Selection on resilience improves disease resistance and tolerance to infections.

Response to infection in animals has 2 main mechanisms: resistance (ability to control pathogen burden) and tolerance (ability to maintain performance given the pathogen burden). Selection on disease resistance and tolerance to infections seems a promising avenue to increase productivity of animals in the presence of disease infections, but it is hampered by a lack of records of pathogen burden of infected animals. Selection on resilience (ability to maintain performance regardless of pathogen burden) may, therefore, be an alternative pragmatic approach, because it does not need records of pathogen burden. Therefore, the aim of this study was to assess response to selection in resistance and tolerance when selecting on resilience compared with direct selection on resistance and tolerance. Monte Carlo simulation was used combined with selection index theory to predict responses to selection. Using EBV for resilience in the absence of records for pathogen burden resulted in favorable responses in resistance and tolerance to infections, with higher responses in tolerance than in resistance. If resistance and tolerance were unfavorably correlated, lower selection responses were obtained, especially in resistance. When the genetic correlation was very unfavorable, the selection response in tolerance became negative. Results showed that lower selection responses in resistance and tolerance were obtained when the frequency of disease outbreaks was 10% rather than 50% of the contemporary groups. The efficiency of selection on EBV for resilience compared with selection on EBV for resistance and tolerance was, however, not affected by the frequency of disease outbreaks. When records on pathogen burden were available, selection responses in resistance, tolerance, and the total breeding goal were 3 to 28%, 66 to 398%, and 2 to 11% higher, respectively, than when using the EBV for resilience, showing a clear benefit of recording pathogen burden. This study shows that selection on resilience is a pragmatic way of increasing disease resistance and tolerance to infections in the absence of records on pathogen burden, but recording pathogen burden would yield higher selection responses in resistance and tolerance.

Genetic variation for farrowing rate in pigs in response to change in photoperiod and ambient temperature.

Seasonal infertility is often observed as anestrus and a lower conception rate resulting in a reduced farrowing rate (FR) during late summer and early autumn. This is often regarded as an effect of heat stress; however, we observed a reduction in the FR of sows even after correcting for ambient temperature in our data. Therefore, we added change in photoperiod in the analysis of FR considering its effect on sow fertility. Change in photoperiod was modeled using the cosine of the day of first insemination within a year. On an average, the FR decreased by 2% during early autumn with decreasing daily photoperiod compared with early summer with almost no change in daily photoperiod. It declined 0.2% per degree Celsius of ambient temperature above 19.2°C. This result is a step forward in disentangling the 2 environmental components responsible for seasonal infertility. Our next aim was to estimate the magnitude of genetic variation in FR in response to change in photoperiod and ambient temperature to explore opportunities for selecting pigs to have a constant FR throughout the year. We used reaction norm models to estimate additive genetic variation in response to change in photoperiod and ambient temperature. The results revealed a larger genetic variation at stressful environments when daily photoperiod decreased and ambient temperatures increased above 19.2°C compared with neutral environments. Genetic correlations between stressful environments and nonstressful environments ranged from 0.90 (±0.03) to 0.46 (±0.13) depending on the severity of the stress, indicating changes in expression of FR depending on the environment. The genetic correlation between responses of pigs to changes in photoperiod and to those in ambient temperature were positive, indicating that pigs tolerant to decreasing daily photoperiod are also tolerant to high ambient temperatures. Therefore, selection for tolerance to decreasing daily photoperiod should also increase tolerance to high ambient temperatures or vice versa.

Pluripotent stem cell derived hepatocytes: using materials to define cellular differentiation and tissue engineering.

Pluripotent stem cell derived liver cells (hepatocytes) represent a promising alternative to primary tissue for biological and clinical applications. To date, most hepatocyte maintenance and differentiation systems have relied upon the use of animal derived components. This serves as a significant barrier to large scale production and application of stem cell derived hepatocytes. Recently, the use of defined biologics has overcome those limitations in two-dimensional monolayer culture. In order to improve the cell phenotype further, three-dimensional culture systems have been employed to better mimic the in vivo situation, drawing upon materials chemistry, engineering and biology. In this review we discuss efforts in the field, to differentiate pluripotent stem cells towards hepatocytes under defined conditions.

