Aptamers based sensing of pregnancy associated glycoproteins (PAG) of bovine for early pregnancy detection.

Tosyl activated magnetic beads were used for aptamer selection against PAG- 7 and 18 proteins of bovine origin. PAG proteins were immobilized on beads with further addition of biotin tagged aptamer library. The recognition of aptamers with PAG was identified by ST-HRP based approach which was colorimetric in nature. The selected aptamers were sequenced and at the same time several new aptamers were identified. Later M-fold structure and G-quadruplex score of aptamers were analyzed for their selection. Those aptamers having high G value and complex structure were chosen. In dot blot assay, aptamers recognized PAG protein in an animal after 42 days of artificial insemination which later given birth to a healthy calf. Further the cross reactivity with serum of 0th day animal (post AI) or with non pregnant animal serum was minimal. Aptamers have also shown interaction with PAG protein of buffalo origin. These selected aptamers have commercial application especially in development of biosensors for early detection of pregnancy in bovine.

Establishment of a Sandwich-ELISA for simultaneous quantification of bovine pregnancy-associated glycoprotein in serum and milk.

Bovine pregnancy-associated glycoproteins (boPAG) are expressed by trophoblast cells in the bovine placenta. The multigene family of boPAG belongs to the group of aspartic proteases. The accumulation and circulation in maternal blood and milk has made boPAG very useful and important for pregnancy diagnosis in cattle. The goal of the present study was to develop and validate a new Sandwich-ELISA which allows the detection of boPAG in maternal serum and whole milk. Therefore, 984 serum and 928 milk samples were collected monthly from 231 Holstein Friesian cows (Bos Taurus) from one week after insemination (p.i.) until six weeks postpartum. The ELISA is able to identify a cow as being pregnant at day 30 p.i. in serum and at day 40 p.i in milk with threshold values of 1.0 ng/ml in serum and 0.0165 ng/ml in milk. The postpartum half-life of boPAG was estimated to be 6.4 days in serum and 7.1 days in milk. The boPAG profile established during pregnancy in serum and milk showed a typical pattern. The amount of boPAG found in milk was 1.5 % of the amount of boPAG present in serum. In conclusion, a Sandwich-ELISA has been developed to quantify boPAG in serum and in whole milk simultaneously with the same test procedure. This is time saving for farmers and more efficient for laboratories.

Comparación de biomarcadores de primer trimestre en gestantes normotensas según su grado de obesidad en el tercer trimestre / Comparison of first trimester biomarkers in normotensive pregnancies according to their level of obesity in the third trimester

INTRODUCCIÓN: Nuestro trabajo tiene como objetivo aumentar la eficiencia del cribado de aneuploidías del primer trimestre de la gestación mediante la creación de modelos predictivos que sirvan para identificar gestantes en riesgo de desarrollar sobrepeso u obesidad en el tercer trimestre e instaurar medidas preventivas de obesidad a partir de ellos. MÉTODOS: Estudio observacional de tipo ambispectivo realizado en atención primaria, en el que se han recogido un total de 380 registros correspondientes a otros tantos embarazos. Se han muestreado 6 centros de salud con las variables siguientes: edad en la gestación, proteína A placentaria asociada al embarazo (PAPP-A) (mU/ml), gonadotropina coriónica humana (b-HCG) (ng/ml), semana de recogida de la muestra para el cribado de primer trimestre, índice de masa corporal (IMC) a las 12 y a las 28 semanas de gestación, TSH a las 12 semanas de gestación, presión arterial sistólica (PAS), presión arterial diastólica (PAD) y presión arterial media (PAM) a las 12 y a las 28 semanas de gestación. Se recodificó la variable IMC a las 28 semanas, clasificando a las embarazadas en peso normal (IMC<25), sobrepeso (IMC 25-29,99) y obesas (IMC≥30). El IMC a las 28 semanas recodificada fue la variable resultado del modelo de regresión logística ordinal. Utilizamos el estudio ANOVA de varios factores para discernir diferencias entre las presiones arteriales. Se aceptó un error alfa del 5%. RESULTADOS: Las medianas de la PAPP-A y de b-HCG medidas en el primer trimestre son menores de manera progresiva en los grupos de gestantes con normopeso, sobrepeso y obesidad observadas en el tercer trimestre. Estos valores son predictores del peso en el tercer trimestre (regresión logística ordinal) (PAPP-A: p = 0,022; b-HCG: p = 0,002). Ninguna gestante desarrolló preeclampsia. Las PAS, PAD y PAM en el tercer trimestre fueron significativamente diferentes (ANOVA de varios factores; p < 0,05). DISCUSIÓN: La regresión logística ordinal demuestra que la disminución de los valores observada de PAPP-A y de b-HCG en el primer trimestre es predictora del grado de obesidad de forma significativa y gradual en una muestra de gestantes normotensas. No hemos querido confeccionar un modelo de regresión ordinal incluyendo el IMC de las 12 semanas por la colinealidad interna que aportaría al estar basada la variable resultado en él. El efecto predictor de la b-HCG es más homogéneo que el de la PAPP-A para el estado de sobrepeso y obesidad

INTRODUCTION: This study aims to improve the efficiency of aneuploidy screening in the first trimester of pregnancy by creating predictive models that serve to identify pregnant women at risk of becoming overweight or obese in the third trimester and to using them to implement preventive measures of obesity. METHODS: An ambispective, observational, primary care study was conducted in which a total of 380 records corresponding to as many pregnancies were collected. Samples were collected from patients of 6 health centres, in order to determine the following variables: age at gestation, pregnancy-associated plasma protein A (PAPP-A) (mU/ml), human chorionic gonadotropin (b-HCG) (ng/ml), sample collection week for first trimester screening, body mass index at 12 and 28 weeks gestation (BMI), TSH at 12 weeks gestation, and systolic, diastolic, and mean arterial blood pressure (SBP, DBP, and MBP, respectively) at 12 and 28 weeks gestation. The BMI variable was recoded at 28 weeks, classifying pregnant women as normal weight (BMI<25), overweight (BMI 25-29.99), or obese (BMI≥30). The recoded BMI at 28 weeks was the variable resulting from the ordinal logistic regression model. An ANOVA study of several factors was used to discern differences between arterial pressures. A 5% alpha error was accepted. RESULTS: The PAPP-A and b-HCG medians measured in the first trimester are progressively lower in the groups of pregnant women with normal weight, overweight, and obesity observed in the third trimester. These values are predictors of the weight in the third trimester (ordinal logistic regression) (PAPP-A: P=.022; b-HCG: P=.002). No pregnant woman developed pre-eclampsia. The SBP, DBP, and MBP in the third trimester were significantly different (ANOVA in several factors; P<.05). DISCUSSION: The ordinal logistic regression demonstrates that the decrease in the observed values of PAPP-A and b-HCG in the first trimester is a predictor of the level of significant and gradual obesity in a sample of normotensive pregnant women. An ordinal regression model including the 12-week BMI was not made due to the internal collinearity that it would provide if the result variable was based on it. The predictive effect of b-HCG is more homogeneous than that of PAPP-A for the level of overweight and obesity

Trypanosoma cruzi Presenilin-Like Transmembrane Aspartyl Protease: Characterization and Cellular Localization.

The increasing detection of infections of Trypanosoma cruzi, the etiological agent of Chagas disease, in non-endemic regions beyond Latin America has risen to be a major public health issue. With an impact in the millions of people, current treatments rely on antiquated drugs that produce severe side effects and are considered nearly ineffective for the chronic phase. The minimal progress in the development of new drugs highlights the need for advances in basic research on crucial biochemical pathways in T. cruzi to identify new targets. Here, we report on the T. cruzi presenilin-like transmembrane aspartyl enzyme, a protease of the aspartic class in a unique phylogenetic subgroup with T. vivax separate from protozoans. Computational analyses suggest it contains nine transmembrane domains and an active site with the characteristic PALP motif of the A22 family. Multiple linear B-cell epitopes were identified by SPOT-synthesis analysis with Chagasic patient sera. Two were chosen to generate rabbit antisera, whose signal was primarily localized to the flagellar pocket, intracellular vesicles, and endoplasmic reticulum in parasites by whole-cell immunofluorescence. The results suggest that the parasitic presenilin-like enzyme could have a role in the secretory pathway and serve as a target for the generation of new therapeutics specific to the T. cruzi.

Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis.

BACKGROUND & AIMS: The intestinal epithelium must be resilient to physiochemical stress to uphold the physiological barrier separating the systemic compartment from the microbial and antigenic components of the gut lumen. Identifying proteins that mediate protection and enhancing their expression is therefore a clear approach to promote intestinal health. We previously reported that oral ingestion of the probiotic Lactobacillus rhamnosus GG not only induced the expression of several recognized cytoprotective factors in the murine colon, but also many genes with no previously described function, including the gene encoding proline-rich acidic protein 1 (PRAP1). PRAP1 is a highly expressed protein in the epithelium of the gastrointestinal tract and we sought to define its function in this tissue. METHODS: Purified preparations of recombinant PRAP1 were analyzed biochemically and PRAP1 antisera were used to visualize localization in tissues. Prap1-/- mice were characterized at baseline and challenged with total body irradiation, then enteroids were generated to recapitulate the irradiation challenge ex vivo. RESULTS: PRAP1 is a 17-kilodalton intrinsically disordered protein with no recognizable sequence homology. PRAP1 expression levels were high in the epithelia of the small intestine. Although Prap1-/- mice presented only mild phenotypes at baseline, they were highly susceptible to intestinal injury upon challenge. After irradiation, the Prap1-/- mice showed accelerated death with a significant increase in apoptosis and p21 expression in the small intestinal epithelium. CONCLUSIONS: PRAP1 is an intrinsically disordered protein highly expressed by the gastrointestinal epithelium and functions at exposed surfaces to protect the barrier from oxidative insult.

Selection, identification, and application of DNA aptamers against bovine pregnancy-associated glycoproteins 4.

The bovine pregnancy-associated glycoproteins (bPAGs) have been widely used as robust markers for early diagnosis of pregnancy in the cattle. The current immune recognition methods for detecting bPAGs are limited and, to a certain extent, are associated with high costs and poor stability of the antibody. Aptamers that are more stable and easily synthesized than antibodies might serve as suitable candidates for the development of rapid detection methods. This paper describes selection and characterization of bPAG4 aptamers and theirs applicability to detect bPAG4 in the serum. In this work, the recombinant bovine pregnancy-associated glycoproteins 4 (bPAG4) with a relative molecular mass of about 48 kDa was successfully expressed in human embryonic kidney 293 (HEK 293) cells. Subsequently, the ssDNA aptamers were selected by systematic evolution of ligands by exponential enrichment (SELEX) using magnetic beads (MB) coated with bPAG4 as target. After 9 rounds of selection, three aptamers with high affinity to bPAG4 (Kd = 11.7~40.2 nM) were identified. The selected aptamers were successfully used in enzyme-linked aptamer assay (ELAA) to detect bPAG4 at a detection limit of 0.09 ng/mL. Meanwhile, it has been successfully applied for the detection of bPAG4 in serum samples. This work demonstrated that the selected aptamers could be used as promising affinity probes in the development of inexpensive, simple, and sensitive analysis methods for detecting bPAGs. Graphical abstract.

Human PZP and common marmoset A2ML1 as pregnancy related proteins.

While pregnancy-related proteins (PRP) are known to contribute to immunotolerance during pregnancy, their significance to development of invasive placenta is unclear. We compared PRP expression in humans and the common marmoset (Callithrix jacchus), a new-world monkey. Invasive placenta was observed at the maternal-foetal interface of marmoset placenta from green fluorescent protein (GFP)-expressing foetus and wild type mother. The pregnancy zone protein (PZP) and alpha-2 macroglobulin-like 1 (A2ML1) proteins exhibited the most prominent increase in expression during the second trimester in humans and marmoset, respectively. In humans, PZP accumulated at the maternal-foetal interface and A2ML1 accumulated in the amnion. Similarly, A2ML1 mRNA was detected in marmoset placenta. These proteins belong to the A2M family of protease inhibitors, and both PZP and A2ML1 share around 90% homology between human and marmoset and have highly conserved structures. However, the protease-reacting bait regions of the proteins had lower homology (56.8-60.7% in proteins) relative to the rest of the sequence. Notably, the cleavage site of a proinflammatory proline-endopeptidase was preserved in human PZP and marmoset A2ML1. These proteins contain multiple sites that are cleaved by proteases involving proline-endopeptidase. Systemic regulation of these A2M family proteins may be important in animals with invasive placenta.

