Advances in the Optimization of Fe Nanoparticles: Unlocking Antifungal Properties for Biomedical Applications.

In recent years, nanotechnology has achieved a remarkable status in shaping the future of biological applications, especially in combating fungal diseases. Owing to excellence in nanotechnology, iron nanoparticles (Fe NPs) have gained enormous attention in recent years. In this review, we have provided a comprehensive overview of Fe NPs covering key synthesis approaches and underlying working principles, the factors that influence their properties, essential characterization techniques, and the optimization of their antifungal potential. In addition, the diverse kinds of Fe NP delivery platforms that command highly effective release, with fewer toxic effects on patients, are of great significance in the medical field. The issues of biocompatibility, toxicity profiles, and applications of optimized Fe NPs in the field of biomedicine have also been described because these are the most significant factors determining their inclusion in clinical use. Besides this, the difficulties and regulations that exist in the transition from laboratory to experimental clinical studies (toxicity, specific standards, and safety concerns) of Fe NPs-based antifungal agents have been also summarized.

Identification of a novel candidate HSD3B2 gene variant for familial hypospadias by whole-exome sequencing.

Introduction: Hypospadias [MIM: 300633] is one of the most frequent congenital malformations of male external genitalia. The spectrum of genetic variants causing hypospadias is varied, with studies commonly implicating genes critical in the fetal steroidogenic pathway. This is the first genetic study on hypospadias from the Yemen ethnicity and the second to report HSD3B2 mutations in more than one affected individual from the same family. Material and methods: Surgical hypospadias repair was performed on two hypospadias-affected siblings from a consanguineous family. Whole-exome sequencing (WES) was performed to identify the potential pathogenic variant for hypospadias, which was later confirmed by Sanger sequencing. The identified variant was further analyzed for its pathogenicity by using in silico tools such as SIFT, PolyPhen-2, MutationAssessor, MutationTaster, FATHMM, and ConSurf. Results: We identified a novel missense mutation (Chr1:119964631T>A, c.507T>A, p. N169K) in 3ß-hydroxysteroid 2-dehydrogenase (HSD3B2) gene by WES. Sanger sequencing confirmed that the variant segregated the disease in the family between the affected and non-affected individuals. Both patients are homozygous, while parents and two unaffected siblings are heterozygous carriers, indicating an autosomal recessive pattern of inheritance. The in silico analysis by all six in silico tools (SIFT, PolyPhen-2, MutationAssessor, MutationTaster, FATHMM, and ConSurf) predicted the variant to be pathogenic/deleterious. Discussion: An abnormal fetal steroidogenic pathway due to genetic influences may affect the development of the male genital tract, including the urethral tract closure and morphogenesis of male genitalia. Furthermore, the pathogenicity of the observed variant in this study, confirmed by multiple in silico tools, characterizes the influence HSD3B2 gene variants may have in the etiology of hypospadias. Conclusion: Understanding of pathogenic manifestation and inheritance of confounding genetic variants in hypospadias is a matter of great concern, especially in familial cases.

Negatively charged food additive dye "Allura Red" rapidly induces SDS-soluble amyloid fibril in beta-lactoglobulin protein.

Recent studies have led to an increased interest to categorize small molecular inhibitors of protein fibrillation. In this study, we used spectroscopy, microscopy and gel electrophoresis techniques that provides an elaborated description of the Allura Red-induced amyloid fibrillation in the ß-LG protein at two pHs (7.4 and 3.5). The spectroscopy results show that ß-LG protein form aggregates in the presence of Allura Red (0.04-15.0mM) at pH 3.5 due to electrostatic and hydrophobic interactions. However, at pH 7.4, the ß-LG does not interact electrostatically with Allura Red and therefore no aggregation occurred. The Allura Red-induced aggregates have an amyloid-like structure that was confirmed by far-UV CD, Congo Red and transmission electron microscopy (TEM). The CD spectrum of ß-LG contains single minima at ∼218nm, which shifts towards higher wavelength minima at ∼225nm in the presence of Allura Red, characteristics of the cross ß-sheet structure. The TEM results suggest that ß-LG form long straight fibril when exposed to Allura Red at pH 3.5. The Allura Red-induced amyloid fibril is SDS-soluble confirmed by SDS-PAGE techniques. A far UV CD result shows the conversion of Allura Red induced cross ß-sheet structure into alpha-helical structure in the presence of increasing concentration of SDS. The results of this study suggest that the electrostatic, as well as hydrophobic interactions play an important role during Allura Red-induced ß-LG fibrillation.

Mitochondrial DNA nucleotide changes in primary congenital glaucoma patients.

