abduction moment was greater in non-replaced limbs compared to controls. Future rehabilitation protocols should consider the replaced knee and also the non-replaced knee and surrounding joints.

EntropyExplorer: an R package for computing and comparing differential Shannon entropy, differential coefficient of variation and differential expression.

BACKGROUND: Differential Shannon entropy (DSE) and differential coefficient of variation (DCV) are effective metrics for the study of gene expression data. They can serve to augment differential expression (DE), and be applied in numerous settings whenever one seeks to measure differences in variability rather than mere differences in magnitude. A general purpose, easily accessible tool for DSE and DCV would help make these two metrics available to data scientists. Automated p value computations would additionally be useful, and are often easier to interpret than raw test statistic values alone. RESULTS: EntropyExplorer is an R package for calculating DSE, DCV and DE. It also computes corresponding p values for each metric. All features are available through a single R function call. Based on extensive investigations in the literature, the Fligner-Killeen test was chosen to compute DCV p values. No standard method was found to be appropriate for DSE, and so permutation testing is used to calculate DSE p values. CONCLUSIONS: EntropyExplorer provides a convenient resource for calculating DSE, DCV, DE and associated p values. The package, along with its source code and reference manual, are freely available from the CRAN public repository at http://cran.r-project.org/web/packages/EntropyExplorer/index.html.

Impact of heat stress on germinal vesicle breakdown and lipolytic changes during in vitro maturation of bovine oocytes.

Two studies were conducted with the overarching goal of determining the extent to which lipolytic changes relate to germinal vesicle breakdown (GVBD) in bovine oocytes matured under thermoneutral or hyperthermic conditions. To this end, cumulus-oocyte complexes underwent in vitro maturation for 0, 2, 4, 6 or 24 h at 38.5 (first study) or 38.5 and 41.0 C (second study; heat stress applied up through first 12 h only, then shifted to 38.5 C). Independent of maturation temperature, triglyceride and phospholipid content decreased markedly by 2 h of in vitro maturation (hIVM; P < 0.0005). Content was lowest at 24 hIVM with no detectable impact of heat stress when exposure occurred during first 12 hIVM. Germinal vesicle breakdown occurred earlier in oocytes experiencing heat stress with effects observed as soon as 4 hIVM (P < 0.0001). Germinal vesicle breakdown was associated with lipolytic changes (R(2) = 0.2123 and P = 0.0030 for triglyceride content; R(2) = 0.2243 and P = 0.0026 for phospholipid content). ATP content at 24 hIVM was higher in oocytes experiencing heat stress (P = 0.0082). In summary, GVBD occurs sooner in heat-stressed oocytes. Although marked decreases in triglyceride and phospholipid content were noted as early as 2 hIVM and preceded GVBD, lipolytic changes such as these are not likely serving as an initial driver of GVBD in heat-stressed oocytes because changes occurred similarly in oocytes matured at thermoneutral conditions.

BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion.

We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of ß-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer.