Identification and Structure Prediction of Human Septin-4 as a Biomarker for Diagnosis of Asthenozoospermic Infertile Patients-Critical Finding Toward Personalized Medicine.

Semen parameters are been found as a key factor to evaluate the count and morphology in the given semen sample. The deep knowledge of male infertility will unravel with semen parameters correlated with molecular and biochemical parameters. The current research study is to identify the motility associated protein and its structure through the in-silico approach. Semen samples were collected and initial analysis including semen parameters was analyzed by using the World Health Organization protocol. Semen biochemical parameters, namely, seminal plasma protein concentration, fructose content, and glucosidase content were calculated and evaluated for correlation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) were carried out for identification of Septin-4 presence in the semen sample. Mascot search was done for protein conformation and in-silico characterization of Septin-4 by structural modeling in Iterative Threading Assembly Refinement (I-TASSER). Twenty-five nanoseconds molecular dynamics (MD) simulations results showed the stable nature of Septin-4 in the dynamic system. Overall, our results showed the presence of motility-associated protein in normospermia and control samples and not in the case of asthenospermia and oligoasthenospermia. Molecular techniques characterized the presence of Septin-4 and as a novel biomarker for infertility diagnosis.

Nanosilver reinforced Parmelia sulcata extract efficiently induces apoptosis and inhibits proliferative signalling in MCF-7 cells.

The Lichen, Parmelia sulcata synthesizes various secondary metabolites, in which phenolic based compounds received much attention due to their importance in biomedical application. Especially the phenolic compound was effective against the cancer treatment. An effective administration of such plant natural product can represent a significant conventional management of cancer in terms of chemoprevention. The nanomedicines are group of agents that selectively interfere the cancer cells which leads to reduction of side effect thereby reducing the doses. Silver nanoparticles is a promising antitumor agent, however, the conventional production of silver nanoparticles have many drawbacks which led to increase in need of eco-friendly biological production methods. In this study, we made an attempt to synthesise a nano silver (Ps-AgNPs) from phenolic extract of lichen Parmelia sulcata extract. The Ps-AgNps was applied for anticancer activity using MCF-7 cells and the effect was characterised by western blotting method. The FTIR, XRD, UV and TEM results confirms the presence of silver nanoparticles in phenolic extract of lichen Parmelia sulcata. The cytotoxicity assay shows that the Ps-AgNPs is toxic against cancer cells (MCF-7) but not to normal cells (NIH3T3), which confirm the selective induction of cell death (apoptosis) against cancer cells. The Western blot analysis also clearly indicates the down regulation of inflammatory genes (TNF-alpha and IL-6) and cell cycle genes (PCNA and Cyclin-D1) thus promoting intrinsic apoptotic pathway. The results suggest that Ps-AgNPs can effectively kill cancer cells and can be used as an alternative therapeutic agent for cancer treatment.

Oxidative Stress-induced Changes in Fertility Status of Cryopreserved Sperm: a Diagnosis Based on Sperm Chromatin Dispersion Assay.

BACKGROUND: Cryopreservation introduces iatrogenic damage to sperm cells due to excess production of reactive oxygen species (ROS) that can damage sperm macromolecules and alter the physiochemical properties of sperm cells. These altered properties can affect the biological potential of sperm cell towards fertility. OBJECTIVE: The study was designed to assess the role of oxidative stress in sperm DNA damage upon cryopreservation. MATERIALS AND METHODS: Semen samples (160) were classified into fertile and infertile on the basis of Computer Assisted Semen Analysis (CASA), and cryopreserved. Thawed samples were analyzed for 8OHdG marker, sperm chromatin dispersion (SCD)-based DNA fragmentation index (SCD-DFI) and ROS levels. Receiver Operating Characteristics (ROC) was performed to find the specificity and sensitivity of SCD-DFI in assessing the sperm DNA integrity. Principle component analysis (PCA) was performed to group semen parameters. RESULTS: SCD-DFI significantly correlates with 8OHdG in infertile samples (r=0.73, p<0.0001) and moderately in fertile samples (r=0.52, p<0.001). Among SCD-DFI, ROS and 8OHdG values, SCD-DFI possess a higher area under the curve (0.83) in ROC, suggesting its potential in representing sperm DNA damage. PCA analysis extracted SCD-DFI and sperm progressive motility as two independent parameters, differentiating fertile and infertile cryopreserved semen. CONCLUSION: The study proves that SCD can potentially represent ROS-induced sperm DNA damage in infertile semen upon cryopreservation.

