Subnational Projections of Lymphatic Filariasis Elimination Targets in Ethiopia to Support National Level Policy.

BACKGROUND: Lymphatic filariasis (LF) is a debilitating, poverty-promoting, neglected tropical disease (NTD) targeted for worldwide elimination as a public health problem (EPHP) by 2030. Evaluating progress towards this target for national programmes is challenging, due to differences in disease transmission and interventions at the subnational level. Mathematical models can help address these challenges by capturing spatial heterogeneities and evaluating progress towards LF elimination and how different interventions could be leveraged to achieve elimination by 2030. METHODS: Here we used a novel approach to combine historical geo-spatial disease prevalence maps of LF in Ethiopia with 3 contemporary disease transmission models to project trends in infection under different intervention scenarios at subnational level. RESULTS: Our findings show that local context, particularly the coverage of interventions, is an important determinant for the success of control and elimination programmes. Furthermore, although current strategies seem sufficient to achieve LF elimination by 2030, some areas may benefit from the implementation of alternative strategies, such as using enhanced coverage or increased frequency, to accelerate progress towards the 2030 targets. CONCLUSIONS: The combination of geospatial disease prevalence maps of LF with transmission models and intervention histories enables the projection of trends in infection at the subnational level under different control scenarios in Ethiopia. This approach, which adapts transmission models to local settings, may be useful to inform the design of optimal interventions at the subnational level in other LF endemic regions.

An Ensemble Framework for Projecting the Impact of Lymphatic Filariasis Interventions Across Sub-Saharan Africa at a Fine Spatial Scale.

BACKGROUND: Lymphatic filariasis (LF) is a neglected tropical disease targeted for elimination as a public health problem by 2030. Although mass treatments have led to huge reductions in LF prevalence, some countries or regions may find it difficult to achieve elimination by 2030 owing to various factors, including local differences in transmission. Subnational projections of intervention impact are a useful tool in understanding these dynamics, but correctly characterizing their uncertainty is challenging. METHODS: We developed a computationally feasible framework for providing subnational projections for LF across 44 sub-Saharan African countries using ensemble models, guided by historical control data, to allow assessment of the role of subnational heterogeneities in global goal achievement. Projected scenarios include ongoing annual treatment from 2018 to 2030, enhanced coverage, and biannual treatment. RESULTS: Our projections suggest that progress is likely to continue well. However, highly endemic locations currently deploying strategies with the lower World Health Organization recommended coverage (65%) and frequency (annual) are expected to have slow decreases in prevalence. Increasing intervention frequency or coverage can accelerate progress by up to 5 or 6 years, respectively. CONCLUSIONS: While projections based on baseline data have limitations, our methodological advancements provide assessments of potential bottlenecks for the global goals for LF arising from subnational heterogeneities. In particular, areas with high baseline prevalence may face challenges in achieving the 2030 goals, extending the "tail" of interventions. Enhancing intervention frequency and/or coverage will accelerate progress. Our approach facilitates preimplementation assessments of the impact of local interventions and is applicable to other regions and neglected tropical diseases.

Characterization of tRNA expression profiles in large offspring syndrome.

