Miracle-dz/UniFAR-FTUs huggingface dataset:
Expression and Regulation of MMP1, MMP3, and MMP9 in the Chicken Ovary in Response to Gonadotropins, Sex Hormones, and TGFB11

ABSTRACT Matrix metalloproteinases (MMPs) are a specific class of proteolytic enzymes that play critical roles in follicular development and luteinization in mammals. However, the role of MMPs in avian ovary remains largely unknown. We found that three MMP genes (MMP1, MMP3, and MMP9) were significantly up-regulated in 23-wk-old (laying phase) chicken ovaries compared with 6-wk-old ovaries (prepubertal phase). In reproductively active chicken ovary, MMP1 expression (both mRNA and protein) remained low in prehierarchical and preovulatory follicles but increased in postovulatory follicles (POFs). Both MMP3 and MMP9 expression levels increased during follicular maturation. MMP3 reached maximal expression in the first largest follicle (F1), while MMP9 levels continued to rise in POF1 and POF2 after ovulation. Immunohistochemistry, Western blot analysis, and zymography experiments indicated that MMP1, MMP3, and MMP9 were synthesized and secreted by granulosa cells of different follicles in the chicken ovary. The mRNA expression of MMP1 and MMP3 in the granulosa cells was stimulated by follicle-stimulating hormone, luteinizing hormone, progesterone, and estrogen but not by transforming growth factor beta 1 (TGFB1). However, the mRNA of MMP9 was induced by TGFB1 but not follicle-stimulating hormone, luteinizing hormone, progesterone, or estrogen. Luciferase reporter and mutagenesis analysis indicated the AP1 and NFkappaB elements located in the promoter region from −1700 to −2400 bp were critical for both basal and TGFB1-induced MMP9 transcription. These data provide the first spatial-temporal expression analysis of MMP system in the chicken ovary.
Structural and Kinetic Analysis of Miscoding Opposite the DNA Adduct 1,N6-Ethenodeoxyadenosine by Human Translesion DNA Polymerase η*

1,N6-Ethenodeoxyadenosine (1,N6-ϵdA) is the major etheno lesion formed in the reaction of DNA with epoxides substituted with good leaving groups (e.g. vinyl chloride epoxide). This lesion is also formed endogenously in DNA from lipid oxidation. Recombinant human DNA polymerase η (hpol η) can replicate oligonucleotide templates containing 1,N6-ϵdA. In steady-state kinetic analysis, hpol η preferred to incorporate dATP and dGTP, compared with dTTP. Mass spectral analysis of incorporation products also showed preferred purine (A, G) incorporation and extensive −1 frameshifts, suggesting pairing of the inserted purine and slippage before further replication. Five x-ray crystal structures of hpol η ternary complexes were determined, three at the insertion and two at the extension stage. Two insertion complexes revealed incoming non-hydrolyzable dATP or dGTP analogs not pairing with but instead in a staggered configuration relative to 1,N6-ϵdA in the anti conformation, thus opposite the 5′-T in the template, explaining the proclivity for frameshift misincorporation. In another insertion complex, dTTP was positioned opposite 1,N6-ϵdA, and the adduct base was in the syn conformation, with formation of two hydrogen bonds. At the extension stage, with either an incorporated dA or dT opposite 1,N6-ϵdA and 2′-deoxythymidine-5′-[(α,β)-imido]triphosphate opposite the 5′-A, the 3′-terminal nucleoside of the primer was disordered, consistent with the tendency not to incorporate dTTP opposite 1,N6-ϵdA. Collectively, the results show a preference for purine pairing opposite 1,N6-ϵdA and for −1 frameshifts.
The Nuclear Receptor NR2F2 Activates Star Expression and Steroidogenesis in Mouse MA-10 and MLTC-1 Leydig Cells1

ABSTRACT Testosterone production is dependent on cholesterol transport within the mitochondrial matrix, an essential step mediated by a protein complex containing the steroidogenic acute regulatory (STAR) protein. In steroidogenic Leydig cells, Star expression is hormonally regulated and involves several transcription factors. NR2F2 (COUP-TFII) is an orphan nuclear receptor that plays critical roles in cell differentiation and lineage determination. Conditional NR2F2 knockout prior to puberty leads to male infertility due to insufficient testosterone production, suggesting that NR2F2 could positively regulate steroidogenesis and Star expression. In this study we found that NR2F2 is expressed in the nucleus of some peritubular myoid cells and in interstitial cells, mainly in steroidogenically active adult Leydig cells. In MA-10 and MLTC-1 Leydig cells, small interfering RNA (siRNA)-mediated NR2F2 knockdown reduces basal steroid production without affecting hormone responsiveness. Consistent with this, we found that STAR mRNA and protein levels were reduced in NR2F2-depleted MA-10 and MLTC-1 cells. Transient transfections of Leydig cells revealed that a −986 bp mouse Star promoter construct was activated 3-fold by NR2F2. Using 5′ progressive deletion constructs, we mapped the NR2F2-responsive element between −131 and −95 bp. This proximal promoter region contains a previously uncharacterized direct repeat 1 (DR1)-like element to which NR2F2 is recruited and directly binds. Mutations in the DR1-like element that prevent NR2F2 binding severely blunted NR2F2-mediated Star promoter activation. These data identify an essential role for the nuclear receptor NR2F2 as a direct activator of Star gene expression in Leydig cells, and thus in the control of steroid hormone biosynthesis.
A functional data analysis approach for continuous 2-D emotion annotations