2D Electrophoretic pattern of bovine placental proteins during early-mid pregnancy.

The Placenta, like every tissue, possesses its own characteristic protein profile, which may change within the course of pregnancy. These changes can be used for the elucidation of the mechanisms related to both physiology of pregnancy and pathological events. The aim of the study was to describe proteinergic profiles of maternal and fetal parts of bovine placenta during early-mid pregnancy by the use of 2D electrophoresis and MALDI TOF/TOF MS identification to evaluate dynamics of the possible changes necessary for placentation. Placental samples were collected from six pregnant cows (3-5 months) in the local abattoir. Placentomes were separated, and proteins were extracted and subjected to 2D electrophoresis and MALDI TOF/TOF identification. Out of 907 spots identified by the statistical analysis of gels, 54 were identified. Out of this number, 36 spots were significantly different between examined samples. Moreover, the obtained patterns differed between maternal and fetal parts of the placenta with regard to the intensity of staining, suggesting quantitative differences in protein content. These preliminary results are unique for this period of pregnancy. Such data are important for further experiments to obtain full protein profiles necessary to understand biochemical mechanisms underlying the attachment between fetal and maternal parts of the placenta during placentation. Moreover, the outcomes may help in elucidating pregnancy biomarkers in the future.

Resetting the ligand binding site of placental protein 13/galectin-13 recovers its ability to bind lactose.

Placental protein 13/galectin-13 (Gal-13) is highly expressed in placenta, where its lower expression is related to pre-eclampsia. Recently, the crystal structures of wild-type Gal-13 and its variant R53H at high resolution were solved. The crystallographic and biochemical results showed that Gal-13 and R53H could not bind lactose. Here, we used site-directed mutagenesis to re-engineer the ligand binding site of wild-type Gal-13, so that it could bind lactose. Of six newly engineered mutants, we were able to solve the crystal structures of four of them. Three variants (R53HH57R, R53HH57RD33G and R53HR55NH57RD33G had the same two mutations (R53 to H, and H57 to R) and were able to bind lactose in the crystal, indicating that these mutations were sufficient for recovering the ability of Gal-13 to bind lactose. Moreover, the structures of R53H and R53HR55N show that these variants could co-crystallize with a molecule of Tris. Surprisingly, although these variants, as well as wild-type Gal-13, could all induce hemagglutination, high concentrations of lactose could not inhibit agglutination, nor could they bind to lactose-modified Sepharose 6b beads. Overall, our results indicate that Gal-3 is not a normal galectin, which could not bind to ß-galactosides. Lastly, the distribution of EGFP-tagged wild-type Gal-13 and its variants in HeLa cells showed that they are concentrated in the nucleus and could be co-localized within filamentary materials, possibly actin.

Genetic and nongenetic profiling of milk pregnancy-associated glycoproteins in Holstein cattle.