Human prostasomes an extracellular vesicle - Biomarkers for male infertility and prostrate cancer: The journey from identification to current knowledge.

Extracellular vesicles (EVs) are gaining attention among the cell biologists and researchers over the last two decades. Prostasomes are considered to be (Evs) secreted by prostate epithelial cells into the semen during emission or ejaculation. Prostasomes contain various proteins required for immune regulation namely, amino and dipeptidyl peptidase; endopeptidase (neutral); decay accelerating factor; angiotensin-converting enzyme. Sperm cells need a few prerequisites in order to fertilize the egg. The role of prostasomes in enhancing the male fertility was reviewed extensively throughout the manuscript. Also, prostasomes have an immunosuppressive, immunomodulatory, antibacterial role in the female reproductive tract, and in some cases they can be used as immunocontraceptive agent to regulate the fertility status. This review will give insights to many active researchers in the field of prostasomal research and male infertility/fertility research. This review will open many unanswered mechanisms of prostasomes with respect to structure-function analysis, fatty acids patterns in diagnosis as well as prognosis of male infertility/fertility. More scientific reports are in need to support the mechanism of prostasomes and its role in immunomodulation. The development of prostasomes as a biomarker for the prostate cancer is still miserable with a lot of controversial results by various researchers.

Improvements in morphology and membrane stability obtained from TPP-TAB, a cryopreservation medium treated infertile smoker sperm cells - An in vitro study.

BACKGROUND: Plants are the major sources of antioxidants, which maintains oxidant: antioxidant state or is to protect from excessive reactive oxygen species in case of cryo medium selection though maintain sperm stability. BOTTLENECK: Cryo preservation of infertile smokers is challengeable with the available medium due to over production of ROS hitches, were cause loss of sperm physiology. Hence, a novel medium is needed to store/protect sperm cells of infertile smokers where they attending/ongoing IVF or oncogenic surgical treatment. AIM: The aim of this study, is to check the stability of sperm cells by TPP-T.arjuna bark (TAB) (E4) cryo medium preserved infertile smoker's against ROS/cryo injury- as a continuous study. MATERIALS AND METHODS: 42 infertile smoker's subjects with 28 control subjects were selected. Surface morphology (acrosome) of sperm by scanning electronic microscope, sperm membrane proteins by colorimetric method, sperm head and tail defects by CASA method and finally sperm cell stability is checking its zeta electric potential charges, were all done with E4 cryo medium treated frozen/thawed selected study subjects. RESULTS: Sperm morphology and zeta potential shows there is no damage along the stability of cells maintained during E4 medium cryopreservation in infertile subjects. SUMMARY: This is the first study is too established for infertile smokers sperm stability was checked for six months with E4 cryo medium.

Biosynthesis and statistical optimization of polyhydroxyalkanoate (PHA) produced by Bacillus cereus VIT-SSR1 and fabrication of biopolymer films for sustained drug release.

