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Contents Equine in vitro fertilization ( IVF) is still inconsistent. In the present work, we studied how modified Whitten's ( MW) medium and Tissue Culture Medium 199 ( TCM) added with Foetal Bovine Serum ( FBS; 10% v/v) or Bovine Serum Albumin ( BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18-20 h (no sperm added). In Ex oocytes, TCM-199 added with FBS or BSA resulted in higher metaphase II ( MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%) or FBS (52.2%; p < 0.05); this was not observed for Cp oocytes. Equine sperm were capacitated in the same media at 10 × 106 sperm/ml for 4 h at 37°C; total motility and protein tyrosine phosphorylation ( PY) were evaluated. While motility remained unchanged, TCM or MW added with FBS enhanced the number of sperm showing PY-stained tails (25 ± 4.8% and 31 ± 6.6%; mean ± SEM, respectively) over BSA supplemented media (3 ± 1.2% and 11.7 ± 1.1%) for TCM and MW (p < 0.05). In view of the previous results, sperm were capacitated in TCM + FBS and MW + BSA (control); IVF trials were run in the same media supplemented with 200 ng/ml of progesterone, but no fertilization occurred. Our results show that TCM + FBS enhances Ex equine oocyte's meiotic competence over MW + BSA and TCM or MW added with FBS successfully induce equine PY over media supplemented with BSA. [ABSTRACT FROM AUTHOR]

Introduction: Cryptorchidism is a hereditary anomaly characterized by the incomplete descent of one or both testicles to the scrotum. One of the challenges of this anomaly is that the retained testicle maintains its endocrine function. As a consequence, cryptorchid animals produce hormone-tainted meat in comparison to castrated animals and are likely to be more aggressive. Cryptorchidism can lead to reduced animal welfare outcomes and cause economic losses. Identifying genetic markers for cryptorchidism is an essential step toward mitigating these negative outcomes and may facilitate genome manipulation to reduce the occurrence of cryptorchidism. Attempts to identify such markers have used genome-wide association studies. Using whole-exome sequencing, we aimed to identify single nucleotide polymorphisms (SNPs) in the coding regions of cryptorchid pigs and to characterize functional pathways concerning these SNPs., Methods: DNA was extracted and sequenced from 5 healthy and 5 cryptorchid animals from the Landrace breed, using the Illumina HiSeq 2500 platform. Data were pre-processed using the SeqyClean tool and further mapped against the swine reference genome (Sus scrofa 11.1) using BWA software. GATK was used to identify polymorphisms (SNPs and InDels), which were annotated using the VEP tool. Network prediction and gene ontology enrichment analysis were conducted using the Cytoscape platform, and STRING software was used for visualization., Results: A total of 63 SNPs were identified across the genes PIGB, CCPG1, COMMD9, LDLRAD3, TRIM44, MYLPF, SEPTIN, ZNF48, TIA1, FAIM2, KRT18, FBP1, FBP2, CTSL, DAPK1, DHX8, GPR179, DEPDC1B, ENSSSCG00000049573, ENSSSCG00000016384, ENSSSCG00000022657, ENSSSCG00000038825, and ENSSSCG00000001229. Using pathway enrichment analyses and network prospection, we have identified the following significant adjusted p value threshold of 0.001 involved with the biological function pathways of estrogen signaling, cytoskeleton organization, and the pentose phosphate pathway., Conclusion: Our data suggest the involvement of new SNPs and genes in developing cryptorchidism in pigs. However, further studies are needed to validate our results in a larger cohort population. Variations in the GPR179 gene, with implications at the protein level, may be associated with the appearance of this anomaly in the swine. Finally, we are showing that the estrogen signaling pathway may be involved in the pathophysiological mechanisms of this congenital anomaly as previously reported in GWAS., (© 2023 S. Karger AG, Basel.)

Controlled ovarian stimulation is a necessary step in some assisted reproductive procedures allowing a higher collection of female gametes. However, consequences of this stimulation for the gamete or the offspring have been shown in several mammals. Most studies used comparisons between oocytes from different donors, which may contribute to different responses. In this work, we use the bovine model in which each animal serves as its own control. DNA methylation profiles were obtained by single-cell whole-genome bisulfite sequencing of oocytes from pre-ovulatory unstimulated follicles compared to oocytes from stimulated follicles. Results show that the global percentage of methylation was similar between groups, but the percentage of methylation was lower for non-stimulated oocytes in the imprinted genes APEG3, MEG3, and MEG9 and higher in TSSC4 when compared to stimulated oocytes. Differences were also found in CGI of imprinted genes: higher methylation was found among non-stimulated oocytes in MEST (PEG1), IGF2R, GNAS (SCG6), KvDMR1 ICR UMD, and IGF2. In another region around IGF2, the methylation percentage was lower for non-stimulated oocytes when compared to stimulated oocytes. Data drawn from this study might help to understand the molecular reasons for the appearance of certain syndromes in assisted reproductive technologies-derived offspring.

The addition of reproductive fluids (RF) to the culture media has shown benefits in different embryonic traits but its long-term effects on the offspring phenotype are still unknown. We aimed to describe such effects in pigs. Blood samples and growth parameters were collected from piglets derived from in vitro-produced embryos (IVP) with or without RF added in the culture media versus those artificially inseminated (AI), from day 0 to month 6 of life. An oral glucose tolerance test was performed on day 45 of life. We show here the first comparative data of the growth of animals produced through different assisted reproductive techniques, demonstrating differences between groups. Overall, there was a tendency to have a larger size at birth and faster growth in animals derived from in vitro fertilization and embryo culture versus AI, although this trend was diminished by the addition of RFs to the culture media. Similarly, small differences in hematological indices and glucose tolerance between animals derived from AI and those derived from IVP, with a sex-dependent effect, tended to fade in the presence of RF. The addition of RF to the culture media could contribute to minimizing the phenotypical differences between the in vitro-derived and AI offspring, particularly in males.

Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Assisted reproductive technologies play a major role in the cattle industry. An increase in the use of in vitro -derived embryos is currently being seen around the globe. But the efficiency and quality of the in vitro -derived embryos are substandard when compared to the in vivo production. Different protocols have been designed to overcome this issue, one of those being the use of reproductive fluids as supplementation to embryo culture media. In this study, in vitro -derived calves produced with reproductive fluids added to their embryo production protocol were followed for the first year of life pairwise with their in vivo control, produced by artificial insemination (AI), and their in vitro control, produced with standard supplementation in embryo production. The objective was to assess if any differences could be found in terms of growth and development as well as hematological and biochemical analytes between the different systems. All the analysed variables (physical, hematological, and biochemical) were within physiological range and very similar between calves throughout the entire experiment. However, differences were more evident between calves derived from standard in vitro production and AI. We concluded that the use of reproductive fluids as a supplementation to the embryo culture media results in calves with closer growth and development patterns to those born by AI than the use of bovine serum albumin as supplementation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lopes, Soriano-Úbeda, París-Oller, Navarro-Serna, Canha-Gouveia, Sarrias-Gil, Cerón and Coy.)

Assisted reproductive technologies (ART), besides solving several reproductive problems, it has also been used as a tool to improve the animal productivity that is required for feeding the human population. One of these techniques, the embryo transfer (ET), has presented limitations in the porcine species, which could constrain its use in the porcine industry. To clarify the potential of this technique, we aimed to compare the impact of using ET or artificial insemination (AI) on the phenotype of the offspring during its first days of age, in terms of growth and blood parameters. At birth, the body weight was higher for ET-females than AI-females, but this difference was no longer observed at day 15. On day 3, it was observed a higher concentration of red blood cells, haemoglobin, and haematocrit in females-ET and a higher concentration of white blood cells in both ET-derived piglets (males and females) when compared to AI groups. On day 3, the biochemical analysis showed a higher level of albumin for ET-derived males, and a lower level of bilirubin for ET-females than AI controls. However, all values were within the normal ranges. Our results indicate that piglets derived from ET seem to be phenotypically similar to those born by AI, which provides preliminary evidence that the ET procedure is a safe technique, but additional studies beyond 15 days of life are requested to conclude its global impact. Furthermore, the presented reference values of blood parameters in this species are interesting data for the pig industry., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Umbilical Hernia (UH) is characterized by the passage of part of the intestine through the umbilical canal forming the herniary sac. There are several potential causes that can lead to the umbilical hernia such as bacterial infections, management conditions and genetic factors. Since the genetic components involved with UH are poorly understood, this study aimed to identify polymorphisms and genes associated with the manifestation of umbilical hernia in pigs using exome and transcriptome sequencing in a case and control design., Results: In the exome sequencing, 119 variants located in 58 genes were identified differing between normal and UH-affected pigs, and in the umbilical ring transcriptome, 46 variants were identified, located in 27 genes. Comparing the two methodologies, we obtained 34 concordant variants between the exome and transcriptome analyses, which were located in 17 genes, distributed in 64 biological processes (BP). Among the BP involved with UH it is possible to highlight cell adhesion, cell junction regulation, embryonic morphogenesis, ion transport, muscle contraction, within others., Conclusions: We have generated the first exome sequencing related to normal and umbilical hernia-affected pigs, which allowed us to identify several variants possibly involved with this disorder. Many of those variants present in the DNA were confirmed with the RNA-Seq results. The combination of both exome and transcriptome sequencing approaches allowed us to better understand the complex molecular mechanisms underlying UH in pigs and possibly in other mammals, including humans. Some variants found in genes and other regulatory regions are highlighted as strong candidates to the development of UH in pigs and should be further investigated., (© 2021. The Author(s).)

Objective: To evaluate the impact of interventions on pain associated with DOMS., Data Sources: PubMed, EMBASE, PEDro, Cochrane, and Scielo databases were searched, from the oldest records until May/2020. Search terms used included combinations of keywords related to "DOMS" and "intervention therapy"., Eligibility Criteria: Healthy participants (no restrictions were applied, e.g., age, sex, and exercise level). To be included, studies should be: 1) Randomized clinical trial; 2) Having induced muscle damage and subsequently measuring the level of pain; 3) To have applied therapeutic interventions (nonpharmacological or nutritional) and compare with a control group that received no intervention; and 4) The first application of the intervention had to occur immediately after muscle damage had been induced., Results: One hundred and twenty-one studies were included. The results revealed that the contrast techniques (p = 0,002 I 2  = 60 %), cryotherapy (p = 0,002 I 2  = 100 %), phototherapy (p = 0,0001 I 2  = 95 %), vibration (p = 0,004 I 2  = 96 %), ultrasound (p = 0,02 I 2  = 97 %), massage (p < 0,00001 I 2  = 94 %), active exercise (p = 0,0004 I 2  = 93 %) and compression (p = 0,002 I 2  = 93 %) have a better positive effect than the control in the management of DOMS., Conclusion: Low quality evidence suggests that contrast, cryotherapy, phototherapy, vibration, ultrasound, massage, and active exercise have beneficial effects in the management of DOMS-related pain., Competing Interests: Declaration of competing interest None declared. The authors declare that the research was conducted with no features that could be construed as a potential conflict of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits., Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group., Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.