Pregnancy-associated glycoproteins (PAG) are secreted by the trophoblast and are detectable in maternal circulation around the time of attachment of the fetal placenta, as well as in blood and milk throughout gestation. The understanding of the genetic mechanisms controlling PAG levels can confer advantages for livestock breeding programs given the precocity and the ease of obtaining this phenotype from routine pregnancy diagnosis. The aim of this study was to characterize PAG levels by estimating genetic parameters and correlations with other dairy traits, and to identify genomic regions and candidate genes associated with PAG levels in milk. The PAG data consisted of pregnancy diagnoses using commercial assays from 2012 to 2017, and genotype data consisted of 54,123 SNP markers for 2,352 individuals (embryos and dams). The model included contemporary group (herd, year, and season) and embryo age as fixed effects, and random embryonic (direct) and maternal (indirect) genetic effects. Using genomic data, the estimated heritability for direct and maternal genetic effects (± standard deviations) were 0.23 ± 0.05 and 0.11 ± 0.05, respectively. The genetic correlation between these effects was almost zero (0.001 ± 0.06). A preliminary analysis revealed low correlations between milk PAG levels and other dairy traits. The genome-wide association analysis was performed using 2 approaches: single-marker and single-step using all markers. Four genomic regions with direct genetic effects were detected on Bos taurus autosome (BTA) 6, BTA7, BTA19, and BTA29 of the embryonic genome. The BTA29 locus was within the bovine PAG gene cluster, suggesting a cis-regulatory quantitative trait locus on the PAG expression. However, other associations, without an obvious link to PAG expression, could be related to the transportation of PAG and their concentration in milk. Only 1 region from the maternal genome, on BTA4, had a significant indirect effect, where WNT2 is a candidate gene related to placenta vascularization and gestation health. Collectively, our results suggest a moderate genetic control of milk PAG levels from both maternal and fetal genomes, but larger studies are needed to fully evaluate the usefulness of milk PAG in the genetic evaluation of fetal growth and cow fertility.

Assessment of the cellular localisation of the annexin A2/S100A10 complex in human placenta.

The AnxA2/S100A10 complex has been implicated in various placental functions but although the localisation of these proteins individually has been studied, there is no information about the localisation of their complex in situ at the cellular level. Using the proximity ligation technique, we have investigated the in situ localisation of AnxA2/S100A10 complex in the placenta and have compared this with the location patterns of the individual proteins. High levels of expression of AnxA2/S100A10 complexes were observed in the amniotic membrane and in blood vessel endothelial cells. Lower levels were detected in the brush border area of the syncytium and in the trophoblasts. Immunohistochemical analysis of AnxA2 and S100A10 individually revealed broadly similar patterns of localisation. The brush border staining pattern suggests that in this location at least some of the AnxA2 is not in complex with S100A10. The formal location of the AnxA2/S100A10 complex is compatible with a role in cell-cell interaction, intracellular transport and secretory processes and regulation of cell surface proteases, implying contributions to membrane integrity, nutrient exchange, placentation and vascular remodelling in different parts of the placenta. Future applications will allow specific assessment of the association of the complex with pathophysiological disorders.

Denaturation and Aggregation of Interferon-τ in Aqueous Solution.

PURPOSE: To evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride. METHODS: Asymmetrical flow field-flow fractionation (AF4) using both UV and multi-angle laser light scattering (MALLS). Flow Microscopy. All subvisible particle imaging measurements were made using a FlowCAM flow imaging system. RESULTS: The two different denaturants provided different estimates of the conformational stability of the protein when extrapolated back to zero denaturant concentration. This suggests that urea and guanidinium hydrochloride (GnHCl) produce different degrees of residual structure in the unfolded state of interferon-τ. The differences were most pronounced at low temperature, suggesting that the residual structure in the denatured state is progressively lost when samples are heated above 25°C. The extent of expansion in the unfolded states was estimated from the m-values and was also measured using AF4. In contrast, the overall size of interferon-τ was determined by AF4 to decrease in the presence of histidine, which is known to bind to the native state, thereby providing conformational stabilization. Addition of histidine as the buffer resulted in formation of fewer subvisible particles over time at 50°C. Finally, the thermal aggregation was monitored using AF4 and the rate constants were found to be comparable to those determined previously by SEC and DLS. The thermal aggregation appears to be consistent with a nucleation-dependent mechanism with a critical nucleus size of 4 ± 1. CONCLUSION: Chemical denaturation of interferon-τ by urea or GnHCl produces differing amounts of residual structure in the denatured state, leading to differing estimates of conformational stability. AF4 was used to determine changes in size, both upon ligand binding as well as upon denaturation with GnHCl. Histidine appears to be the preferred buffer for interferon-τ, as shown by slower formation of soluble aggregates and reduced levels of subvisible particles when heated at 50°C.

Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA.

In the presented assay, we elaborated a method for distinguishing sequences that are genetically closely related to each other. This is particularly important in a situation where a fine balance of the allele abundance is a point of research interest. We developed a peptide nucleic acid (PNA) strand invasion technique for the differentiation between multiple sclerosis-associated retrovirus (MSRV) and ERVWE1 sequences, both molecularly similar, belonging to the human endogenous retrovirus HERV-W family. We have found that this method may support the PCR technique in screening for minor alleles which, in certain conditions, may be undetected by the standard PCR technique. We performed the analysis of different ERVWE1 and MSRV template mixtures ranging from 0 to 100% of ERVWE1 in the studied samples, finding the linear correlation between template composition and signal intensity of final reaction products. Using the PNA strand invasion assay, we were able to estimate the relative ERVWE1 expression level in human specimens such as U-87 MG, normal human astrocytes cell lines and placental tissue. The results remained in concordance with those obtained by semi-quantitative or quantitative PCR.

The ability to predict pregnancy loss in cattle with ELISAs that detect pregnancy associated glycoproteins is antibody dependent.

The concentration of circulating pregnancy associated glycoproteins (PAGs) early in pregnancy may serve as markers to predict late embryonic mortality or fetal mortality in cattle. In this study, pregnancies were established in dairy cows, by either fixed-time AI (FTAI) or fixed-time embryo transfer (FTET) with in vitro produced embryos. Circulating PAGs were measured with different combinations of antibodies in either a laboratory-based ELISA or a commercial ELISA. For the in-house ELISA, three monoclonal 'trapping' antibodies (A6, J2, and L4) and two polyclonal 'detection' antisera (antibodies F2 or 45) were used to quantify PAGs in serum from the same cows. The different assays were identified as follows: 'Mix-45' (A6, J2, and L4 with 45), 'Mix-F2' (A6, J2, and L4 with F2), and 'L4-F2': (L4 with F2); the commercial assay was from IDEXX. Ovulation was synchronized and FTAI or FTET was performed on day 0 or 7, respectively. Ultrasound-based diagnosis of pregnancy and serum collections occurred on day 30. The proportion of cows that subsequently experienced pregnancy loss between days 30 and 60 was 23% (43 of 183) and 16% (21 of 131) for the FTAI or FTET groups, respectively. In the FTAI group, mean serum concentration of PAGs detected with Mix-45 was higher in cows that maintained pregnancy (9.2 ±â€¯0.4 ng/ml; mean ±â€¯SEM) compared with cows that experienced pregnancy failure (3.9 ±â€¯0.6 ng/ml) between day 30-60 (P < .001). However, there was no difference (P > .69) in circulating concentrations of PAGs between cows that experienced loss or survival between days 30 and 60 when Mix-F2 or L4-F2 were used in an in-house ELISA. Likewise, a commercial assay also did not result in measurable differences in PAG concentrations between those animals that experienced loss or survival. Following FTET, circulating concentrations of PAGs on day 30 were lower (P < .001) in cows that experienced pregnancy failure compared to cows that maintained pregnancy when the Mix-45 and the commercial assay were used, but not with the other antibody combinations. A receiver operating characteristic curve showed that only the Mix-45 antibody combination was predictive (95% accuracy) of pregnancy loss but not the other antibody combinations following FTAI. However, both Mix-45 and the commercial assay were predictive of losses following FTET. In summary, although multiple PAG assay formats have been shown to accurately detect pregnancy, the ability to predict embryo survival during early gestation appears to be antibody dependent.

Short communication: The effect of storage conditions and storage duration on milk ELISA results for pregnancy diagnosis.