Polyhydroxyalkanoates are a fascinating group of biological polymers, which have attracted extensive attention. An effectual PHB accumulating Bacillus cereus strain VIT-SSR1 was isolated from industrial effluent contaminated site. Plackett-Burman design was used to screen the critical factors affecting PHA production. Response surface method was employed to evaluate the interactive effects between the independent variables. The Central composite design was performed to optimize the concentrations of Molasses, ammonium sulphate and initial pH. A PHA content of (1.42 ±â€¯0.01) g/L and a maximum PHA yield of (40.3 ±â€¯0.77) % was obtained on implementing the optimized conditions. The produced biopolymer was analysed using Fourier transform infrared spectroscopy (FT-IR) and Gas chromatography-mass spectrometry analysis(GC-MS) and the thermal properties of the biopolymer were evaluated by Thermogravimetric analysis (TGA), and from the Nuclear magnetic Resonance (NMR) analysis the biopolymer was identified to be polyhydroxybutyrate (PHB). PHB-chitosan blends were prepared and tested for biocompatibility by MTT assay on L929 mouse fibroblast cell lines. Curcumin, a well-known drug was loaded onto PHB-chitosan films and their drug release pattern was observed. Curcumin showed a sustained mode of drug release. PHB films are biocompatible and can act as a potent polymer matrix for drug release studies.

Human sperm DNA damage inhibition and antioxidant activity of T. arjuna bark: an in vitro study.

Complimentary or natural antioxidant type of alternative medicine is developed worldwide to treat male infertility. The aim of this study is to the extraction of T. arjuna bark and activity against human sperm DNA damage in asthenoteratospermic smoker's subjects-an in vitro study. All preliminary and antioxidant assays (DPPH, H2O2, and total antioxidant, reducing power activity) were done. T. arjuna bark metal analysis was done with AAS. On the other hand, patients were asked to fill a direct questionnaire about smoking history; 25 infertile smokers were identified as asthenoteratospermic; 34 fertile non-smokers (control) were assessed for semen parameters by CASA, seminal plasma Zinc analysis by AAS, DNA fragmentation by colorimetric method and semen genomic DNA damage inhibition by modified non-enzymatic salting out extraction method. Most of the antioxidants are highly present in the aqueous extract; meanwhile, the major content in this extract is zinc 16 µg/g (Ca = 0.5 µg/g; Se = 2.2 µg/g and Mg = 1.6 µg/g) along with FT-IR peaks which also confirmed the metal presence. The semen parameters in smokers that were noticed are low sperm count and morphological changes. Meanwhile, in the seminal plasma of smokers, zinc and DNA fragmentation results were positively correlated with sperm morphology (p < 0.001). Repaired DNA bands were noticed in the in vitro study of aqueous T. arjuna bark, in smokers' semen. T. arjuna bark will act as cryo protector as well as great zinc supplementary to maintain sperm motility and morphology in smokers.

Tea polyphenol-T. arjuna bark as sperm antioxidant extender in infertile smokers.

BACKGROUND: Antioxidants protect spermatozoa against lipid peroxidation during freezing. OBJECTIVE: The study is designed to elucidate the suitable extender to preserve infertile semen of smokers against ROS damage using natural Tea polyphenol (T. arjuna bark extract). MATERIALS AND METHODS: Forty-two infertile subjects with smoking habit and 28 fertile subjects without smoking habit were considered for the study. Four semen extenders including our naturally derived antioxidant component were prepared and used to preserve semen sample from the study subjects for a period of one month. Standard semen parameters, biochemical and sperm DNA damage marker with inhibition were measured before and after cryopreservation. RESULTS: The motility and morphology of sperm cells were maintained better in E4 extender, and DNA damage is reduced. CONCLUSION: Extender recipe with natural antioxidants (E3 and E4) was found to be apt for infertile semen preservation.

Optimization of Human Semen Extender Components for Cryopreservation Using Statistical Tools.

　　BACKGROUND: Human sperm cell preservation is an important part of assisted reproductive technology (ART). OBJECTIVE: This study aimed to find the essential and significant components in semen preservation extender required to prolong the shelf life of human spermatozoa. MATERIALS AND METHODS: By using the statistical tool 'Plackett-Burman design' the significant components present in E4 extender (formulated in our previous study) was determined by reducing the unacceptable large number of trial experiments from the full factorial method. RESULTS: It was found that vitamin E, taurine and vitamin C were highly significant in maintaining the stability of sperm cells; and egg yolk, vitamin C and glucose were highly significant in sustaining the motility of the sperm cells. R2 values for the models were 0.9950 and 0.9960 respectively. In the optimized E4 extender 75 % and 81 % of the total motility was retained by the sperm cells from infertile and fertile samples respectively after cryopreservation. Also an increase in zeta potential was observed indicating a reduction in stability in both fertile and infertile sample (4 % and 18 % respectively) after cryopreservation in E4 medium, which was much less when compared with the sample preserved only with glycerol as cryoprotectant (11 % and 69 % for infertile and fertile samples respectively). CONCLUSION: The major components present in E4 semen extender was successfully optimized for further use.

Evolving trends in cryopreservation and parameters influencing semen extender preparation - a prospective review.

BACKGROUND: Cryopreservation is a technique by which, semen can be preserved to subzero temperature, usually at -196° C. The freezing of semen desires vitrification mediators that diminish wreck to the cells (spermatozoan) during the freeze and thaw process. Using cryopreservation, the quality of the semen has been increased in the latest years, by which the achievement rate for the insemination techniques has increased in an agreed way. The area need to be focused is to enhance the quality of the semen extender preparation before cryopreservation. Many researchers are working in the area of cryopreservation of human semen with different semen extenders. Several parameters influence the properties of semen extender essential for better post thaw results. This review is mainly focused on a range of parameters which influence the best semen extender for cryopreservation that includes glycerol and its importance, buffer and novel usage of antimicrobial peptides as antimicrobial agents.

Validation of artificial neural network models for predicting biochemical markers associated with male infertility.

UNLABELLED: Seminal fluid is the secretion from many glands comprised of several organic and inorganic compounds including free amino acids, proteins, fructose, glucosidase, zinc, and other scavenging elements like Mg(2+), Ca(2+), K(+), and Na(+). Therefore, in the view of development of novel approaches and proper diagnosis to male infertility, overall understanding of the biochemical and molecular composition and its role in regulation of sperm quality is highly desirable. Perhaps this can be achieved through artificial intelligence. This study was aimed to elucidate and predict various biochemical markers present in human seminal plasma with three different neural network models. A total of 177 semen samples were collected for this research (both fertile and infertile samples) and immediately processed to prepare a semen analysis report, based on the protocol of the World Health Organization (WHO [2010]). The semen samples were then categorized into oligoasthenospermia (n=35), asthenospermia (n=35), azoospermia (n=22), normospermia (n=34), oligospermia (n=34), and control (n=17). The major biochemical parameters like total protein content, fructose, glucosidase, and zinc content were elucidated by standard protocols. All the biochemical markers were predicted by using three different artificial neural network (ANN) models with semen parameters as inputs. Of the three models, the back propagation neural network model (BPNN) yielded the best results with mean absolute error 0.025, -0.080, 0.166, and -0.057 for protein, fructose, glucosidase, and zinc, respectively. This suggests that BPNN can be used to predict biochemical parameters for the proper diagnosis of male infertility in assisted reproductive technology (ART) centres. ABBREVIATIONS: AAS: absorption spectroscopy; AI: artificial intelligence; ANN: artificial neural networks; ART: assisted reproductive technology; BPNN: back propagation neural network model; DT: decision tress; MLP: multilayer perceptron; PESA: percutaneous epididymal sperm spiration; RBFN: radical basis function network; SRNN: simple recurrent neural network; SVM: support vector machines; TSE: testicular sperm extraction; WHO: World Health Organization.

Screening, production, optimization and characterization of ß-glucosidase using microbes from shellfish waste.