This study investigated the effect of bovine oviductal fluid from late follicular (LF) and early luteal (EL) phases on bull sperm functionality under non-capacitating (NCAP) and capacitating (CAP) conditions. Frozen-thawed semen samples from five bulls were thawed and incubated (0, 1 or 2 h) in NCAP and CAP media supplemented with 1% bovine oviductal fluid (LF and EL groups) and in absence of fluid (C group). Motion parameters were assessed by CASA; sperm viability, acrosomal integrity and membrane lipid disorder parameters were evaluated by flow cytometry; and sperm DNA fragmentation was evaluated by the Comet assay. Finally, in vitro fertilization with sperm treated under CAP conditions was performed and further embryo culture results evaluated. In NCAP medium, addition of LF and EL fluid increased the total and progressive motility, and LF fluid improved the stability of sperm DNA. However, under CAP conditions addition of LF and EL fluid decreased some sperm motion parameters and some parameters of sperm DNA stability. Proportion of viable sperm cells with low lipid disorder was higher in NCAP than CAP medium and addition of LF fluid markedly increased the proportion of viable spermatozoa with high lipid disorder and acrosome alteration (spontaneous acrosome reaction). Under current conditions, incubation of bull sperm with oviductal fluid before insemination did not affect detrimentally the IVF results nor embryo development, being blastocyst rate similar between CAP-LF, CAP-EL and control groups. In conclusion, oviductal fluid positively influences sperm functionality and modulate in vitro capacitation., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The increasing use of in vitro embryo production (IVP) followed by embryo transfer (ET), alongside with cryopreservation of embryos, has risen concerns regarding the possible altered pregnancy rates, calving or even neonatal mortality. One of the hypotheses for these alterations is the current culture conditions of the IVP. In an attempt to better mimic the physiological milieu, embryos were produced with female reproductive fluids (RF) as supplements to culture medium, and another group of embryos were supplemented with bovine serum albumin (BSA) as in vitro control. Embryos were cryopreserved and transferred while, in parallel, an in vivo control (artificial insemination, AI) with the same bull used for IVP was included. An overview on pregnancy rates, recipients' hormonal levels, parturition, and resulting calves were recorded. Results show much similarity between groups in terms of pregnancy rates, gestation length and calves' weight. Nonetheless, several differences on hormonal levels were noted between recipients carrying AI embryos especially when compared to BSA. Some calving issues and neonatal mortality were observed in both IVP groups. In conclusion, most of the parameters studied were similar between both types of IVP derived embryos and the in vivo-derived embryos, suggesting that the IVP technology used was efficient enough for the safe production of calves.

Preimplantation embryos experience profound resetting of epigenetic information inherited from the gametes. Genome-wide analysis at single-base resolution has shown similarities but also species differences between human and mouse preimplantation embryos in DNA methylation patterns and reprogramming. Here, we have extended such analysis to two key livestock species, the pig and the cow. We generated genome-wide DNA methylation and whole-transcriptome datasets from gametes to blastocysts in both species. In oocytes from both species, a distinctive bimodal methylation landscape is present, with hypermethylated domains prevalent over hypomethylated domains, similar to human, while in the mouse the proportions are reversed.An oocyte-like pattern of methylation persists in the cleavage stages, albeit with some reduction in methylation level, persisting to blastocysts in cow, while pig blastocysts have a highly hypomethylated landscape. In the pig, there was evidence of transient de novo methylation at the 8-16 cell stages of domains unmethylated in oocytes, revealing a complex dynamic of methylation reprogramming. The methylation datasets were used to identify germline differentially methylated regions (gDMRs) of known imprinted genes and for the basis of detection of novel imprinted loci. Strikingly in the pig, we detected a consistent reduction in gDMR methylation at the 8-16 cell stages, followed by recovery to the blastocyst stage, suggesting an active period of imprint stabilization in preimplantation embryos. Transcriptome analysis revealed absence of expression in oocytes of both species of ZFP57, a key factor in the mouse for gDMR methylation maintenance, but presence of the alternative imprint regulator ZNF445. In conclusion, our study reveals species differences in DNA methylation reprogramming and suggests that porcine or bovine models may be closer to human in key aspects than in the mouse model.

Umbilical hernia (UH) is one of the most frequent defects affecting pig production, however, it also affects humans and other mammals. UH is characterized as an abnormal protrusion of the abdominal contents to the umbilical region, causing pain, discomfort and reduced performance in pigs. Some genomic regions associated to UH have already been identified, however, no study involving RNA sequencing was performed when umbilical tissue is considered. Therefore, here, we have sequenced the umbilical ring transcriptome of five normal and five UH-affected pigs to uncover genes and pathways involved with UH development. A total of 13,216 transcripts were expressed in the umbilical ring tissue. From those, 230 genes were differentially expressed (DE) between normal and UH-affected pigs (FDR <0.05), being 145 downregulated and 85 upregulated in the affected compared to the normal pigs. A total of 68 significant biological processes were identified and the most relevant were extracellular matrix, immune system, anatomical development, cell adhesion, membrane components, receptor activation, calcium binding and immune synapse. The results pointed out ACAN, MMPs, COLs, EPYC, VIT, CCBE1 and LGALS3 as strong candidates to trigger umbilical hernias in pigs since they act in the extracellular matrix remodeling and in the production, integrity and resistance of the collagen. We have generated the first transcriptome of the pig umbilical ring tissue, which allowed the identification of genes that had not yet been related to umbilical hernias in pigs. Nevertheless, further studies are needed to identify the causal mutations, SNPs and CNVs in these genes to improve our understanding of the mechanisms of gene regulation., Competing Interests: The authors have declared that no competing interests exist.