BACKGROUND: Assisted Reproductive Technologies (ART) use can increase the risk of congenital overgrowth syndromes, such as large offspring syndrome (LOS) in ruminants. Epigenetic variations are known to influence gene expression and differentially methylated regions (DMRs) were previously determined to be associated with LOS in cattle. We observed DMRs overlapping tRNA clusters which could affect tRNA abundance and be associated with tissue specificity or overgrowth. Variations in tRNA expression have been identified in several disease pathways suggesting an important role in the regulation of biological processes. Understanding the role of tRNA expression in cattle offers an opportunity to reveal mechanisms of regulation at the translational level. We analyzed tRNA expression in the skeletal muscle and liver tissues of day 105 artificial insemination-conceived, ART-conceived with a normal body weight, and ART-conceived bovine fetuses with a body weight above the 97th percentile compared to Control-AI. RESULTS: Despite the centrality of tRNAs to translation, in silico predictions have revealed dramatic differences in the number of tRNA genes between humans and cattle (597 vs 1,659). Consistent with reports in human, only a fraction of predicted tRNA genes are expressed. We detected the expression of 474 and 487 bovine tRNA genes in the muscle and liver with the remainder being unexpressed. 193 and 198 unique tRNA sequences were expressed in all treatment groups within muscle and liver respectively. In addition, an average of 193 tRNA sequences were expressed within the same treatment group in different tissues. Some tRNA isodecoders were differentially expressed between treatment groups. In the skeletal muscle and liver, we categorized 11 tRNA isoacceptors with undetected expression as well as an isodecoder that was unexpressed in the liver (SerGGA). Our results identified variation in the proportion of tRNA gene copies expressed between tissues and differences in the highest contributing tRNA anticodon within an amino acid family due to treatment and tissue type. Out of all amino acid families, roughly half of the most highly expressed tRNA isoacceptors correlated to their most frequent codon in the bovine genome. CONCLUSION: Although the number of bovine tRNA genes is nearly triple of that of the tRNA genes in human, there is a shared occurrence of transcriptionally inactive tRNA genes in both species. We detected differential expression of tRNA genes as well as tissue- and treatment- specific tRNA transcripts with unique sequence variations that could modulate translation during protein homeostasis or cellular stress, and give rise to regulatory products targeting genes related to overgrowth in the skeletal muscle and/or tumor development in the liver of LOS individuals. While the absence of certain isodecoders may be relieved by wobble base pairing, missing tRNA species could increase the likelihood of mistranslation or mRNA degradation.

Progress towards the elimination of hepatitis B in children in Colombia: A novel two-phase study approach.

The World Health Organization (WHO) has established a target to eliminate mother-to-child-transmission (EMTCT) of hepatitis B virus (HBV), defined as a prevalence of hepatitis B surface antigen (HBsAg) of ≤0.1% among children, by 2030. Using nationally representative serosurveys to verify achievement of this target requires large sample sizes and significant resources. We assessed the feasibility of a potentially more efficient two-phase method to verify EMTCT of HBV in Colombia. In the first phase, we conducted a risk assessment to identify municipalities at the highest risk of ongoing HBV transmission. We ranked the 1122 municipalities of Colombia based on the reports of HBV infection in pregnant women per 1000 population. Municipalities with ≥0.3 reports per 1000 persons (equating to the top quartile) were further assessed based on health facility birth rates, coverage with three doses of hepatitis B vaccine (HepB3) and seroprevalence data. Hepatitis B risk was considered to be further increased for municipalities with HepB3 coverage or health facility birth rate <90%. In the second phase, we conducted a multistage household serosurvey of children aged 5-10 years in 36 municipalities with the highest assessed HBV risk. HBsAg was not detected in any of 3203 children tested, yielding a 90% upper confidence bound of <0.1% prevalence. Coverage with HepB3 and hepatitis B birth dose was high at 97.5% and 95.6%, respectively. These results support the conclusion that Colombia has likely achieved EMTCT of HBV.

Predicted Impact of COVID-19 on Neglected Tropical Disease Programs and the Opportunity for Innovation.

Due to the COVID-19 pandemic, many key neglected tropical disease (NTD) activities have been postponed. This hindrance comes at a time when the NTDs are progressing towards their ambitious goals for 2030. Mathematical modelling on several NTDs, namely gambiense sleeping sickness, lymphatic filariasis, onchocerciasis, schistosomiasis, soil-transmitted helminthiases (STH), trachoma, and visceral leishmaniasis, shows that the impact of this disruption will vary across the diseases. Programs face a risk of resurgence, which will be fastest in high-transmission areas. Furthermore, of the mass drug administration diseases, schistosomiasis, STH, and trachoma are likely to encounter faster resurgence. The case-finding diseases (gambiense sleeping sickness and visceral leishmaniasis) are likely to have fewer cases being detected but may face an increasing underlying rate of new infections. However, once programs are able to resume, there are ways to mitigate the impact and accelerate progress towards the 2030 goals.