An extracellular ß-glucosidase was isolated from Proteus mirabilis VIT117 found to be growing on prawn shells. The enzyme production was found to be enhanced (14.58 U/ml) when the culture was maintained at pH 9 and provided with sorbitol as carbon source, yeast extract as nitrogen source and incubated at 37 °C for approximately 72 h. Statistical methods like Plackett-Burman and RSM were also applied here to study the effects of different combinations of growth parameters for the bacteria, where the most significant parameters were found to be inoculum size, pH, yeast extract, incubation time and sorbitol. The optimum concentrations of inoculum size, pH and yeast extract determined by RSM were 2 %, 9 and 2 %, respectively. Partial purification of the protein was done by ammonium sulfate precipitation, followed by dialysis, gel filtration chromatography and SDS-PAGE. The enzyme was found to have a molecular weight of approximately 50 kDa and was observed to be most active at 37 °C in pH 9, with a sharp decline in the enzyme activity when temperature or the pH was increased. Enzyme kinetics study was performed to understand the catalytic behavior of the enzyme and it was found that our ß-glucosidase had 5.613 U/ml and 0.082 mM as V max and K m values, respectively.

EFFECTS OF VARIOUS SEMEN EXTENDERS ON SEMEN PARAMETERS FOR THE PURPOSE OF HUMAN MALE FERTILITY PRESERVATION.

BACKGROUND: Cryopreservation enables semen to be preserved at subzero temperatures, usually at -196 degrees C. There is a need in preparing good extender for the semen to be cryopreserved until use, especially in the field of assisted reproduction. OBJECTIVE: To elucidate the apt extender for preserving both infertile and fertile samples for a minimum period and to check the post thaw results for various extenders used. MATERIALS AND METHODS: A total of 103 samples were collected for this research, and after semen analysis the semen samples were categorized into oligospermia (n = 20), oligoasthenospermia (n = 22), asthenospermia (n = 24), normospermia (n = 28), and control (n = 9). RESULTS: The extender supplemented with various antioxidants yields better results when compared to all the other extenders in case of fertile and infertile samples. CONCLUSION: Supplementing semen extender with antioxidants and various ingredients is the concern in designing an apt semen extender recipe. This research prescribes antioxidant extender (E4) to preserve the infertile and fertile semen samples for the purpose of research and also for doing assisted reproduction.

EFFECT OF SEMEN EXTENDER ON PROTEIN CONCENTRATION IN EACH FRACTION OF CRYOPRESERVED HUMAN SEMEN.

BACKGROUND: Semen extender is liquid diluents which are added to semen to preserve its fertilizing ability. The addition of extender to semen protects the sperm against possible damage by toxic seminal plasma, as well as providing nutrients. AIM AND OBJECTIVE: The hypotheses of the study are to evaluate, if there is any significant change in the protein concentration in each fractions of human semen before and after freezing with prepared semen extender. Then we are going to correlate the semen parameters with thee fresh semen total protein concentration. RESULTS: After preserving with the extender, all the semen samples show statistically significant (p < 0.001) in case of protein concentration. CONCLUSION: There is a significant different (p < 0.001) in protein concentration in various fractions before and after freezing. Also the prepared semen extender can be used in Assisted Reproductive centres (ART).

Prediction of Zn concentration in human seminal plasma of Normospermia samples by Artificial Neural Networks (ANN).

PURPOSE: There has been an increasing interest in the evaluation of metal ion concentration, present in different body fluids. It is known that metal ions, especially zinc play vital role in the fertility of human semen. OBJECTIVE: The main objective of the study is to evaluate the Zn concentration in Normospermia samples by Atomic absorption spectroscopy (AAS) and to predict the same by artificial neural network (ANN). MATERIALS AND METHODS: Normospermia semen samples were collected from the patients who came to attend semen analysis at Bangalore assisted conception centre, Bangalore, India. Semen analysis was done according to World Health Organization (WHO) guidance. Atomic absorption spectroscopy was used to estimate the total Zn in these samples, while the Back propagation neural network algorithm (BPNN) was used to predict the Zn levels in these samples. RESULTS: Zinc concentration obtained by AAS and BPNN indicated that there was a good correlation between the estimated and predicted values and was also found to be statistically significant. CONCLUSION: The BPNN algorithm developed in this study could be used for the prediction of Zn concentration in human Normospermia samples. FUTURE PERSPECTIVE: The algorithm could be further developed to predict the concentration of all the trace elements present in human seminal plasma of different infertile categories.