Social living animals have to adjust their behavior to rapid changes in the social environment. It has been hypothesized that the expression of social behavior is better explained by the activity pattern of a diffuse social decision-making network (SDMN) in the brain than by the activity of a single brain region. In this study, we tested the hypothesis that it is the assessment that individuals make of the outcome of the fights, rather than the expression of aggressive behavior per se , that triggers changes in the pattern of activation of the SDMN which are reflected in socially driven behavioral profiles (e.g., dominant vs. subordinate specific behaviors). For this purpose, we manipulated the perception of the outcome of an agonistic interaction in an African cichlid fish ( Oreochromis mossambicus ) and assessed if either the perception of outcome or fighting by itself was sufficient to trigger rapid changes in the activity of the SDMN. We have used the expression of immediate early genes ( c-fos and egr-1 ) as a proxy to measure the neuronal activity in the brain. Fish fought their own image on a mirror for 15 min after which they were allocated to one of three conditions for the two last minutes of the trial: (1) they remained fighting the mirror image (no outcome treatment); (2) the mirror was lifted and a dominant male that had just won a fight was presented behind a transparent partition (perception of defeat treatment); and (3) the mirror was lifted and a subordinate male that had just lost a fight was presented behind a transparent partition (perception of victory treatment). Results show that these short-term social interactions elicit distinct patterns in the SDMN and that the perception of the outcome was not a necessary condition to trigger a SDMN response as evidenced in the second treatment (perception of defeat treatment). We suggest that the mutual assessment of relative fighting behavior drives these acute changes in the state of the SDMN., (Copyright © 2019 Almeida, Félix, Oliveira, Lopes and Oliveira.)

Bovine follicular fluid (bFF) is the natural milieu for oocyte growth and development. However, its value as supplementation to in vitro maturation medium is still questioned due to inconsistent results. In this study we hypothesized that adding 10% of follicular fluid as well as heat treating it to inhibit the complement system, would produce higher quality embryos. To do so, experiments were conducted to compare the effect of bFF and heat-treated bFF (bFFin) on oocyte competence assessed by different parameters such as nuclear and cytoplasmic maturation, IVF efficiency, in vitro embryo development and embryo survivability post-vitrification. No differences on nuclear maturation nor cortical granules migration were observed but differences were found on oocyte's cumulus cell expansion, with bFF group having the highest increase (79.0 ± 3.7%). bFFin had a negative impact on IVF efficiency (58.6 ± 3.2%), but no differences were found between bFF (62.9 ± 3.2%) and control (72.8 ± 3.0%). Although the cleavage and blastocyst rate were similar between groups, the day 6 embryo development rate was higher in bFFin group, suggesting an accelerated developmental kinetics. Hatched blastocysts from the bFF group showed a higher cell count than the control group (241.3 ± 20.1 and 185.8 ± 10.0, respectively), and bFFin embryos showed values in between (214.9 ± 14.0). No difference on survivability post-vitrification was found between groups, although the blastocyst stage had a significant impact on the survival rate across all groups. In conclusion, using bFF as supplementation to maturation medium showed a higher benefit when comparing to the standard supplementation by having oocytes with higher cumulus expansion rate, faster development of embryos and higher number of cells per embryo. Inactivation of bFF lowered IVF efficiency but didn't compromise blastocyst development and quality., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Animals communicate by exchanging signals frequently in the proximity of other conspecifics that may detect and intercept signals not directed to them. There is evidence that the presence of these bystanders modulates the signaling behavior of interacting individuals, a phenomenon that has been named audience effect. Research on the audience effect has predominantly focused on its function rather than on its proximate mechanisms. Here, we have investigated the physiological and neuromolecular correlates of the audience effect in a cichlid fish (Mozambique tilapia, Oreochromis mossambicus ). A male was exposed to a territorial intrusion in the presence or absence of a female audience. Results showed that the presence of the female audience increased territorial defense, but elicited a lower androgen and cortisol response to the territorial intrusion. Furthermore, analysis of the expression of immediate early genes, used as markers of neuronal activity, in brain areas belonging to the social decision-making network (SDMN) revealed different patterns of network activity and connectivity across the different social contexts (i.e., audience × intrusion). Overall, these results suggest that socially driven plasticity in the expression of territorial behavior is accommodated in the central nervous system by rapid changes in functional connectivity between nodes of relevant networks (SDMN) rather than by localized changes of activity in specific brain nuclei.

Setting global strategies and targets for disease prevention and control often involves mathematical models. Model structure is typically subject to intense scrutiny, such as confrontation with empirical data and alternative formulations, while a less frequently challenged aspect is the widely adopted reduction of parameters to their average values. Focusing on endemic diseases, we use a general transmission model to explain how mean field approximations decrease the estimated R0 from prevalence data, while threshold phenomena - such as the epidemic and reinfection thresholds - remain invariant. This results in an underestimation of the effort required to control disease, which may be particularly severe when the approximation inappropriately places transmission estimates below important thresholds. These concepts are widely applicable across endemic pathogen systems., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Group living animals must be able to express different behavior profiles depending on their social status. Therefore, the same genotype may translate into different behavioral phenotypes through socially driven differential gene expression. However, how social information is translated into a neurogenomic response and what are the specific cues in a social interaction that signal a change in social status are questions that have remained unanswered. Here, we show for the first time, to our knowledge, that the switch between status-specific neurogenomic states relies on the assessment of fight outcome rather than just on self- or opponent-only assessment of fighting ability. For this purpose, we manipulated the perception of fight outcome in male zebrafish and measured its impact on the brain transcriptome using a zebrafish whole genome gene chip. Males fought either a real opponent, and a winner and a loser were identified, or their own image on a mirror, in which case, despite expressing aggressive behavior, males did not experience either a victory or a defeat. Massive changes in the brain transcriptome were observed in real opponent fighters, with losers displaying both a higher number of differentially expressed genes and of coexpressed gene modules than winners. In contrast, mirror fighters expressed a neurogenomic state similar to that of noninteracting fish. The genes that responded to fight outcome included immediate early genes and genes involved in neuroplasticity and epigenetic modifications. These results indicate that, even in cognitively simple organisms such as zebrafish, neurogenomic responses underlying changes in social status rely on mutual assessment of fighting ability.

Public information is widely available at low cost to animals living in social groups. For instance, bystanders may eavesdrop on signaling interactions between conspecifics and use it to adapt their subsequent behavior towards the observed individuals. This social eavesdropping ability is expected to require specialized mechanisms such as social attention, which selects social information available for learning. To begin exploring the genetic basis of social eavesdropping, we used a previously established attention paradigm in the lab to study the brain gene expression profile of male zebrafish (Danio rerio) in relation to the attention they paid towards conspecifics involved or not involved in agonistic interactions. Microarray gene chips were used to characterize their brain transcriptomes based on differential expression of single genes and gene sets. These analyses were complemented by promoter region-based techniques. Using data from both approaches, we further drafted protein interaction networks. Our results suggest that attentiveness towards conspecifics, whether interacting or not, activates pathways linked to neuronal plasticity and memory formation. The network analyses suggested that fos and jun are key players on this response, and that npas4a, nr4a1 and egr4 may also play an important role. Furthermore, specifically observing fighting interactions further triggered pathways associated to a change in the alertness status (dnajb5) and to other genes related to memory formation (btg2, npas4b), which suggests that the acquisition of eavesdropped information about social relationships activates specific processes on top of those already activated just by observing conspecifics.

According to the social decision-making (SDM) network hypothesis, SDM is encoded in a network of forebrain and midbrain structures in a distributed and dynamic fashion, such that the expression of a given social behaviour is better reflected by the overall profile of activation across the different loci rather than by the activity of a single node. This proposal has the implicit assumption that SDM relies on integration across brain regions, rather than on regional specialization. Here we tested the occurrence of functional localization and of functional connectivity in the SDM network. For this purpose we used zebrafish to map different social behaviour states into patterns of neuronal activity, as indicated by the expression of the immediate early genes c-fos and egr-1, across the SDM network. The results did not support functional localization, as some loci had similar patterns of activity associated with different social behaviour states, and showed socially driven changes in functional connectivity. Thus, this study provides functional support to the SDM network hypothesis and suggests that the neural context in which a given node of the network is operating (i.e. the state of its interconnected areas) is central to its functional relevance., (© 2015 The Author(s).)

Tuberculosis (TB) is a leading cause of human mortality due to infectious disease. Treatment default is a relevant factor which reduces therapeutic success and increases the risk of resistant TB. In this work we analyze the relation between treatment default and treatment length along with its consequence on the disease spreading. We use a stylized model structure to explore, systematically, the effects of varying treatment duration and compliance. We find that shortening treatment alone may not reduce TB prevalence, especially in regions where transmission intensity is high, indicating the necessity of complementing this action with increased compliance. A family of default functions relating the proportion of defaulters to the treatment length is considered and adjusted to a particular dataset. We find that the epidemiological benefits of shorter treatment regimens are tightly associated with increases in treatment compliance and depend on the epidemiological background., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Garcinielliptone FC (GFC) isolated from hexanic fraction seed extract of species Platonia insignis Mart. It is widely used in folk medicine to treat skin diseases in both humans and animals as well as the seed decoction has been used to treat diarrheas and inflammatory diseases. However, there is no research on GFC effects in the central nervous system of rodents. The present study aimed to evaluate the GFC effects at doses of 25, 50 or 75 mg/kg on seizure parameters to determine their anticonvulsant activity and its effects on amino acid (γ-aminobutyric acid (GABA), glutamine, aspartate and glutathione) levels as well as on acetylcholinesterase (AChE) activity in mice hippocampus after seizures. GFC produced an increased latency to first seizure, at doses 25mg/kg (20.12 ± 2.20 min), 50mg/kg (20.95 ± 2.21 min) or 75 mg/kg (23.43 ± 1.99 min) when compared with seized mice. In addition, GABA content of mice hippocampus treated with GFC75 plus P400 showed an increase of 46.90% when compared with seized mice. In aspartate, glutamine and glutamate levels detected a decrease of 5.21%, 13.55% and 21.80%, respectively in mice hippocampus treated with GFC75 plus P400 when compared with seized mice. Hippocampus mice treated with GFC75 plus P400 showed an increase in AChE activity (63.30%) when compared with seized mice. The results indicate that GFC can exert anticonvulsant activity and reduce the frequency of installation of pilocarpine-induced status epilepticus, as demonstrated by increase in latency to first seizure and decrease in mortality rate of animals. In conclusion, our data suggest that GFC may influence in epileptogenesis and promote anticonvulsant actions in pilocarpine model by modulating the GABA and glutamate contents and of AChE activity in seized mice hippocampus. This compound may be useful to produce neuronal protection and it can be considered as an anticonvulsant agent., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The biological effects of interventions to control infectious diseases typically depend on the intensity of pathogen challenge. As much as the levels of natural pathogen circulation vary over time and geographical location, the development of invariant efficacy measures is of major importance, even if only indirectly inferrable. Here a method is introduced to assess host susceptibility to pathogens, and applied to a detailed dataset generated by challenging groups of insect hosts (Drosophila melanogaster) with a range of pathogen (Drosophila C Virus) doses and recording survival over time. The experiment was replicated for flies carrying the Wolbachia symbiont, which is known to reduce host susceptibility to viral infections. The entire dataset is fitted by a novel quantitative framework that significantly extends classical methods for microbial risk assessment and provides accurate distributions of symbiont-induced protection. More generally, our data-driven modeling procedure provides novel insights for study design and analyses to assess interventions.

Androgens are known to respond to social challenges and to control the expression of social behavior and reproductive traits, such as gonadal maturation and sperm production, expression of secondary sex characters and reproductive behaviors. According to the challenge hypothesis variation in androgen levels above a breeding baseline should be explained by the regime of social challenges faced by the individual considering the trade-offs of androgens with other traits (e.g. parental care). One prediction that can be derived from the challenge hypothesis is that androgen levels should increase in response to social instability. Moreover, considering that a tighter association of relevant traits is expected in periods of environmental instability, we also predict that in unstable environments the degree of correlations among different behaviors should increase and hormones and behavior should be associated. These predictions were tested in a polygamous cichlid fish (Mozambique tilapia, Oreochromis mossambicus) with exclusive maternal care. Social instability was produced by swapping dominant males among groups. Stable treatment consisted in removing and placing back dominant males in the same group, in order to control for handling stress. Cortisol levels were also measured to monitor stress levels involved in the procedure and their relation to the androgen patterns and behavior. As predicted androgen levels increased in males in response to the establishment of a social hierarchy and presence of receptive females. However, there were no further differential increases in androgen levels over the social manipulation phase between social stable and social unstable groups. As predicted behaviors were significantly more correlated among themselves in the unstable than in the stable treatment and an associated hormone-behavior pattern was only observed in the unstable treatment., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Archaeplastida (=Kingdom Plantae) are primary plastid-bearing organisms that evolved via the endosymbiotic association of a heterotrophic eukaryote host cell and a cyanobacterial endosymbiont approximately 1,400 Ma. Here, we present analyses of cyanobacterial and plastid genomes that show strongly conflicting phylogenies based on 75 plastid (or nuclear plastid-targeted) protein-coding genes and their direct translations to proteins. The conflict between genes and proteins is largely robust to the use of sophisticated data- and tree-heterogeneous composition models. However, by using nucleotide ambiguity codes to eliminate synonymous substitutions due to codon-degeneracy, we identify a composition bias, and dependent codon-usage bias, resulting from synonymous substitutions at all third codon positions and first codon positions of leucine and arginine, as the main cause for the conflicting phylogenetic signals. We argue that the protein-coding gene data analyses are likely misleading due to artifacts induced by convergent composition biases at first codon positions of leucine and arginine and at all third codon positions. Our analyses corroborate previous studies based on gene sequence analysis that suggest Cyanobacteria evolved by the early paraphyletic splitting of Gloeobacter and a specific Synechococcus strain (JA33Ab), with all other remaining cyanobacterial groups, including both unicellular and filamentous species, forming the sister-group to the Archaeplastida lineage. In addition, our analyses using better-fitting models suggest (but without statistically strong support) an early divergence of Glaucophyta within Archaeplastida, with the Rhodophyta (red algae), and Viridiplantae (green algae and land plants) forming a separate lineage., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Tuberculosis remains a high burden for Human society despite considerable investments in its control. Unique features in the history of infection and transmission dynamics of tuberculosis pose serious limitations on the direct interpretation of surveillance data and call for models that incorporate latent processes and simulate specific interventions., Methods: A transmission model was adjusted to the dataset of active tuberculosis cases reported in Portugal between 2002 and 2009. We estimated key transmission parameters from the data (i.e. time to diagnosis, treatment length, default proportion, proportion of pulmonary TB cases). Using the adjusted model to the Portuguese case, we estimated the total burden of tuberculosis in Portugal. We further performed sensitivity analysis to heterogeneities in susceptibility to infection and exposure intensity., Results: We calculated a mean time to diagnose of 2.81 months and treatment length of 8.80 months in Portugal. The proportion defaulting treatment was calculated as 0.04 and the proportion of pulmonary cases as 0.75. Using these values, we estimated a TB burden of 1.6 million infected persons, corresponding to more than 15% of the Portuguese population. We further described the sensitivity of these estimates to heterogeneity., Conclusions: We showed that the model reproduces well the observed dynamics of the Portuguese data, thus demonstrating its adequacy for devising control strategies for TB and predicting the effects of interventions.