Conditions of embryo culture from days 5 to 7 of development alter the DNA methylome of the bovine fetus at day 86 of gestation.

PURPOSE: We tested whether in vitro production (IVP) causes changes in DNA methylation in fetal liver and skeletal muscle and if exposure of cultured embryos to colony-stimulating factor 2 (CSF2) alters DNA methylation. METHODS: Female fetuses were produced by artificial insemination or transfer of an IVP embryo. Embryos were treated from days 5 to 7 after fertilization with CSF2 or vehicle. DNA methylation in fetal liver and skeletal muscle was determined by post-bisulfite adaptor tagging-based sequencing. The degree of DNA methylation for CpG sites in 50-bp windows of the promoter region 500 bp upstream of the transcriptional start site was compared between treatments. RESULTS: For liver, there were 12 genes (6% of those analyzed) in which DNA methylation was affected by treatment, with one 50-bp window per gene affected by treatment. For muscle, the degree of DNA methylation was affected by treatment for 32 windows (19% of the total windows analyzed) representing 28 distinct genes (23% of analyzed genes). For 19 of the 28 genes in muscle, the greatest deviation in DNA methylation was for the CSF2 group. CONCLUSION: Results are consistent with alterations in the methylome being one of the mechanisms by which IVP can result in altered fetal development and postnatal function in the resultant offspring. In addition, results indicate that maternally derived cell-signaling molecules can regulate the pattern of DNA methylation.

Modeling allele-specific expression at the gene and SNP levels simultaneously by a Bayesian logistic mixed regression model.

BACKGROUND: High-throughput sequencing experiments, which can determine allele origins, have been used to assess genome-wide allele-specific expression. Despite the amount of data generated from high-throughput experiments, statistical methods are often too simplistic to understand the complexity of gene expression. Specifically, existing methods do not test allele-specific expression (ASE) of a gene as a whole and variation in ASE within a gene across exons separately and simultaneously. RESULTS: We propose a generalized linear mixed model to close these gaps, incorporating variations due to genes, single nucleotide polymorphisms (SNPs), and biological replicates. To improve reliability of statistical inferences, we assign priors on each effect in the model so that information is shared across genes in the entire genome. We utilize Bayesian model selection to test the hypothesis of ASE for each gene and variations across SNPs within a gene. We apply our method to four tissue types in a bovine study to de novo detect ASE genes in the bovine genome, and uncover intriguing predictions of regulatory ASEs across gene exons and across tissue types. We compared our method to competing approaches through simulation studies that mimicked the real datasets. The R package, BLMRM, that implements our proposed algorithm, is publicly available for download at https://github.com/JingXieMIZZOU/BLMRM . CONCLUSIONS: We will show that the proposed method exhibits improved control of the false discovery rate and improved power over existing methods when SNP variation and biological variation are present. Besides, our method also maintains low computational requirements that allows for whole genome analysis.

Vector Preference Annihilates Backward Bifurcation and Reduces Endemicity.

We propose and analyze a mathematical model of a vector-borne disease that includes vector feeding preference for carrier hosts and intrinsic incubation in hosts. Analysis of the model reveals the following novel results. We show theoretically and numerically that vector feeding preference for carrier hosts plays an important role for the existence of both the endemic equilibria and backward bifurcation when the basic reproduction number [Formula: see text] is less than one. Moreover, by increasing the vector feeding preference value, backward bifurcation is eliminated and endemic equilibria for hosts and vectors are diminished. Therefore, the vector protects itself and this benefits the host. As an example of these phenomena, we present a case of Andean cutaneous leishmaniasis in Peru. We use parameter values from previous studies, primarily from Peru to introduce bifurcation diagrams and compute global sensitivity of [Formula: see text] in order to quantify and understand the effects of the important parameters of our model. Global sensitivity analysis via partial rank correlation coefficient shows that [Formula: see text] is highly sensitive to both sandflies feeding preference and mortality rate of sandflies.

Consequences of assisted reproductive techniques on the embryonic epigenome in cattle.

