Histomorphology and Chemical Constituents of Interdigital Gland of Vembur Sheep, Ovis aries.

The interdigital gland is a specialized skin gland located between the digits of Artiodactyla (i.e., even-toed ungulates). Its secretion participates in semiochemical communication, and protects from ultraviolet radiation as well as fungal and bacterial infections of the feet. The present study aimed at finding if there are male-female differences in the anatomy, morphology, and volatile compounds of the interdigital gland of the South Indian breed of Vembur sheep. A total of 24 sheep (12 each of male and female) were spotted at the slaughterhouse and the interdigital gland was removed for examination. The anatomical examination revealed it to resemble a tobacco pipe and to consist of a body, flexure, and excretory duct with an external orifice located at the cleft of the digits. Morphometrically, the interdigital glands differed between males and females. The gland possesses a distinct fibrous capsule, epidermis, and dermis. The fibrous capsule contains several parallel bundles of collagen fibers, nerve fibers, and blood vessels, etc. The epidermis consists of keratinized squamous epithelium formed of stratum basale, stratum granulosum and stratum spinosum. The dermis consists of hair follicles, nerve plexuses, arrector pili muscles, and apocrine and sebaceous glandular lobules. The latter, lined by a simple cuboidal epithelium, are arranged in clusters of acini in the upper portion of the dermis. The apocrine secretory lobules, made up of parenchymal cells, are found in the lower portion of the dermis. The density and diameter of the apocrine and sebaceous secretory lobules were significantly higher in the males than females. Scanning electron microscopic (SEM) analysis confirmed the apocrine and sebaceous secretory components. Twenty-three major compounds were identified in the interdigital gland postings of male and female sheep, among which butanoic acid, 2-methylpropanoic acid, 1-heptanol and octadecanoic acid were present only in the male glandular post, whereas octane, 7-hexyl-tridecane, tetradecane, heptadecane and decanoic acid were present only in the female glandular post. Tetradecanol, tetradecanoic acid and hexadecanol peaks, reportedly antibacterial compounds in pronghorn antelopes, were highly prominent in both male and female sheep. Thus, the interdigital gland of Vembur sheep has two major secretory lobules, namely, sebaceous and apocrine, larger in males than females, which secrete a variety chemical compounds that may serve as chemical communication systems and protect the sheep from foot-borne diseases.

The Exoproteome of Staphylococcus pasteuri Isolated from Cervical Mucus during the Estrus Phase in Water Buffalo (Bubalus bubalis).

Bacterial extracellular proteins participate in the host cell communication by virtue of the modulation of pathogenicity, commensalism and mutualism. Studies on the microbiome of cervical mucus of the water buffalo (Bubalus bubalis) have shown the occurrence of Staphylococcus pasteuri and that the presence of this bacterium is indicative of various physiological and reproductive states in the host. Recently, S. pasteuri has been isolated from the cervical mucus of the buffalo during the different phases of estrous cycle, and has proved to be much more pronounced during the estrus phase. The basis underlying the availability of a significantly increased S. pasteuri population, specifically during the estrus phase, is not known. Consequently, it is important to determine the significance of the specific abundance of S. pasteuri during the estrus phase of the buffalo host, particularly from the perspective of whether this bacterial species is capable of contributing to sexual communication via its extracellular proteins and volatiles. Therefore, the relevance of S. pasteuri exoproteome in the buffalo cervical mucus during the estrus phase was analyzed using LC-MS/MS. As many as 219 proteins were identified, among which elongation factor Tu (EF-Tu), 60-kDa chaperonin (Cpn60), enolase, fructose-bisphosphate aldolase class 1 (FBP aldolase), enoyl-[acyl-carrier-protein] reductase [NADPH] (ENR) and lipoprotein (Lpp) were the functionally important candidates. Most of the proteins present in the exoproteome of S. pasteuri were those involved in cellular-metabolic functions, as well as catalytic- and binding activities. Moreover, computational studies of Lpp have shown enhanced interaction with volatiles such as acetic-, butanoic-, isovaleric- and valeric acids, which were identified in the cervical mucus S. pasteuri culture supernatant. The present findings suggest that S. pasteuri extracellular proteins may play an important role in buffalo sexual communication during the estrus phase.

Mollification of Doxorubicin (DOX)-Mediated Cardiotoxicity Using Conjugated Chitosan Nanoparticles with Supplementation of Propionic Acid.

Doxorubicin is an extensively prescribed antineoplastic agent. It is also known for adverse effects, among which cardiotoxicity tops the list. The possible mechanism underlying doxorubicin (DOX)-mediated cardiotoxicity has been investigated in this study. Further, to reduce the DOX-mediated cardiotoxicity, DOX was conjugated with Chitosan Nanoparticles (DCNPs) and supplemented with propionic acid. Initially, the drug loading efficacy and conjugation of DOX with chitosan was confirmed by UV-Visible Spectroscopy (UV) and Fourier Transform Infrared Spectroscopy (FTIR). The average sizes of the synthesized Chitosan Nanoparticles (CNPs) and DCNPs were measured by Dynamic Light Scattering (DLS) analysis as 187.9 ± 1.05 nm and 277.3 ± 8.15 nm, respectively, and the zeta potential values were recorded as 55.2 ± 0.7 mV and 51.9 ± 1.0 mV, respectively. The size and shape of CNPs and DCNPs were recorded using a High-Resolution Electron Microscopy (HRTEM). The particles measured <30 nm and 33-84 nm, respectively. The toxic effects of DCNPs and propionic acid were evaluated in rat model. The data from the electrocardiogram (ECG), cardiac biomarkers, Peroxisome proliferator-activated receptor gamma (PPARγ) and histological observations indicated evidence of DOX-mediated cardiotoxicity, whereas the administration of DCNPs, as well as Propionic Acid (PA), brought about a restoration to normalcy and offered protection in the context of DOX-induced cardiotoxicity.

Salivary Proteome Profile of Women during Fertile Phase of Menstrual Cycle as Characterized by Mass Spectrometry.

OBJECTIVES: Ovulation is such a critical physiological process that its noninvasive detection based on salivary constituents has several advantages in humans. Hence, the present study is proposed to identify the ovulatory-specific proteins in saliva in order to detect ovulation phase. MATERIALS AND METHODS: Samples were collected from women volunteers. The procedure adopted was approved by the Institutional Human Ethical Committee (DM/2014/101/38), Bharathidasan University. The saliva samples were collected from thirty healthy female volunteers, with a prior written consent. One-way analysis of variance was used to calculate protein concentration and band intensity using SPSS 16 software (SPSS Inc., Cary, NC, USA). The salivary protein expression pattern during different phases of menstrual cycle was analyzed using gel-based high resolution-liquid chromatography-mass spectrometry/mass spectrometry and matrix-assisted laser desorption ionization-time of flight/time of flight. Further, bioinformatics tools were adopted to annotate the proteins identified at various phases of menstrual cycle. RESULTS: As many as 530 proteins showed up in the saliva during ovulatory phase, whereas there were only 251 proteins identified during postovulatory phase. The functional annotation of salivary proteins revealed that the proteins got assigned to the class of "extracellular proteins" which are concerned with regulatory functions. The 16 unique and/or differentially expressed protein spots appeared during ovulatory phase, among which Cystatin-S, Prolactin-inducible protein, Cystatin-A, Cystatin-SN, BPI fold-containing family A member 2, Alpha-tubulin N-acetyltransferase 1, Carbonic anhydrase-6, Protein LEG1 homolog, Hemoglobin subunit beta, and Pancreatic alpha-amylase were identified. CONCLUSION: Total salivary proteome profile has been listed with respect to various phases of menstrual cycle. Among the protein listed, Cystatin-S offers a biomarker protein and/or indicator of ovulatory phase. However, extensive validation is required before arriving to a candidate bio-marker protein.

A novel method to detect bovine sex pheromones using l-tyrosine-capped silver nanoparticles: Special reference to nanosensor based estrus detection.

In the present study, a simple and a selective colorimetric method for pheromone detection to diagnose estrus in cattle was established based on the l-tyrosine functionalized silver nanoparticles (l-TyrAgNPs). The synthesized silver nanoparticles was spotted by color change (colorless to pale yellow) due to surface plasmon resonance (SPR). In order to confirm, Ag nanoparticles was characterized by field emission scanning electron microscopy (FESEM), dynamic light scattering (DLS) and zeta potential, X-ray diffraction (XRD) and UV- Vis spectrophotometer. It was found that the pre-colored Ag colloids could be turned from yellow to reddish-brown by the addition of the sex pheromones such as acetic acid or propionic acid, which may have potential application in the colorimetric sensor. The augmented optical nature of nanoparticles furnishes a suitable base to develop a colorimetric sensor for bovine sex pheromones detection. In addition, the computational analyses are critically required to validate residual interactions of bovine odorant-binding protein (OBP) with pheromones. The method was successfully applied to the detection of acetic acid or propionic acid using a biological molecule l-Tyr AgNPs. These results clearly indicate that the biosynthesis of l-Tyr AgNPs can be used as a promising colorimetric sensor for accurate time of estrus prediction in bovine.

Author Correction: Buffalo nasal odorant-binding protein (bunOBP) and its structural evaluation with putative pheromones.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Buffalo nasal odorant-binding protein (bunOBP) and its structural evaluation with putative pheromones.

Pheromones are odoriferous volatile chemical cues produced by animals for communication among conspecifics so as to regulate their social behaviors. In general, the odor compounds are recognized by receptors in the nasal cavity. Odorant-binding protein (OBP), a lipocalin family protein, mediates the air-borne odor cues to nasal receptors through nasal mucus. The presence of OBP in several mammalian species is well documented but to-date there is no report of a nasal OBP in buffalo. Hence, the present study was undertaken to investigate if OBP is present in buffalo nasal mucus. Uni- and two-dimensional gel electrophoresis of the nasal mucus suggested the presence of OBP, which was confirmed using mass spectrometry. In silico homology model of the OBP was generated and its structural similarity with other mammalian OBPs was assessed. Finally, molecular-docking and -dynamics simulations analysis revealed the efficiency of buffalo nasal OBP (bunOBP) to bind with buffalo pheromones as well as other reported chemical cues. Taken together, the occurrence of nasal OBP in buffalo and its putative role in odor binding are reported for the first time. The potential association of this protein with estrus-specific volatiles could be taken to advantage for non-invasive detection of estrus in buffaloes.

Proteomic analysis of human saliva: An approach to find the marker protein for ovulation.

Human saliva contains numerous molecules that play a variety of roles. Among them there are proteins which serve as biomarkers of various physiological and/or pathological conditions. Compared to other body fluids, saliva is the most convenient material for investigations, and especially for monitoring the disease conditions. Presently, there is an increasing need to develop a noninvasive method to identify the time of ovulation in humans to ensure successful fertilization, and for evolving strategies for family planning. The present investigation has been an attempt to identify one or more proteins in the human saliva that would be an indicator(s) of ovulation. SDS-PAGE of salivary proteins showed seven prominent bands during the different phases of the menstrual cycle. Particularly, the 14.5kDa band was highly expressed during the ovulatory phase. Eleven proteins were identified in this band of which ten were highly specific to the ovulatory phase. Among those proteins the intense expression of Cystatin-S was validated using immunoblot analysis (p<0.05). The functional annotation of salivary proteins revealed a high percentage of proteins that engage in binding and regulatory activities. The present results indicate that salivary proteins, particularly those present during the ovulatory phase, might be used as biomarkers for impending ovulation.

Structural elucidation of estrus urinary lipocalin protein (EULP) and evaluating binding affinity with pheromones using molecular docking and fluorescence study.

Transportation of pheromones bound with carrier proteins belonging to lipocalin superfamily is known to prolong chemo-signal communication between individuals belonging to the same species. Members of lipocalin family (MLF) proteins have three structurally conserved motifs for delivery of hydrophobic molecules to the specific recognizer. However, computational analyses are critically required to validate and emphasize the sequence and structural annotation of MLF. This study focused to elucidate the evolution, structural documentation, stability and binding efficiency of estrus urinary lipocalin protein (EULP) with endogenous pheromones adopting in-silico and fluorescence study. The results revealed that: (i) EULP perhaps originated from fatty acid binding protein (FABP) revealed in evolutionary analysis; (ii) Dynamic simulation study shows that EULP is highly stable at below 0.45 Å of root mean square deviation (RMSD); (iii) Docking evaluation shows that EULP has higher binding energy with farnesol and 2-iso-butyl-3-methoxypyrazine (IBMP) than 2-naphthol; and (iv) Competitive binding and quenching assay revealed that purified EULP has good binding interaction with farnesol. Both, In-silico and experimental studies showed that EULP is an efficient binding partner to pheromones. The present study provides impetus to create a point mutation for increasing longevity of EULP to develop pheromone trap for rodent pest management.

Examining and elucidation of human weight cycle model adopting e-cell simulation system.

Cellular rhythms regulate various physiological functions in circadian oscillatory mechanisms. Weight cycling or 'yo-yo' dieting is an evitable process in human, because of subsequent loss and regain of body weight due to irregular diet. Human weight cycle (HWC) is the major factor for causing global epidemic diseases in human beings. Understanding the HWC process would provide potent additional knowledge to prevent obesity. However till date, there is no study dealing with examine the HWC model using virtual cell simulation based on system biological approach. Therefore, the present study was designed to develop a computational HWC model, which was simulated using E-cell system v3.0. The developed model has the cyclic feedback reactions of three significant variables (the consecutive cycles of weight loss in continuous food intake (Q) and regain of body weight (P) at highest threshold point of cognitive restraint (R)) which are obtained by mathematical modelling. The dynamic plot results supported that the PQR variables depicted sustained oscillation with reversible modification due to protein diet. By contrast, the virtual model simulation would provide extensive information on HWC, which might provide knowledge to develop HWC linked with obesity pathway. The presents study concludes that optimization of body weight is essential to prevent the obesity based diseases.

Exploration of salivary proteins in buffalo: an approach to find marker proteins for estrus.

Saliva is considered as the best source of biological material for biomarker discovery studies since it is noninvasive in comparison to other body sources. Usually buffalo cannot precisely express estrus signals. Hence, there is a need for concise methods to detect the time of estrus to ensure the success of artificial insemination. Therefore, we have established a reference proteome map on the whole saliva of buffalo during their estrous cycle with special reference to estrus. Nearly 12 bands have been observed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole saliva. Collectively, 179 proteins are identified with respect to different phases of the estrous cycle using mass spectrometry. On the whole, 37 proteins are exclusively expressed in the estrus phase, which include ß-enolase, Toll-like receptor (TLR) 4, clusterin, lactoperoxidase, serotransferrin, TGM3, UBA6, and transducin. Among the proteins, ß-enolase and TLR 4 were validated, and their specific expression was found during estrus as compared to other phases using immunoblot. The functional annotation reveals many as binding proteins in the estrus saliva when compared to the other phases. The present findings conclude that the proteomic approach adopted to identify the proteins from buffalo saliva around the estrous cycle may provide a new tool for screening the estrus phase. The results further conclude that the specific expression of ß-enolase and TLR 4 can be taken as the indicator of estrus in buffalo.

DOR - a Database of Olfactory Receptors - Integrated Repository for Sequence and Secondary Structural Information of Olfactory Receptors in Selected Eukaryotic Genomes.

Olfaction is the response to odors and is mediated by a class of membrane-bound proteins called olfactory receptors (ORs). An understanding of these receptors serves as a good model for basic signal transduction mechanisms and also provides important clues for the strategies adopted by organisms for their ultimate survival using chemosensory perception in search of food or defense against predators. Prior research on cross-genome phylogenetic analyses from our group motivated the addressal of conserved evolutionary trends, clustering, and ortholog prediction of ORs. The database of olfactory receptors (DOR) is a repository that provides sequence and structural information on ORs of selected organisms (such as Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, and Homo sapiens). Users can download OR sequences, study predicted membrane topology, and obtain cross-genome sequence alignments and phylogeny, including three-dimensional (3D) structural models of 100 selected ORs and their predicted dimer interfaces. The database can be accessed from http://caps.ncbs.res.in/DOR. Such a database should be helpful in designing experiments on point mutations to probe into the possible dimerization modes of ORs and to even understand the evolutionary changes between different receptors.

Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis.

Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

Identification of p-cresol as an estrus-specific volatile in buffalo saliva: comparative docking analysis of buffalo OBP and ß-lactoglobulin with p-cresol.

Assessment of salivary volatile compounds adopting gas chromatography-linked mass spectrometry (GC-MS) analysis revealed the presence of a total of 11 compounds in the buffalo saliva irrespective of the stages in the reproductive cycle. p-cresol was identified as an estrus-specific volatile compound in the saliva. In addition, modeling of odorant-binding protein (OBP) and ß-lactoglobulin revealed that OBP is highly stable and has strong binding affinity with p-cresol. Hydrogen bond interactions indicated that OBP is responsible for pheromone release through saliva. In contrast, ß-lactoglobulin, which belongs to the same lipocalin family as OBP, possesses less affinity to p-cresol than OBP, suggesting that it is not involved in p-cresol binding and transport. Phylogenetic characterization revealed that bovine family of OBP is separately clustered. It is suggested that p-cresol has the potential to be developed as a biomarker to detect the reproductive status in the buffalo and for behavioral manipulations.

Effect of mutation on aggregation propensity in homology model structures of syntaxin-3 from Homo sapiens.

Perception of molecular mechanism would provide potent additional knowledge on mammalian membrane proteins involved in causing diseases. In human, syntaxin-3 (STX3) is a significant apical targeting protein in the epithelial membrane and in exocytosis process; it also acts as a vesicle transporter by cellular receptor in neutrophils, which is crucial for protein trafficking event. Structurally, syntaxin-3 has hydrophobic domain at carboxyl terminus that directs itself to intra-cellular compartments. In addition, the experimental structure of STX3 is not available and no mutational study has been carried out with natural variants of proteins. Moreover, there is no evidence so far for the natural variant Val286 of STX3 causing any diseases. Hence, in the present study, analyses of residue-based properties of the homology model STX3 were carried out along with mutations at carboxyl terminus of STX3 by implementing protein engineering and in silico approaches. The model structure of STX3 was constructed adopting Modeller v9.11 and the aggregation propensity was analyzed with BioLuminate tool. The results showed that there was reduction in aggregation propensity with point mutation at Val286, instead of Ile, resulting into increasing the structural stability of STX3. In conclusion, the Ccap exposed residue would be a suitable position for further mutational studies, particularly with Val286 of STX3 in human. This approach could gainfully be applied to STX3 for efficient drug designing which would be a valuable target in the cancer treatment.

Urinary lipocalin protein in a female rodent with correlation to phases in the estrous cycle: an experimental study accompanied by in silico analysis.

Male urinary lipocalin family proteins, practically odorant-binding proteins but also could be pheromones by themselves, in rodents act as a shuttle for chemosignal communication and facilitate delivery of the signals for access to congeners. However, presence of this protein in urine of female rodents has not yet been reported. Therefore, the present investigation was carried out to find if lipocalin family protein is present in the urine of female house rat and, if so, to find whether its expression differs between the phases in the estrous cycle. The rat urinary protein was separated in single dimensional gel electrophoresis. A 14.5 kDa lipocalin protein appeared in the urine prominently during the estrus and metestrus phases compared to proestrus and diestrus phases. The expression of this protein in the urine was very low in ovariectomized rats. MALDI-TOF/MS analysis affirmed the 14.5 kDa protein as a lipocalin family protein. Analysis adopting bio-informatics tools further proved the protein as a lipocalin family member. Thus, this study for the first time demonstrated the presence of a lipocalin family protein in the urine of a female rodent and it was highly expressed during estrus phase. This lipocalin protein in female rat urine may facilitate a chemosignal function independently of a pheromone or in association with a specific pheromone.