The estimation of parameters in molecular evolution may be biased when some processes are not considered. For example, the estimation of selection at the molecular level using codon-substitution models can have an upward bias when recombination is ignored. Here we address the joint estimation of recombination, molecular adaptation and substitution rates from coding sequences using approximate Bayesian computation (ABC). We describe the implementation of a regression-based strategy for choosing subsets of summary statistics for coding data, and show that this approach can accurately infer recombination allowing for intracodon recombination breakpoints, molecular adaptation and codon substitution rates. We demonstrate that our ABC approach can outperform other analytical methods under a variety of evolutionary scenarios. We also show that although the choice of the codon-substitution model is important, our inferences are robust to a moderate degree of model misspecification. In addition, we demonstrate that our approach can accurately choose the evolutionary model that best fits the data, providing an alternative for when the use of full-likelihood methods is impracticable. Finally, we applied our ABC method to co-estimate recombination, substitution and molecular adaptation rates from 24 published human immunodeficiency virus 1 coding data sets.

Multiple sclerosis (MS) is one of the leading causes of neurologic deficits in young adults and can lead to physical, intellectual and emotional problems. Approved treatments are expensive and are among the 10 highest budgets of the Brazilian Health Ministry. Given the diverse prevalence of MS among Brazilian regions, it is important to determine prevalence rates across the country. Seven studies have assessed MS in Brazil and reported rates ranging from 15 cases to 18 cases per 100,000 inhabitants. It has been hypothesized that this rate is even higher in southern Brazil, which has a high proportion of European heritage (mostly German and Italian) immigrants. Here, we report that the prevalence of MS in the city of Santa Maria, Rio Grande do Sul, Brazil, is 27.2 cases/100,000 inhabitants.

Human tuberculosis is an infectious disease caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). Although spoligotyping and MIRU-VNTR are standard methodologies in MTBC genetic epidemiology, recent studies suggest that Single Nucleotide Polymorphisms (SNP) are advantageous in phylogenetics and strain group/lineages identification. In this work we use a set of 79 SNPs to characterize 1987 MTBC isolates from Portugal and 141 from Northeast Brazil. All Brazilian samples were further characterized using spolygotyping. Phylogenetic analysis against a reference set revealed that about 95% of the isolates in both populations are singly attributed to bacterial lineage 4. Within this lineage, the most frequent strain groups in both Portugal and Brazil are LAM, followed by Haarlem and X. Contrary to these groups, strain group T showed a very different prevalence between Portugal (10%) and Brazil (1.5%). Spoligotype identification shows about 10% of mis-matches compared to the use of SNPs and a little more than 1% of strains unidentifiability. The mis-matches are observed in the most represented groups of our sample set (i.e., LAM and Haarlem) in almost the same proportion. Besides being more accurate in identifying strain groups/lineages, SNP-typing can also provide phylogenetic relationships between strain groups/lineages and, thus, indicate cases showing phylogenetic incongruence. Overall, the use of SNP-typing revealed striking similarities between MTBC populations from Portugal and Brazil., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: Oral myiasis is a rare infection for which treatment protocol has not yet been established. This article presents 2 cases treated with a combination of topical application of sulfuric ether and surgery. The reasons for the use of surgical therapy, as well as the possible advantages and disadvantages of drug-based treatments, are discussed., Case Report: Two cases of oral myiasis are described, the first being observed in a 9-year-old child with hypotonic cerebral palsy, and the second in a 52-year-old adult, alcohol-dependent, both showing infection in the gingival sulcus. Both cases were successfully treated in a process that involved topical application of sulfuric ether, mechanical removal of larvae, and surgical debridement., Conclusions: Oral myiasis can be treated effectively with surgery after topical application of sulfuric ether. The use of drugs may suggest a therapeutic alternative, but still requires further study and experience to be implemented, especially in individuals with neurological disorders., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Recurrent episodes of tuberculosis (TB) can be due to relapse of latent infection or exogenous reinfection, and discrimination is crucial for control planning. Molecular genotyping of Mycobacterium tuberculosis isolates offers concrete opportunities to measure the relative contribution of reinfection in recurrent disease. Here, a mathematical model of TB transmission is fitted to data from 14 molecular epidemiology studies, enabling the estimation of relevant epidemiological parameters. Meta-analysis reveals that rates of reinfection after successful treatment are higher than rates of new TB, raising an important question about the underlying mechanism. We formulate two alternative mechanisms within our model framework: (i) infection increases susceptibility to reinfection or (ii) infection affects individuals differentially, thereby recruiting high-risk individuals to the group at risk for reinfection. The second mechanism is better supported by the fittings to the data, suggesting that reinfection rates are inflated through a population phenomenon that occurs in the presence of heterogeneity in individual risk of infection. As a result, rates of reinfection are higher when measured at the population level even though they might be lower at the individual level. Finally, differential host recruitment is modulated by transmission intensity, being less pronounced when incidence is high.