Procedures used in assisted reproduction have been under constant scrutiny since their inception with the goal of improving the number and quality of embryos produced. However, invitro production of embryos is not without complications because many fertilised oocytes fail to become blastocysts, and even those that do often differ in the genetic output compared with their invivo counterparts. Thus only a portion of those transferred complete normal fetal development. An unwanted consequence of bovine assisted reproductive technology (ART) is the induction of a syndrome characterised by fetal overgrowth and placental abnormalities, namely large offspring syndrome; a condition associated with inappropriate control of the epigenome. Epigenetics is the study of chromatin and its effects on genetic output. Establishment and maintenance of epigenetic marks during gametogenesis and embryogenesis is imperative for the maintenance of cell identity and function. ARTs are implemented during times of vast epigenetic reprogramming; as a result, many studies have identified ART-induced deviations in epigenetic regulation in mammalian gametes and embryos. This review describes the various layers of epigenetic regulation and discusses findings pertaining to the effects of ART on the epigenome of bovine gametes and the preimplantation embryo.

The effects of superovulation and reproductive aging on the epigenome of the oocyte and embryo.

A societal preference of delaying maternal age at first childbirth has increased reliance on assisted reproductive technologies/therapies (ART) to conceive a child. Oocytes that have undergone physiologic aging (≥35 years for humans) are now commonly used for ART, yet evidence is building that suboptimal reproductive environments associated with aging negatively affect oocyte competence and embryo development-although the mechanisms underlying these relationship are not yet well understood. Epigenetic programming of the oocyte occurs during its growth within a follicle, so the ovarian stimulation protocols that administer exogenous hormones, as part of the first step for all ART procedures, may prevent the gamete from establishing an appropriate epigenetic state. Therefore, understanding how oocyte. Therefore, understanding how hormone stimulation and oocyte physiologic age independently and synergistically physiologic age independently and synergistically affect the epigenetic programming of these gametes, and how this may affect their developmental competence, are crucial to improved ART outcomes. Here, we review studies that measured the developmental outcomes affected by superovulation and aging, focusing on how the epigenome (i.e., global and imprinted DNA methylation, histone modifications, and epigenetic modifiers) of gametes and embryos acquired from females undergoing physiologic aging and exogenous ovarian stimulation is affected.

Characterization of global loss of imprinting in fetal overgrowth syndrome induced by assisted reproduction.

Embryos generated with the use of assisted reproductive technologies (ART) can develop overgrowth syndromes. In ruminants, the condition is referred to as large offspring syndrome (LOS) and exhibits variable phenotypic abnormalities including overgrowth, enlarged tongue, and abdominal wall defects. These characteristics recapitulate those observed in the human loss-of-imprinting (LOI) overgrowth syndrome Beckwith-Wiedemann (BWS). We have recently shown LOI at the KCNQ1 locus in LOS, the most common epimutation in BWS. Although the first case of ART-induced LOS was reported in 1995, studies have not yet determined the extent of LOI in this condition. Here, we determined allele-specific expression of imprinted genes previously identified in human and/or mouse in day ∼105 Bos taurus indicus × Bos taurus taurus F1 hybrid control and LOS fetuses using RNAseq. Our analysis allowed us to determine the monoallelic expression of 20 genes in tissues of control fetuses. LOS fetuses displayed variable LOI compared with controls. Biallelic expression of imprinted genes in LOS was associated with tissue-specific hypomethylation of the normally methylated parental allele. In addition, a positive correlation was observed between body weight and the number of biallelically expressed imprinted genes in LOS fetuses. Furthermore, not only was there loss of allele-specific expression of imprinted genes in LOS, but also differential transcript amounts of these genes between control and overgrown fetuses. In summary, we characterized previously unidentified imprinted genes in bovines and identified misregulation of imprinting at multiple loci in LOS. We concluded that LOS is a multilocus LOI syndrome, as is BWS.

Colony-stimulating factor 2 acts from days 5 to 7 of development to modify programming of the bovine conceptus at day 86 of gestation†.

Colony-stimulating factor 2 (CSF2) is an embryokine that improves competence of the embryo to establish pregnancy and which may participate in developmental programming. We tested whether culture of bovine embryos with CSF2 alters fetal development and alleviates abnormalities associated with in vitro production (IVP) of embryos. Pregnancies were established by artificial insemination (AI), transfer of an IVP embryo (IVP), or transfer of an IVP embryo treated with 10 ng/ml CSF2 from day 5 to 7 of development (CSF2). Pregnancies were produced using X-sorted semen. Female singleton conceptuses were collected on day 86 of gestation. There were few morphological differences between groups, although IVP and CSF2 fetuses were heavier than AI fetuses. Bicarbonate concentration in allantoic fluid was lower for IVP than for AI or CSF2. Expression of 92 genes in liver, placenta, and muscle was determined. The general pattern for liver and placenta was for IVP to alter expression and for CSF2 to sometimes reverse this effect. For muscle, CSF2 affected gene expression but did not generally reverse effects of IVP. Levels of methylation for each of the three tissues at 12 loci in the promoter of insulin-like growth factor 2 (IGF2) and five in the promoter of growth factor receptor bound protein 10 were unaffected by treatment except for CSF2 effects on two CpG for IGF2 in placenta and muscle. In conclusion, CSF2 can act as a developmental programming agent but alone is not able to abolish the adverse effects of IVP on fetal characteristics.

Hes4: A potential prognostic biomarker for newly diagnosed patients with high-grade osteosarcoma.

BACKGROUND: Prognostic biomarkers for osteosarcoma (OS) at the time of diagnosis are lacking. Necrotic response of OS to preoperative chemotherapy correlates with survival and is determined 3-4 months after diagnosis. The purpose of this study is to identify biomarkers that will stratify patients into good or poor responders to chemotherapy at diagnosis and determine the role of potential biomarkers in OS pathogenesis. PROCEDURE: Because OS may be caused by disruptions of osteogenic differentiation, and the Notch pathway is one regulator of bone development, we examined the link between Notch effectors, OS differentiation, and OS outcome. We probed the R2: Genomics Analysis and Visualization Platform for RNA expression levels of Notch targets in mixed high-grade OS pretreatment biopsies. We used human OS cell lines in vitro and in mice to determine the role of the Notch target hairy/enhancer of split 4 (Hes4) in OS. RESULTS: We found that in OS patients, high expression of Hes4 is correlated with decreased metastasis-free and overall survival. Human OS cells that overexpress Hes4 are more immature and have an increased invasive capacity in vitro. This was not universal to all Notch effectors, as Hes1 overexpression induced opposing effects. When injected into NSG mice, Hes4-overexpressing OS cells produced significantly larger, more lytic tumors and significantly more metastases than did control cells. CONCLUSIONS: Hes4 overexpression promotes a more aggressive tumor phenotype by preventing osteoblastic differentiation of OS cells. Hes4 expression may allow for the stratification of patients into good or poor responders to chemotherapy at diagnosis.

Detection of Missing Proteins Using the PRIDE Database as a Source of Mass Spectrometry Evidence.

The current catalogue of the human proteome is not yet complete, as experimental proteomics evidence is still elusive for a group of proteins known as the missing proteins. The Human Proteome Project (HPP) has been successfully using technology and bioinformatic resources to improve the characterization of such challenging proteins. In this manuscript, we propose a pipeline starting with the mining of the PRIDE database to select a group of data sets potentially enriched in missing proteins that are subsequently analyzed for protein identification with a method based on the statistical analysis of proteotypic peptides. Spermatozoa and the HEK293 cell line were found to be a promising source of missing proteins and clearly merit further attention in future studies. After the analysis of the selected samples, we found 342 PSMs, suggesting the presence of 97 missing proteins in human spermatozoa or the HEK293 cell line, while only 36 missing proteins were potentially detected in the retina, frontal cortex, aorta thoracica, or placenta. The functional analysis of the missing proteins detected confirmed their tissue specificity, and the validation of a selected set of peptides using targeted proteomics (SRM/MRM assays) further supports the utility of the proposed pipeline. As illustrative examples, DNAH3 and TEPP in spermatozoa, and UNCX and ATAD3C in HEK293 cells were some of the more robust and remarkable identifications in this study. We provide evidence indicating the relevance to carefully analyze the ever-increasing MS/MS data available from PRIDE and other repositories as sources for missing proteins detection in specific biological matrices as revealed for HEK293 cells.

Superovulation induces alterations in the epigenome of zygotes, and results in differences in gene expression at the blastocyst stage in mice.

Gamete and embryo manipulations can result in alterations to the epigenome, and are associated with altered gene expression. The initial objective of this study was to determine the transcript level of several epigenetic modifiers in embryos that had been cultured from the 2-cell stage until the late-blastocyst stage in four culture conditions. Cultured embryos were compared to control, in vivo-produced late blastocysts to ascertain if differences in gene expression existed among the culture conditions; none were observed. As all of the embryos used were produced in females that had undergone superovulation, we next compared the transcript level of the same epigenetic modifiers between superovulated, in vivo-produced embryos and embryos produced from natural ovulation. Following in vitro culturing, expression of the genes analyzed was increased in all superovulation groups. We therefore hypothesized that the superovulation procedure-used to increase the number of embryos obtained for experimentation-may have caused an inappropriate acquisition of epigenetic modifications in the maternal genome prior to ovulation, which in turn caused misexpression of genes at the blastocyst stage. To test this hypothesis, we compared the level of global DNA methylation and histone 3 lysine-9 or -14 acetylation in zygotes obtained by natural- or superovulation. Indeed, superovulation decreased global DNA methylation on the maternal pronucleus of zygotes, which inversely correlated with H3K9/14 acetylation. In conclusion, superovulation alters the epigenome of the oocyte, resulting in the dysregulation of gene expression at the blastocyst stage.

What are the minimum requirements for ketogenic diet services in resource-limited regions? Recommendations from the International League Against Epilepsy Task Force for Dietary Therapy.

Despite the increasing use of dietary therapies for children and adults with refractory epilepsy, the availability of these treatments in developing countries with limited resources remains suboptimal. One possible contributory factor may be the costs. There is often reported a significant perceived need for a large ketogenic diet team, supplements, laboratory studies, and follow-up visits to provide this treatment. The 2009 Epilepsia Consensus Statement described ideal requirements for a ketogenic diet center, but in some situations this is not feasible. As a result, the International League Against Epilepsy (ILAE) Task Force on Dietary Therapy was asked to convene and provide practical, cost-effective recommendations for new ketogenic diet centers in resource-limited regions of the world.

Effects of the use of assisted reproduction and high-caloric diet consumption on body weight and cardiovascular health of juvenile mouse offspring.

Maternal obesity and the use of assisted reproductive technologies (ART) are two suboptimal developmental environments that can lead to offspring obesity and cardiovascular disease. We hypothesized that these environments independently and synergistically adversely affect the offspring's weight and cardiovascular performance at ~7 weeks of age. Mice were fed either 24% fat and 17.5% high-fructose (HF) corn syrup or maintenance chow (5% fat; low-fat, no-fructose (LF)). Dams were subdivided into no ART and ART groups. ART embryos were cultured in Whitten's medium and transferred into pseudopregnant recipients consuming the same diet as the donor. Offspring were fed the same diet as the mother. Body weights (BW) were measured weekly and mean arterial pressure (MAP) was collected through carotid artery catheterization at killing (55±0.5 days old). Expression of genes involved in cardiovascular remodeling was measured in thoracic aorta using qRT-PCR, and levels of reactive oxygen species (ROS) were measured intracellularly and extracellularly in mesenteric resistance arteries. ART resulted in increased BW at weaning. This effect decreased over time and diet was the predominant determinant of BW by killing. Males had greater MAP than females (P=0.002) and HF consumption was associated with greater MAP regardless of sex (P<0.05). Gene expression was affected by sex (P<0.05) and diet (P<0.1). Lastly, the use of ART resulted in offspring with increased intracellular ROS (P=0.05). In summary, exposure to an obesogenic diet pre- and/or post-natally affects weight, MAP, and gene expression while ART increases oxidative stress in mesenteric resistance arteries of juvenile offspring, no synergistic effects were observed.

Heterogeneous distribution of histone methylation in mature human sperm.

PURPOSE: To analyze the presence of various histone modifications in ejaculated human spermatozoa METHODS: In this prospective study, seminal ejaculates from 39 normozoospermic individuals were evaluated for semen analysis and the presence of histone modifications in isolated nuclei. RESULTS: We observed heterogeneous presence of histone methylation in normal mature human sperm. We observed that 12 to 30 % of the nuclei of normal sperm contain a heterogeneous distribution of the marks H3K4Me1, H3K9Me2, H3K4Me3, H3K79Me2, and H3K36Me3. Moreover, the presence of these marks is higher in the poor motile fraction of the ejaculate, which is associated with poor morphology and functional quality. In contrast, we did not observe histone acetylation (H3K4Ac and H4K5Ac) in normal or abnormal mature human sperm CONCLUSIONS: Defects in the process of spermatogenesis may alter the correct epigenetic programming in mature sperm. Further studies are required to evaluate the impact of these findings in human infertility.

Cathepsin L regulates oocyte meiosis and preimplantation embryo development.

Early embryonic loss, caused by reduced embryo developmental competence, is the major cause of subfertility in humans and animals. This embryo developmental competence is determined during oocyte maturation and the first embryo divisions. Therefore, it is essential to identify the underlying molecules regulating these critical developmental stages. Cathepsin L (CTSL), a lysosomal cysteine protease, is involved in regulating cell cycle progression, proliferation and invasion of different cell types. However, CTSL role in mammalian embryo development is unknown. Using bovine in vitro maturation and culture systems, we show that CTSL is a key regulator for embryo developmental competence. We employed a specific CTSL detection assay in live cells to show that CTSL activity correlates with meiotic progression and early embryo development. Inhibiting CTSL activity during oocyte maturation or early embryo development significantly impaired oocyte and embryo developmental competence as evidenced by lower cleavage, blastocyst and hatched blastocyst rates. Moreover, enhancing CTSL activity, using recombinant CTSL (rCTSL), during oocyte maturation or early embryo development significantly improved oocyte and embryo developmental competence. Importantly, rCTSL supplementation during oocyte maturation and early embryo development significantly improved the developmental competence of heat-shocked oocytes/embryos which are notoriously known for reduced quality. Altogether, these results provide novel evidence that CTSL plays a pivotal role in regulating oocyte meiosis and early embryonic development.

Locus-specific DNA methylation reprogramming during early porcine embryogenesis.

During early mammalian embryogenesis, there is a wave of DNA demethylation postfertilization and de novo methylation around implantation. The paternal genome undergoes active DNA demethylation, whereas the maternal genome is passively demethylated after fertilization in most mammals except for sheep and rabbits. However, the emerging genome-wide DNA methylation landscape has revealed a regulatory and locus-specific DNA methylation reprogramming pattern in mammalian preimplantation embryos. Here we optimized a bisulfite sequencing protocol to draw base-resolution DNA methylation profiles of several selected genes in gametes, early embryos, and somatic tissue. We observed locus-specific DNA methylation reprogramming in early porcine embryos. First, some pluripotency genes (POU5F1 and NANOG) followed a typical wave of DNA demethylation and remethylation, whereas CpG-rich regions of SOX2 and CDX2 loci were hypomethylated throughout development. Second, a differentially methylated region of an imprint control region in the IGF2/H19 locus exhibited differential DNA methylation which was maintained in porcine early embryos. Third, a centromeric repeat element retained a moderate DNA methylation level in gametes, early embryos, and somatic tissue. The diverse DNA methylation reprogramming during early embryogenesis is thought to be possibly associated with the multiple functions of DNA methylation in transcriptional regulation, genome stability and genomic imprinting. The latest technology such as oxidative bisulfite sequencing to identify 5-hydroxymethylcytosine will further clarify the DNA methylation reprogramming during porcine embryonic development.