The standard paradigm in Affective Computing involves acquiring one/several markers (e.g., physiological signals) of emotions and training models on these to predict emotions. However, due to the internal nature of emotions, labelling/annotation of emotional experience is done manually by humans using specially developed annotation tools. To effectively exploit the resulting subjective annotations for developing affective systems, their quality needs to be assessed. This entails, (i) evaluating the variations in annotations, across different subjects and emotional stimuli, to detect spurious/unexpected patterns; and (ii) developing strategies to effectively combine these subjective annotations into a ground truth annotation. This article builds on our previous work by presenting a novel Functional Data Analysis based approach to assess the quality of annotations. Specifically, the bivariate annotation time-series are transformed into functions, such that each resulting functional annotation then becomes a sample element for analysis like Multivariate Functional Principal Component Analysis (MFPCA) that evaluate variation across all annotations. The resulting scores from MFPCA provide interesting insights into annotation patterns and facilitate the use of multivariate statistical techniques to address both (i) and (ii). Given the presented efficacy of these methods, we believe they offer an exciting new approach to assessing the quality of annotations.
Attenuation of Blood-Brain Barrier Breakdown and Hyperpermeability by Calpain Inhibition*

Blood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability followed by brain edema are hallmark features of several brain pathologies, including traumatic brain injuries (TBI). Recent studies indicate that pro-inflammatory cytokine interleukin-1β (IL-1β) that is up-regulated following traumatic injuries also promotes BBB dysfunction and hyperpermeability, but the underlying mechanisms are not clearly known. The objective of this study was to determine the role of calpains in mediating BBB dysfunction and hyperpermeability and to test the effect of calpain inhibition on the BBB following traumatic insults to the brain. In these studies, rat brain microvascular endothelial cell monolayers exposed to calpain inhibitors (calpain inhibitor III and calpastatin) or transfected with calpain-1 siRNA demonstrated attenuation of IL-1β-induced monolayer hyperpermeability. Calpain inhibition led to protection against IL-1β-induced loss of zonula occludens-1 (ZO-1) at the tight junctions and alterations in F-actin cytoskeletal assembly. IL-1β treatment had no effect on ZO-1 gene (tjp1) or protein expression. Calpain inhibition via calpain inhibitor III and calpastatin decreased IL-1β-induced calpain activity significantly (p < 0.05). IL-1β had no detectable effect on intracellular calcium mobilization or endothelial cell viability. Furthermore, calpain inhibition preserved BBB integrity/permeability in a mouse controlled cortical impact model of TBI when studied using Evans blue assay and intravital microscopy. These studies demonstrate that calpain-1 acts as a mediator of IL-1β-induced loss of BBB integrity and permeability by altering tight junction integrity, promoting the displacement of ZO-1, and disorganization of cytoskeletal assembly. IL-1β-mediated alterations in permeability are neither due to the changes in ZO-1 expression nor cell viability. Calpain inhibition has beneficial effects against TBI-induced BBB hyperpermeability.
Rice Endosperm Starch Phosphorylase (Pho1) Assembles with Disproportionating Enzyme (Dpe1) to Form a Protein Complex That Enhances Synthesis of Malto-oligosaccharides*

Starch synthesis in cereal grain endosperm is dependent on the concerted actions of many enzymes. The starch plastidial phosphorylase (Pho1) plays an important role in the initiation of starch synthesis and in the maturation of starch granule in developing rice seeds. Prior evidence has suggested that the rice enzyme, OsPho1, may have a physical/functional interaction with other starch biosynthetic enzymes. Pulldown experiments showed that OsPho1 as well as OsPho1 devoid of its L80 region, a peptide unique to higher plant phosphorylases, captures disproportionating enzyme (OsDpe1). Interaction of the latter enzyme form with OsDpe1 indicates that the putative regulatory L80 is not responsible for multienzyme assembly. This heterotypic enzyme complex, determined at a molar ratio of 1:1, was validated by reciprocal co-immunoprecipitation studies of native seed proteins and by co-elution chromatographic and co-migration electrophoretic patterns of these enzymes in rice seed extracts. The OsPho1-OsDpe1 complex utilized a broader range of substrates for enhanced synthesis of larger maltooligosaccharides than each individual enzyme and significantly elevated the substrate affinities of OsPho1 at 30 °C. Moreover, the assembly with OsDpe1 enables OsPho1 to utilize products of transglycosylation reactions involving G1 and G3, sugars that it cannot catalyze directly.
Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling*

Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the β2-adrenergic receptor (β2-AR)-Gαs and V1A receptor (V1AR)-Gαq. The Gαs and Gαq peptides adopt different orientations in β2-AR and V1AR, respectively. The β2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with β2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions.
Estimated Diversity of Messenger RNAs in Each Murine Spermatozoa and Their Potential Function During Early Zygotic Development1

ABSTRACT To study the diversity of mRNAs in murine spermatozoa and their potential function during zygotic development, total RNAs in murine spermatozoa were sequenced via RNA-Seq and analyzed through bioinformatics techniques. The delivery and translation of sperm-borne mRNA in fertilized oocyte were detected using RT-PCR (reverse transcription-polymerase chain reaction), Western blot, and immunofluorescence. A total of 35 288 825 reads matching 33 039 transcripts, including 27 310 coding transcripts, were obtained. Based on our analyses, we hypothesized that the transcripts with RPKM (reads per kilobase of exon model per million mapped reads) higher than six may exist in each sperm cell as consistently retained transcripts. There were 4885 consistent transcripts in each sperm, and the remainder were randomly retained. If the baseline RPKM increased, the remaining coding transcripts were more likely related to reproduction and development. The sperm-borne transcripts Wnt4 and Foxg1 were delivered into fertilized oocytes on fertilization. Furthermore, Wnt4 was translated into protein in zygotes, whereas Foxg1 was not translated. In conclusion, approximately 4885 mRNAs were present in each murine spermatozoon, and the spermatozoal mRNAs related to reproduction and development were more likely retained. The sperm-borne mRNA Wnt4 was delivered into the fertilized oocyte and translated, evidence of a paternal effect on zygotic development.
Thrombospondin-1 (TSP-1), a new bone morphogenetic protein-2 and -4 (BMP-2/4) antagonist identified in pituitary cells

Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation, suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). Surface plasmon resonance and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C domain of TSP-1 is likely responsible for this BMP-2/4-binding activity, an assertion based on sequence similarity that TSP-1 shares with the von Willebrand type C domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented a structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action.
The Steroid Hormone Environment During Primordial Follicle Formation in Perinatal Mouse Ovaries1

ABSTRACT Primordial follicle assembly is essential for reproduction in mammalian females. Oocytes develop in germ cell cysts that in late fetal development begin break down into individual oocytes and become surrounded by pregranulosa cells, forming primordial follicles. As they separate, many oocytes are lost by apoptosis. Exposure to steroid hormones delays cyst breakdown, follicle formation, and associated oocyte loss in some species. One model for regulation of follicle formation is that steroid hormones in the maternal circulation keep cells in cysts and prevent oocyte death during fetal development but that late in pregnancy hormone levels drop, triggering cyst breakdown and associated oocyte loss. However, herein we found that, while maternal circulating levels of progesterone drop during late fetal development, maternal estradiol levels remain high. We hypothesized that fetal ovaries were the source of hormones and that late in fetal development their production stops. To test this, mRNA and protein levels of steroidogenic enzymes required for estradiol and progesterone synthesis were measured. We found that aromatase and 3-beta-hydroxysteroid dehydrogenase mRNA levels drop before cyst breakdown. The 3-beta-hydroxysteroid dehydrogenase protein levels also dropped, but we did not detect a change in aromatase protein levels. The steroid content of perinatal ovaries was assayed, and both estradiol and progesterone were detected in fetal ovaries before cyst breakdown. To determine the role of steroid hormones in oocyte development, we examined the effects of blocking steroid hormone production in organ culture and found that the number of oocytes was reduced, supporting our model that steroid hormones are important for fetal oocyte survival.
Isolation and Partial Characterisation of Thermophilic Cellulolytic Bacteria from North Malaysian Tropical Mangrove Soil

This study reports the biodiversity of thermophilic cellulolytic bacterial strains that present in the north Malaysian mangrove ecosystem. Soil samples were collected at the four most northern state of Malaysia (Perak, Pulau Pinang, Kedah and Perlis). The samples obtained were first enriched in nutrient broth at 45°C and 55°C prior culturing in the carboxymethylcellulose (CMC) agar medium. Repeated streaking was performed on the CMC agar to obtain a pure culture of each isolate prior subjecting it to hydrolysis capacity testing. The isolates that showing the cellulolytic zone (halozone) were sent for 16S rRNA sequencing. Total seven isolates (two from Perak, three from Kedah, another two were from Perlis and Penang each) showed halozone. The isolate (KFX-40) from Kedah exhibited highest halozone of 3.42 ± 0.58, meanwhile, the one obtained from Perak (AFZ-0) showed the lowest hydrolysis capacity (2.61 ± 0.10). Based on 16S rRNA sequencing results, 5 isolates (AFY-40, AFZ-0, KFX-40, RFY-20, and PFX-40) were determined to be Anoxybacillus sp. The other two isolates were identified as Bacillus subtilis (KFY-40) and Paenibacillus dendritiformis (KFX-0). Based on growth curve, doubling time of Anoxybacillus sp. UniMAP-KB06 was calculated to be 32.3 min. Optimal cellulose hydrolysis temperature and pH of this strain were determined to be 55°C and 6.0 respectively. Addition of Mg2+ and Ca2+ were found to enhance the cellulase activity while Fe3+ acted as an enzyme inhibitor.
Cryptorchidism in the Orl Rat Is Associated with Muscle Patterning Defects in the Fetal Gubernaculum and Altered Hormonal Signaling1

ABSTRACT Cryptorchidism, or undescended testis, is a common male genital anomaly of unclear etiology. Hormonal stimulation of the developing fetal gubernaculum by testicular androgens and insulin-like 3 (INSL3) is required for testicular descent. In studies of the orl fetal rat, one of several reported strains with inherited cryptorchidism, we studied hormone levels, gene expression in intact and hormone-stimulated gubernaculum, and imaging of the developing cremaster muscle facilitated by a tissue clearing protocol to further characterize development of the orl gubernaculum. Abnormal localization of the inverted gubernaculum was visible soon after birth. In the orl fetus, testicular testosterone, gubernacular androgen-responsive transcript levels, and muscle-specific gene expression were reduced. However, the in vitro transcriptional response of the orl gubernaculum to androgen was largely comparable to wild type (wt). In contrast, increases in serum INSL3, gubernacular INSL3-responsive transcript levels, expression of the INSL3 receptor, Rxfp2, and the response of the orl gubernaculum to INSL3 in vitro all suggest enhanced activation of INSL3/RXFP2 signaling in the orl rat. However, DNA sequence analysis did not identify functional variants in orl Insl3. Finally, combined analysis of the present and previous studies of the orl transcriptome confirmed altered expression of muscle and cellular motility genes, and whole mount imaging revealed aberrant muscle pattern formation in the orl fetal gubernaculum. The nature and prevalence of developmental muscle defects in the orl gubernaculum are consistent with the cryptorchid phenotype in this strain. These data suggest impaired androgen and enhanced INSL3 signaling in the orl fetus accompanied by defective cremaster muscle development.
Urban colonization through multiple genetic lenses: The city‐fox phenomenon revisited

Abstract Urbanization is driving environmental change on a global scale, creating novel environments for wildlife to colonize. Through a combination of stochastic and selective processes, urbanization is also driving evolutionary change. For instance, difficulty in traversing human‐modified landscapes may isolate newly established populations from rural sources, while novel selective pressures, such as altered disease risk, toxicant exposure, and light pollution, may further diverge populations through local adaptation. Assessing the evolutionary consequences of urban colonization and the processes underlying them is a principle aim of urban evolutionary ecology. In the present study, we revisited the genetic effects of urbanization on red foxes (Vulpes vulpes) that colonized Zurich, Switzerland. Through use of genome‐wide single nucleotide polymorphisms and microsatellite markers linked to the major histocompatibility complex (MHC), we expanded upon a previous neutral microsatellite study to assess population structure, characterize patterns of genetic diversity, and detect outliers associated with urbanization. Our results indicated the presence of one large evolutionary cluster, with substructure evident between geographic sampling areas. In urban foxes, we observed patterns of neutral and functional diversity consistent with founder events and reported increased differentiation between populations separated by natural and anthropogenic barriers. We additionally reported evidence of selection acting on MHC‐linked markers and identified outlier loci with putative gene functions related to energy metabolism, behavior, and immunity. We concluded that demographic processes primarily drove patterns of diversity, with outlier tests providing preliminary evidence of possible urban adaptation. This study contributes to our overall understanding of urban colonization ecology and emphasizes the value of combining datasets when examining evolutionary change in an increasingly urban world.