Does the risk of metabolic disorders increase among women with polycystic ovary morphology? A population-based study.

STUDY QUESTION: Is polycystic ovary morphology (PCOM) associated with metabolic syndrome (MS), insulin resistance (IR) and dyslipidemia? SUMMARY ANSWER: No associations between PCOM and metabolic disorders were found. WHAT IS KNOWN ALREADY: Polycystic ovary morphology has a prevalence of 21-63% in healthy women of reproductive age. Results of studies focusing on metabolic abnormalities among females with PCOM, are insufficient and controversial. STUDY DESIGN, SIZE, DURATION: This was a cross-sectional population-based study from five provinces in Iran. A standard questionnaire was filled out during face-to-face interviews and clinical examinations were done. All study subjects were invited to undergo blood sampling and ultrasonographic assessment. PARTICIPANTS/MATERIALS, SETTING, METHODS: From a total of 1772 women, 809 participants met the inclusion criteria of this study, i.e. non-pregnant, reproductive-age, ovulatory, normo-androgenic, without hyperprolactinemia/thyroid dysfunction. Participants were divided into two groups; 126 women with PCOM on ultrasound assessment, as the case and 683 women with normal ovarian morphology, as the control groups. The association of PCOM with MS, IR and dyslipidemia were analyzed using logistic regression models, adjusted for confounding variables. MAIN RESULTS AND THE ROLE OF CHANCE: Mean systolic blood pressure (SBP), high density lipoprotein (HDL) and androstenedione (A4) serum levels of women with PCOM were significantly higher than in the normal group (P = 0.04, 0.05 and 0.008, respectively). Comparison between groups revealed dyslipidemia to be higher among controls. However the results of logistic regression models, after adjustment for possible confounding variables showed that there were no significant association between prevalence of MS, IR and dyslipidemia with PCOM. LIMITATIONS, REASONS FOR CAUTION: Due to the study being cross-sectional, blood samples were collected only once thus we did not measure serum concentrations of progesterone in the luteal phase, which determines subclinical anovulation. Moreover, due to budget limitations, enzyme immunoassay was used for androgenic measurements while mass spectrometry-based assays have been known as the gold standard method. However we defined our groups very strictly to overcome these limitations. WIDER IMPLICATIONS OF THE FINDINGS: It seems that biochemical and metabolic characteristics of women with PCOM do not differ significantly to those of normal women. To clarify the association between PCOM and metabolic characteristics, longitudinal studies investigating long-term metabolic disorders among women with PCOM are highly recommended. STUDY FUNDING/COMPETING INTEREST: No external funding was used for this study. No conflicts of interest are declared.

Estimation of genetic parameters and breeding values across challenged environments to select for robust pigs.

Robustness is an important issue in the pig production industry. Since pigs from international breeding organizations have to withstand a variety of environmental challenges, selection of pigs with the inherent ability to sustain their productivity in diverse environments may be an economically feasible approach in the livestock industry. The objective of this study was to estimate genetic parameters and breeding values across different levels of environmental challenge load. The challenge load (CL) was estimated as the reduction in reproductive performance during different weeks of a year using 925,711 farrowing records from farms distributed worldwide. A wide range of levels of challenge, from favorable to unfavorable environments, was observed among farms with high CL values being associated with confirmed situations of unfavorable environment. Genetic parameters and breeding values were estimated in high- and low-challenge environments using a bivariate analysis, as well as across increasing levels of challenge with a random regression model using Legendre polynomials. Although heritability estimates of number of pigs born alive were slightly higher in environments with extreme CL than in those with intermediate levels of CL, the heritabilities of number of piglet losses increased progressively as CL increased. Genetic correlations among environments with different levels of CL suggest that selection in environments with extremes of low or high CL would result in low response to selection. Therefore, selection programs of breeding organizations that are commonly conducted under favorable environments could have low response to selection in commercial farms that have unfavorable environmental conditions. Sows that had experienced high levels of challenge at least once during their productive life were ranked according to their EBV. The selection of pigs using EBV ignoring environmental challenges or on the basis of records from only favorable environments resulted in a sharp decline in productivity as the level of challenge increased. In contrast, selection using the random regression approach resulted in limited change in productivity with increasing levels of challenge. Hence, we demonstrate that the use of a quantitative measure of environmental CL and a random regression approach can be comprehensively combined for genetic selection of pigs with enhanced ability to maintain high productivity in harsh environments.

Estimating challenge load due to disease outbreaks and other challenges using reproduction records of sows.

A method was developed and tested to estimate challenge load due to disease outbreaks and other challenges in sows using reproduction records. The method was based on reproduction records from a farm with known disease outbreaks. It was assumed that the reduction in weekly reproductive output within a farm is proportional to the magnitude of the challenge. As the challenge increases beyond certain threshold, it is manifested as an outbreak. The reproduction records were divided into 3 datasets. The first dataset called the Training dataset consisted of 57,135 reproduction records from 10,901 sows from 1 farm in Canada with several outbreaks of porcine reproductive and respiratory syndrome (PRRS). The known disease status of sows was regressed on the traits number born alive, number of losses as a combination of still birth and mummified piglets, and number of weaned piglets. The regression coefficients from this analysis were then used as weighting factors for derivation of an index measure called challenge load indicator. These weighting factors were derived with i) a two-step approach using residuals or year-week solutions estimated from a previous step, and ii) a single-step approach using the trait values directly. Two types of models were used for each approach: a logistic regression model and a general additive model. The estimates of challenge load indicator were then compared based on their ability to detect PRRS outbreaks in a Test dataset consisting of records from 65,826 sows from 15 farms in the Netherlands. These farms differed from the Canadian farm with respect to PRRS virus strains, severity and frequency of outbreaks. The single-step approach using a general additive model was best and detected 14 out of the 15 outbreaks. This approach was then further validated using the third dataset consisting of reproduction records of 831,855 sows in 431 farms located in different countries in Europe and America. A total of 41 out of 48 outbreaks detected using data analysis were confirmed based on diagnostic information received from the farms. Among these, 30 outbreaks were due to PRRS while 11 were due to other diseases and challenging conditions. The results suggest that proposed method could be useful for estimation of challenge load and detection of challenge phases such as disease outbreaks.

Tissue engineered bone using select growth factors: A comprehensive review of animal studies and clinical translation studies in man.

There is a growing socio-economic need for effective strategies to repair damaged bone resulting from disease, trauma and surgical intervention. Bone tissue engineering has received substantial investment over the last few decades as a result. A multitude of studies have sought to examine the efficacy of multiple growth factors, delivery systems and biomaterials within in vivo animal models for the repair of critical-sized bone defects. Defect repair requires recapitulation of in vivo signalling cascades, including osteogenesis, chondrogenesis and angiogenesis, in an orchestrated spatiotemporal manner. Strategies to drive parallel, synergistic and consecutive signalling of factors including BMP-2, BMP-7/OP-1, FGF, PDGF, PTH, PTHrP, TGF-ß3, VEGF and Wnts have demonstrated improved bone healing within animal models. Enhanced bone repair has also been demonstrated in the clinic following European Medicines Agency and Food and Drug Administration approval of BMP-2, BMP-7/OP-1, PDGF, PTH and PTHrP. The current review assesses the in vivo and clinical data surrounding the application of growth factors for bone regeneration. This review has examined data published between 1965 and 2013. All bone tissue engineering studies investigating in vivo response of the growth factors listed above, or combinations thereof, utilising animal models or human trials were included. All studies were compiled from PubMed-NCBI using search terms including 'growth factor name', 'in vivo', 'model/animal', 'human', and 'bone tissue engineering'. Focus is drawn to the in vivo success of osteoinductive growth factors incorporated within material implants both in animals and humans, and identifies the unmet challenges within the skeletal regenerative area.

Evaluation of skeletal tissue repair, part 1: assessment of novel growth-factor-releasing hydrogels in an ex vivo chick femur defect model.

Current clinical treatments for skeletal conditions resulting in large-scale bone loss include autograft or allograft, both of which have limited effectiveness. In seeking to address bone regeneration, several tissue engineering strategies have come to the fore, including the development of growth factor releasing technologies and appropriate animal models to evaluate repair. Ex vivo models represent a promising alternative to simple in vitro systems or complex, ethically challenging in vivo models. We have developed an ex vivo culture system of whole embryonic chick femora, adapted in this study as a critical size defect model to investigate the effects of novel bone extracellular matrix (bECM) hydrogel scaffolds containing spatio-temporal growth factor-releasing microparticles and skeletal stem cells on bone regeneration, to develop a viable alternative treatment for skeletal degeneration. Alginate/bECM hydrogels combined with poly (d,l-lactic-co-glycolic acid) (PDLLGA)/triblock copolymer (10-30% PDLLGA-PEG-PDLLGA) microparticles releasing VEGF, TGF-ß3 or BMP-2 were placed, with human adult Stro-1+ bone marrow stromal cells, into 2mm central segmental defects in embryonic chick femurs. Alginate/bECM hydrogels loaded with HSA/VEGF or HSA/TGF-ß3 demonstrated a cartilage-like phenotype, with minimal collagen I deposition, comparable to HSA-only control hydrogels. The addition of BMP-2 releasing microparticles resulted in enhanced structured bone matrix formation, evidenced by increased Sirius red-stained matrix and collagen expression within hydrogels. This study demonstrates delivery of bioactive growth factors from a novel alginate/bECM hydrogel to augment skeletal tissue formation and the use of an organotypic chick femur defect culture system as a high-throughput test model for scaffold/cell/growth factor therapies for regenerative medicine.

Evaluation of skeletal tissue repair, part 2: enhancement of skeletal tissue repair through dual-growth-factor-releasing hydrogels within an ex vivo chick femur defect model.

There is an unmet need for improved, effective tissue engineering strategies to replace or repair bone damaged through disease or injury. Recent research has focused on developing biomaterial scaffolds capable of spatially and temporally releasing combinations of bioactive growth factors, rather than individual molecules, to recapitulate repair pathways present in vivo. We have developed an ex vivo embryonic chick femur critical size defect model and applied the model in the study of novel extracellular matrix (ECM) hydrogel scaffolds containing spatio-temporal combinatorial growth factor-releasing microparticles and skeletal stem cells for bone regeneration. Alginate/bovine bone ECM (bECM) hydrogels combined with poly(d,l-lactic-co-glycolic acid) (PDLLGA)/triblock copolymer (10-30% PDLLGA-PEG-PLDLGA) microparticles releasing dual combinations of vascular endothelial growth factor (VEGF), chondrogenic transforming growth factor beta 3 (TGF-ß3) and the bone morphogenetic protein BMP2, with human adult Stro-1+bone marrow stromal cells (HBMSCs), were placed into 2mm central segmental defects in embryonic day 11 chick femurs and organotypically cultured. Hydrogels loaded with VEGF combinations induced host cell migration and type I collagen deposition. Combinations of TGF-ß3/BMP2, particularly with Stro-1+HBMSCs, induced significant formation of structured bone matrix, evidenced by increased Sirius red-stained matrix together with collagen expression demonstrating birefringent alignment within hydrogels. This study demonstrates the successful use of the chick femur organotypic culture system as a high-throughput test model for scaffold/cell/growth factor therapies in regenerative medicine. Temporal release of dual growth factors, combined with enriched Stro-1+HBMSCs, improved the formation of a highly structured bone matrix compared to single release modalities. These studies highlight the potential of a unique alginate/bECM hydrogel dual growth factor release platform for bone repair.

Variation among sows in response to porcine reproductive and respiratory syndrome.

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease with negative impacts on reproduction of sows. Genetic selection to improve the response of sows to PRRS could be an approach to control the disease. Determining sow response to PRRS requires knowing pathogen burden and sow performance. In practice, though, records of pathogen burden are unavailable. We develop a statistical method to distinguish healthy and disease phases and to develop a method to quantify sows' responses to PRRS without having individual pathogen burden. We analyzed 10,910 sows with 57,135 repeated records of reproduction performance. Disease phases were recognized as strong deviation of herd-year-week estimates for reproduction traits using two methods: Method 1 used raw weekly averages of the herd; Method 2 used a linear model with fixed effects for seasonality, parity, and year, and random effects for herd-year-week and sow. The variation of sows in response to PRRS was quantified using 2 models on the traits number of piglets born alive (NBA) and number of piglets born dead (LOSS): 1) bivariate model considering the trait in healthy and disease phases as different traits, and 2) reaction norm model modeling the response of sows as a linear regression of the trait on herd-year-week estimates of NBA. The linear model for NBA had the highest sensitivity (78%) for disease phases. Residual variances of both were more than doubled in the disease phase compared with the healthy phase. Trait correlations between healthy and disease phases deviated from unity (0.57 ± 0.13 - 0.87 ± 0.18). In the bivariate model, repeatabilities were lower in disease phase compared with healthy phase (0.07 ± 0.027 and 0.16 ± 0.005 for NBA; 0.07 ± 0.027 and 0.09 ± 0.004 for LOSS). The reaction norm model fitted the data better than the bivariate model based on Akaike's information criterion, and had also higher predictive ability in disease phase based on cross validation. Our results show that the linear model is a practical method to distinguish between healthy and disease phases in farm data. We showed that there is variation among sows in response to PRRS, implying possibilities for selection, and the reaction norm model is a good model to study the response of animals toward diseases.

A cross-sectional study of the seroprevalence and flock-level factors associated with ovine and caprine brucellosis in southeastern Iran.

This cross-sectional study was conducted to estimate seroprevalence and to identify flock-level factors associated with seropositivity to brucellosis in small ruminants in Kerman province, southeastern Iran. In October-November 2011, serum samples were randomly collected from 1767 sheep and 1233 goats, older than 18 months, from 300 flocks. The sera were initially screened for the presence of anti-Brucella antibodies using the Rose-Bengal test; those found to be positive were then examined by Wright and 2-mercaptoethanol Brucella agglutination tests. A questionnaire was used to collect data on flock-level factors likely associated with the within flock seroprevalence of brucellosis. The associations were statistically evaluated for significance in multivariable logistic models. Sixty three flocks (21.00%; 95% CI: 16.80-26.60) had at least one seropositive animal. The mean within-flock seroprevalence was 3.10% (95% CI: 2.60-3.90). The presence of newly purchased animals (OR=3.42; 95% CI: 1.35-8.65) was significantly associated with seropositivity. Our findings highlight the role of animal movement among flocks in the epidemiology of brucellosis in this region. Thus, a control program for brucellosis in the region is suggested to impose appropriate restrictions on animal trade and improve knowledge of livestock owners about quarantine principles for newly purchased animals.

Transient serum exposure regimes to support dual differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (MSCs), which can generate both osteoblasts and chondrocytes, represent an ideal resource for orthopaedic repair using tissue-engineering approaches. One major difficulty for the development of osteochondral constructs using undifferentiated MSCs is that serum is typically used in culture protocols to promote differentiation of the osteogenic component, whereas existing chondrogenic differentiation protocols rely on the use of serum-free conditions. In order to define conditions which could be compatible with both chondrogenic and osteogenic differentiation in a single bioreactor, we have analysed the efficiency of new biphasic differentiation regimes based on transient serum exposure followed by serum-free treatment. MSC differentiation was assessed either in serum-free medium or with a range of transient exposure to serum, and compared to continuous serum-containing treatment. Although osteogenic differentation was not supported in the complete absence of serum, marker expression and extensive mineralization analyses established that 5 days of transient exposure triggered a level of differentiation comparable to that observed when serum was present throughout. This initial phase of serum exposure was further shown to support the successful chondrogenic differentiation of MSCs, comparable to controls maintained in serum-free conditions throughout. This study indicates that a culture based on temporal serum exposure followed by serum-free treatment is compatible with both osteogenic and chondrogenic differentiation of MSCs. These results will allow the development of novel strategies for osteochondral tissue engineering approaches using MSCs for regenerative medicine.

Passionflower in the treatment of generalized anxiety: a pilot double-blind randomized controlled trial with oxazepam.

OBJECTIVE: Passionflower (Passiflora incarnata) is a folk remedy for anxiety. A double-blind randomized trial compared the efficacy of Passiflora incarnata extract with oxazepam in the treatment of generalized anxiety disorder. METHODS: The study was performed on 36 out-patients diagnosed with GAD using DSM IV criteria. Patients were allocated in a random fashion: 18 to the Passiflora extract 45 drops/day plus placebo tablet group, and 18 to oxazepam 30 mg/day plus placebo drops for a 4-week trial. RESULTS: Passiflora extract and oxazepam were effective in the treatment of generalized anxiety disorder. No significant difference was observed between the two protocols at the end of trial. Oxazepam showed a rapid onset of action. On the other hand, significantly more problems relating to impairment of job performance were encountered with subjects on oxazepam. CONCLUSION: The results suggest that Passiflora extract is an effective drug for the management of generalized anxiety disorder, and the low incidence of impairment of job performance with Passiflora extract compared to oxazepam is an advantage. A large-scale trial is justified.

Sequence motifs determine structure and Ca++-binding by EF-hand proteins.

Prediction of protein structural and functional characteristics based on specific motif interactions could serve as a powerful tool in many facets of the biological sciences. Such improvements in protein modeling will be instrumental in the enhancement of drug design. A new approach to a sequence description of EF-hand motifs with more than one EF-hand domain is presented here; this permits precise insight into the structural and functional properties of many members of the EF-hand superfamily of calcium-binding proteins. Three separate regular expressions, or signatures, are used to describe an EF-hand motif, and specific relationships must exist between the two sequence motifs for the two neighboring EF-hands in a given calcium-binding domain. Specifically, each of the sequence motifs has a conserved phenylalanine. These two phenylalanine residues are separated by 57+/-10 amino acid residues but interact closely with each other in the tertiary structure of the calcium-binding domain. Changes in conserved residues in the sequence motifs have been shown experimentally to decrease or eliminate the ability of the protein to bind calcium. This new approach of use of multiple sequence motifs, with motif interrelationships, yields a highly specific and robust tool for the prediction of structural and functional properties of new and novel proteins.

Inhibition by lithium of gentamicin-induced release of N-acetyl-beta-D-glucosaminidase in rat submandibular saliva.

1. The effects of gentamicin, lithium and concurrent treatment of these drugs on concentration of total protein and activity of the enzyme N-acetyl-beta-D-glucosaminidase (NAG) in rat submandibular saliva were studied. 2. Pure submandibular saliva was collected intraorally by micropolyethylene canula from anesthetized rats with the use of pilocarpine as secretagogue. 3. Daily intraperitoneal injection of gentamicin (50 mg kg/day) for 5 consecutive days caused a marked increase in total protein concentration and the NAG activity. 4. Chronic treatment of rats for 10 days with lithium chloride solution (1,200 mg/l) caused a significant decrease in total protein concentration but did not affect the NAG activity. 5. Concurrent treatment of lithium with gentamicin caused the total protein concentration and the NAG activity of submandibular saliva to reach to those of controls. 6. In this regard, the protective effects of lithium may be due to interference of this ion with the phosphoinositide cycle.