Objectives: To report the results from the Brazilian database on multiple sclerosis (MS) and pregnancy., Methods: Retrospective data from MS patients who became pregnant at any time of their disease were sent to a Brazilian database, using a specific file for this purpose., Results: Data on 128 women (142 pregnancies) from 30 neurologists working in 21 cities in Brazil were collected. Patients' average age at pregnancy was 29.8 years (range 16-42). EDSS at start of pregnancy was 1.5±1.4; and the relapse rate in the year preceding pregnancy was 1.2±1.5. Exposure to medication at any time during pregnancy was high (69.7%): 48.6% to interferon beta; 14.1% to glatiramer acetate; and 7% to other immunomodulatory and immunosuppressive drugs. There was a significant decrease in relapse rate during pregnancy. The prevalence of complications was relatively low, with 4.9% of obstetric and 1.4% neonatal unfavorable outcomes., Conclusions: Our patients had low degrees of disability, short histories of disease, high drug exposure, and relatively high relapse rate in the year previous to pregnancy. Obstetric and neonatal outcomes were successful in over 90% of our patients., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Interferon (IFN) beta is a safe and efficient drug for treating multiple sclerosis (MS). It is widely accepted that previously depressed patients may get worse when using IFN-beta. There are few reports on the association of IFN-beta and severe depression among patients without previous psychiatric history., Methods: Discussion of a case of a patient with MS who developed severe depression and attempted suicide while using IFN-beta encouraged us to review the subject. A group of neurologists in Brazil retrospectively gathered together their similar cases for the present paper., Results: The present paper reports on 11 cases of severe depression with suicide attempts or ideation among patients with MS who were using IFN-beta. These patients had no previous history of any psychiatric disease. Nine patients developed the symptoms over a relatively short period (4 months, on average). Two patients developed severe depression after more than 1 year of treatment with IFN-beta. Phobic, aggressive, behavioral, psychotic, and manic symptoms also were observed in these patients, thus suggesting the existence of a complex mood-behavior disorder associated with this drug. Interferon beta withdrawal led to complete remission of symptoms. The Naranjo algorithm established a highly probable association between IFN-beta and this adverse reaction in these patients., Conclusions: Although uncommon, severe depression with suicide ideation or attempts may be observed during treatment of MS with IFN-beta. This association should not discourage the use of this drug, but physicians need to be aware of this possible adverse event from IFN-beta.

Approximate Bayesian computation (ABC) is a recently developed technique for solving problems in Bayesian inference. Although typically less accurate than, for example, the frequently used Markov Chain Monte Carlo (MCMC) methods, they have greater flexibility because they do not require the specification of a likelihood function. For this reason considerable amounts of data can be analysed and more complex models can be used providing, thereby, a potential better fit of the model to the data. Since its first applications in the late 1990s its usage has been steadily increasing. The framework was originally developed to solve problems in population genetics. However, as its efficiency was recognized its popularity increased and, consequently, it started to be used in fields as diverse as phylogenetics, ecology, conservation, molecular evolution and epidemiology. While the ABC algorithm is still being greatly studied and alterations to it are being proposed, the statistical approach has already reached a level of maturity well demonstrated by the number of related computer packages that are being developed. As improved ABC algorithms are proposed, the expansion of the use of this method can only increase. In this paper we are going to depict the context that led to the development of ABC focusing on the field of infectious disease epidemiology. We are then going to describe its current usage in such field and present its most recent developments., (Copyright 2009 Elsevier B.V. All rights reserved.)

The clinical picture of dengue is characterized by a maximum duration of 14 days despite frequent complaints of longer symptoms. This study evaluated the occurrence of persistent symptoms (> 14 days) and its impact on daily life. A hundred eighteen patients were interviewed, and the main symptoms at diagnosis were mialgia (98.3%), fever (97.5%) and weakness (95.8%). The presence of at least a persistent symptom was related by 77 (65.2%) individuals of wich 10 (8.5%) described it as intense and lasting for 30 days or more. The most persistent symptoms mentioned were weakness (58 cases), hiporexia (49) and sleepiness (40), occurring mostly in women, with odds ratio: 5.4 (IC95%: 2.3-12.3). A significant association between the persistence of the symptoms and the history of extra expenses (p = 0,02) was found, as well as a delay to return to normal activities (p < 0.001). Thus, it was verified that dengue presented a relevant impact on every day life, even after 14 days, a fact wich was associated with the presence of persistent symptoms of the illness.

Unlabelled: PopABC is a computer package for inferring the pattern of demographic divergence of closely related populations and species. The software performs coalescent simulation in the framework of approximate Bayesian computation (ABC). PopABC can also be used to perform Bayesian model choice to discriminate between different demographic scenarios. The program can be used either for research or for education and teaching purposes., Availability and Implementation: Source code and binaries are freely available at http://www.reading.ac.uk/ approximately sar05sal/software.htm. The program was implemented in C and can run on UNIX, MacOSX and Windows operating systems.

A fifty-nine year old man, known to have hypertrophic cardiomyopathy, presented worsening of angina. Multivessel coronary artery disease was diagnosed, and he underwent myocardial revascularization (mammary and two safenous grafts were implanted) with good evolution and reduction of left atrium dimension.

We report right ventricular involvement in apical hypertrophic cardiomyopathy (HCMP) in two women and one man, aged from 19 to 44 years-old, who presented different signs from those with left ventricular disease (who have good clinical evolution), with signs and symptoms of severe diastolic restriction, showing great atrial enlargement and two with atrial fibrillation and death due to thromboembolism. The right ventricular involvement in apical HCMP leads to a worst prognosis than isolated left ventricular disease, does not have the typical electrocardiographic changes and presents with ventricular restriction and tricuspid regurgitation.

The effect of three anti-depressive psychotropes (Clorimipramine, Doxepine and Dibenzepine) was studied in 107 depressed patients. In each patient the mean value of twelve symptoms was evaluated and compared weekly (for 4 weeks), by statistical methods. In addition, the effect of each drug was analysed in personality stratus. A thymeretic and thymoanaleptic rapid action on 'corporality' and 'endotimic-vital' layer was found with Clorimipramine. Doxepine acts rapidly with sedative and anxiolitic actions on reactive symptoms related with personality super-structures having long term anti-depressive effects. Dibenzepine has a thymeretic rapid and intensive action and a slow thymoanaleptic effect on the same personality stratus of Clorimipramine.