The objective of this study was to evaluate the effect of storage temperature and time from sample collection to analysis on test classification of a commercially available ELISA for diagnosis of pregnancy using the measurement of pregnancy-associated glycoproteins (PAG) in milk samples from dairy cows. Few studies have evaluated the effects of sample handling on milk PAG results. Using a repeated-measures study design, we evaluated sample storage at 5 temperatures: 37°C, 22°C, 4°C, -20°C, or -80°C. Sample aliquots from 45 cows (20 with a pregnant test result, 10 open, and 15 recheck) were stored for 4, 7, 14, 28, 60, 90, or 365 d. The measured PAG level was influenced by storage duration and condition. Samples stored for 365 d had a slightly increased PAG level, whereas samples stored for all other durations showed a slight decline in PAG level compared with the initial result. The reason for an increase in PAG level following long-term storage is not known. This will not affect dairy producers using the test but may be important in samples stored for research applications. The changes in PAG level were small and within the expected variation for this test. Fewer than 6% of samples changed in classification and, as expected, they were samples near the test interpretation cut-points.

Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2).

Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a ~45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM. Graphical Abstract ᅟ.

Effect of early pregnancy on the expression of progesterone receptor and progesterone-induced blocking factor in ovine lymph node.

Lymph nodes are the sites where the immune reaction or suppression takes place. Progesterone (P4) exerts an essential effect of the immunomodulation on the maternal uterus during early pregnancy in ruminants. At present study, the inguinal lymph nodes were obtained at day 16 of non-pregnancy, days 13, 16 and 25 of pregnancy (n = 3 for each group) in ewes, and RT-PCR assay, western blot and immunohistochemistry analysis were used to analyze to the effect of early pregnancy on the expression of P4 receptor (PGR) and progesterone-induced blocking factor (PIBF) in the lymph nodes. Our results showed that the PGR and PIBF mRNA were up-regulated in the lymph nodes in pregnant ewes, and the PGR isoform (60 kDa) and the PIBF variant (75 kDa) were expressed constantly in the lymph nodes. However, there was no expression of the PGR isoform (40 kDa) and the PIBF variant (48 kDa) at day 16 of the estrous cycle. The immunohistochemistry results confirmed that the PGR and PIBF proteins were limited to the subcapsular sinus and trabeculae in the cortex, medullary sinuses, and were localized in the cytoplasm of the specific cells. This paper reports for the first time that early pregnancy exerts its effect on the specific cells in the lymph nodes through P4, which results in the up-regulated expression of the PGR mRNA and 40 kDa isoform, the PIBF mRNA and 48 kDa variant, and is involved in the immunoregulation of the lymph nodes through a cytosolic pathway in ewes.

Implantación del cociente sFlt-1/PlGF para la predicción y el diagnóstico de la preeclampsia en embarazos únicos: implicaciones para la práctica clínica / Implantation of the sFlt-1 / PlGF ratio for the prediction and diagnosis of Preeclampsia in single pregnancies: implications for clinical practice

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Label-free proteomic analysis of placental proteins during Toxoplasma gondii infection.

Toxoplasma gondii is a ubiquitous, obligate intracellular parasite capable of crossing the placental barrier and causing spontaneous abortion, preterm labor, or significant disease in the surviving neonate. To better understand molecular mechanisms underlying abnormal pregnancy outcomes caused by T. gondii, placental proteins extracted from T. gondii-infected and -uninfected mice were comparatively analyzed using label-free liquid chromatography-tandem mass spectrometry. Significant difference was observed in the expression of 58 out of 792 proteins in infected placentas (p<0.05) compared with that in uninfected placentas. Quantitative real-time polymerase chain reaction, western blotting, and immunohistochemical staining were used to validate the results of the proteomic analysis. Some placental proteins differentially expressed in infected and uninfected mice were found to be associated with several different biological processes of pregnancy, particularly with trophoblast invasion and placental development. The results provide possible novel insights into the molecular mechanisms for abnormal pregnancy outcomes associated with T. gondii infection. SIGNIFICANCE: In order to further explore the mechanisms of abnormal pregnant outcomes caused by T. gondii infection, we first applied label-free proteomic technology to analyze the differentially expressed host placental proteins with T. gondii infection. The results showed that some differential proteins are associated with trophoblast invasion and placenta development. The findings provide a systemic view of the altered placental proteins and help to declare the molecular mechanisms of abnormal pregnancy outcomes caused by T. gondii infection.

An effective placental cotyledons proteins extraction method for 2D gel electrophoresis.

Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen.