A decline in avian cytokine expression with age revealed by commercially available cytokine array.

Cytokines are secreted immunomodulators that are key regulators of the avian immune response. Currently, the most commonly used method to follow cytokine expression is qPCR, which measures cellular levels of mRNA, rather their extracellular circulating levels. Here we present a commercially available cytokine array designed to assay circulating expression levels of multiple cytokines and immunomodulators simultaneously. Upon minor modifications to the manufacturer protocol, background noise was reduced, leading to a significant increase in the sensitivity of the device. Our data indicate that the array is reliable and produce consistent data between biological repeats. We tested the reproducibility of the array in a biologically relevant context by assessing age-related changes in circulating cytokines. While individual features did not show a consistent pattern, our data revealed a consistent decline in the median of all cytokine values, supporting the validity of the array in studying biological processes.

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.

Time-Restricted Feeding in Commercial Layer Chickens Improves Egg Quality in Old Age and Points to Lack of Adipostat Activity in Chickens.

In mammals, time-restricted feeding (TRF) with no caloric restriction provides health benefits and extends longevity, usually with a minor (∼3%) or no reduction in total food consumption. In the current study, a TRF regimen of 6 h free access to food (08:00-14:00 h) was applied to Leghorn chickens from 25 to 86 weeks of age; control birds ate freely during the light hours (06:00-20:00 h). Unexpectedly, the TRF-treated birds consumed, on average, 11.7% less food than the controls. This was manifested by an average reduction of 9.6% in body weight, 2.6-fold in visceral fat accumulation, and 6.5% in egg weight. Hen-housed egg production was reduced by 3.6% in the TRF group compared with the control, along the first 40 weeks of the follow-up (P < 0.05), and changed into a tendency of 0.7% higher egg production thereafter. Several parameters of egg quality showed significant improvement (P < 0.05) in the TRF group compared with the controls. A comparison of diurnal patterns of feed consumption revealed a higher rate of hourly consumption in the TRF group and increased consumption before dark in the control group. In conclusion, the reduced feed intake in response to the TRF treatment and loss in visceral fat accumulation supports the lack of a strong adipostat activity in chickens and different appetite regulation mechanisms compared with mammals. Therefore, future TRF studies in chickens should be adjusted by extending the ad libitum time window. The lower feed intake by the TRF-treated chickens compared with the ad libitum-fed controls seems to reduce the efficiency of egg production. Nevertheless, the improved egg quality and persistence of egg lay at the older age suggest that similarly to mammals, the TRF treatment delayed at least some of the negative impacts associated with advanced age.

Salivary luteinizing hormone: An open window to detect oestrous period in buffalo.

Silent oestrus is an unsurmountable problem in the management of buffalo reproduction. In addressing this issue, we have earlier reported variation in the levels of urinary luteinizing hormone (LH) through the different phases of oestrous cycle with an extended window during the mid-oestrous phase. Based on this report, the present study is designed to assess the salivary LH levels in buffalo during the different phases of oestrous cycle. Bovine LH ELISA kit was used to determine the level of salivary LH. We observed a notable variation in salivary LH levels during the different phases of oestrous cycle. The maximum LH level, 39.07 mIU/ml, observed during oestrus, which was significantly (p < .05) higher than other consecutive phases. Altogether, the results showed a significant (p < .05) fold variation during oestrus compared with other phases. Therefore, the study convincingly shows that salivary LH has the potential of application in development of a modality for non-invasive oestrous detection in buffalo.

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.

Ultrastructural and physico-chemical characterization of saliva during menstrual cycle in perspective of ovulation in human.

Human saliva is a potential diagnostic fluid and any alteration in body might be reflected in saliva so that saliva is considered as "mirror of the body". Variations in salivary hormone level, ultra structure, pH, flow rate, buffering capacity and electrolytes level are found during menstrual cycle in regard to ovulation. Thirty healthy volunteers were used for the assessment of physico-chemical changes in saliva. Reproductive cycle was categorized as pre-ovulation phase (5 to 12 days), ovulation phase (13 or 14 days) and post-ovulation phase (15 to 25 days) according to salivary arborization test and hormonal analysis. Estradiol and luteinizing hormone was gradually increased and attained peak at the level of 2.28 ± 0.20 pg/mL and 1.35 ± 0.41 mIU/mL respectively during the ovulation phase. The electrolytes result clearly indicates that the influx of common electrolytes is important for crystallization and help to induce clear ferning pattern in ovulation phase. Sodium (Na) and chloride (Cl) were found to be high during ovulation phase only. Average salivary pH was 7.5, 7.1, and 7.3 during ovulation, pre- and post-ovulation phases respectively. Buffering capacity of saliva was normal during pre- and post- ovulation phases. In contrast, in ovulation phase the buffer capacity was slightly higher. At the first time, the scanning electron microscopy (SEM) studies revealed the ultra structure difference of saliva during menstrual cycle. During ovulation phase a compact network-shaped mesh was appeared; such structure was not appeared in pre- and post ovulation phases. Additionally, we observed the saliva is arrayed as a fine mosaic-like structure during ovulation. Based on physico-chemical properties and hormonal levels may lead to develop a detection kit/sensor for detecting the ovulation phase in human.

Marine sponge-associated bacteria as a potential source for polyhydroxyalkanoates.

Marine sponges are filter feeding porous animals and usually harbor a remarkable array of microorganisms in their mesohyl tissues as transient and resident endosymbionts. The marine sponge-microbial interactions are highly complex and, in some cases, the relationships are thought to be truly symbiotic or mutualistic rather than temporary associations resulting from sponge filter-feeding activity. The marine sponge-associated bacteria are fascinating source for various biomolecules that are of potential interest to several biotechnological industries. In recent times, a particular attention has been devoted to bacterial biopolymer (polyesters) such as intracellular polyhydroxyalkanoates (PHAs) produced by sponge-associated bacteria. Bacterial PHAs act as an internal reserve for carbon and energy and also are a tremendous alternative for fossil fuel-based polymers mainly due to their eco-friendliness. In addition, PHAs are produced when the microorganisms are under stressful conditions and this biopolymer synthesis might be exhibited as one of the survival mechanisms of sponge-associated or endosymbiotic bacteria which exist in a highly competitive and stressful sponge-mesohyl microenvironment. In this review, we have emphasized the industrial prospects of marine bacteria for the commercial production of PHAs and special importance has been given to marine sponge-associated bacteria as a potential resource for PHAs.

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.

Does salivary protein(s) act an ovulation indicator for women? A hypothesis.

Ovulation is an important physiological process in human, and its effect may reflect in body fluids via secretion of biomolecules such as proteins, amino acids, antioxidants, antimicrobial peptides and so on. Recently, the non-invasive sampling approaches are used to diagnose disease status and access health condition of human. Saliva comprises various proteins which are secreted through salivary glands. The proteins present in the saliva may vary in their expression according to the hormonal level and physiological nature of the body which are said to be hormone receptors, stress proteins and antimicrobial peptides. Therefore, it is postulated that saliva can be used in the detection of ovulation time in human using specific protein(s) expression and which can be considered as a best non-invasive method. The identification of these proteins by adopting LC-MS/MS followed by Western blot analysis are possible to identify a promising biomarker for ovulation detection in human.

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.

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.