PURPOSE: Primary congenital glaucoma (PCG) is the second most common cause of blindness, accounting for 0.01%-0.04% of total blindness worldwide. Most congenital glaucoma cases are mapped to the GLC3A locus, and many aspects of PCG are still unknown. Recent studies have reported an increased frequency of mitochondrial DNA (mtDNA) sequence changes in primary open-angle glaucoma, primary angle-closure glaucoma, and pseudoexfoliation glaucoma compared to controls. Thus, this study was planned with the aim of detecting mitochondrial DNA variations in PCG cases. METHODS: Twenty primary congenital glaucoma cases were selected from Dr. R. P. Centre for Ophthalmic Sciences of All India Institute of Medical Sciences (AIIMS), New Delhi, India. DNA was isolated from whole blood samples. The entire coding region of the mitochondrial genome was amplified by PCR in 20 patients and 20 controls. The full mtDNA genome was sequenced and analyzed against mitochondrial reference sequence NC_012920. RESULTS: MtDNA sequencing revealed a total of 195 nucleotide variations in PCG patients and 58 in controls. Of the 195 changes, 43 (22.05%) were nonsynonymous, 82 (42.05%) were synonymous, and 30 were in RNA genes. A total of 39/195 (20.00%) variations were observed in the D-loop (hypervariable region), 19/195 (9.74%) in different ribosomal RNA (rRNAs), 11/195 (5.64%) in transfer RNA (tRNAs), 66/195 (33.84%) in complex I, 17/195 (8.71%) in complex III, 27/195 (13.84%) in complex IV, and 15/195 (7.69%) in complex V. Of 58 variations in the controls, 14 were nonsynonymous changes. The Sorting Intolerant from Tolerant and Polymorphism Phenotyping analyses of all nonsynonymous changes from patients revealed two pathogenic changes in NADH-ubiquinone oxidoreductase chain 2 (ND2) and cytochrome oxidase subunit III (COXIII) subunits. In one of the patients, the insertion of cytosine introduced a frame shift change (p.Ile104AsnfsX26) in the cytochrome b (CYB) subunit of the electron transport chain. In another patient, a variation (G8572A) in ATP synthase 8 (ATpase8) led to the introduction of a stop codon or termination at amino acid position 69. Haplogroup/phylogenetic analysis of mtDNA showed that primary congenital glaucoma patients belong to three macrohaplogroups: M (4), N (15), and L (1). Fifty percent of the patients belonged to the H2a2a lineage of the N-derived haplogroup. CONCLUSIONS: Although several mutations were found at a higher frequency among our population, there is a need to complement this study with functional studies and to analyze a large number of samples in different populations of different haplogroups, as penetrance varies among haplogroups.

Mitochondrial DNA variations in ova and blastocyst: implications in assisted reproduction.

Mitochondrial DNA (mtDNA) of oocyte is critical for its function, embryo quality and development. Analysis of complete mtDNA of 49 oocytes and 18 blastocysts from 67 females opting for IVF revealed 437 nucleotide variations. 40.29% samples had either disease associated or non-synonymous novel or pathogenic mutation in evolutionarily conserved regions. Samples with disease associated mtDNA mutations had low fertilization rate and poor embryo quality, however no difference in implantation or clinical pregnancy rate was observed. Screening mtDNA from oocyte/blastocyst is a simple, clinically reliable method for diagnostic evaluation of female infertility and may reduce risk of mtDNA disease transmission.

Herbo-mineral supplementation in men with idiopathic oligoasthenoteratospermia : A double blind randomized placebo-controlled trial.

INTRODUCTION: There is insufficient scientific data on the medical management options for idiopathic oligoasthenoteratospermia (iOATs). We conducted a double blind, randomized, placebo-controlled trial to assess the efficacy and safety of the herbo-mineral supplement, Addyzoa(®), in infertile men with iOATs. We also evaluated its effect on semen reactive oxygen species (ROS) levels, total antioxidant capacity (TAC) and DNA fragmentation index. MATERIALS AND METHODS: Fifty infertile men with iOATS were recruited into an institutional ethics committee approved protocol from April to August 2009. Randomization was done using numbered, identical containers. Baseline semen samples were evaluated for routine parameters, ROS level, DNA fragmentation index and TAC. Drug/placebo was administered at a dose of two capsules twice a day for 3 months. All parameters were reassessed at 3 months and clinical side-effects were recorded. The study was registered with the Clinical Trials Registry of India and is available at www.ctri.in as study protocol number CTRI/2009/091/000551. RESULTS: Forty-four subjects completed the study, 21 in the drug arm and 23 in the placebo arm. There was no difference in baseline parameters between the two groups. Men in the drug group had significant improvement in mean total motility from 23.2 ± 17.3% to 33.4 ± 23.2% (P-value: 0.008) and mean progressive (Type A+B) motility from 15.7 ± 12.6% to 22.6 ± 18.0% (P-value: 0.024). ROS, TAC and DFI did not change significantly in either group and did not show any correlation with other semen parameters. CONCLUSIONS: Treatment with Addyzoa resulted in a significant improvement in total and progressive motility in the semen of men with iOATs after 3 months of therapy. There was no change in the sperm concentration, ROS, DFI or TAC levels.

Clinical significance of sperm DNA damage threshold value in the assessment of male infertility.

Sperm DNA integrity is a prerequisite for normal spermatozoal function. The aim of the study was to evaluate the role of sperm chromatin damage, its cut-off level and its effect on sperm parameters in men with idiopathic infertility by analyzing 100 idiopathic infertile men and 50 fertile controls. Semen samples were analyzed as per WHO 1999 guidelines and sperm chromatin structure assay (SCSA) was applied to measure DNA fragmentation index (DFI) in sperm. The mean DFI of infertile men (35.75) was significantly (P < .0001) higher as compared to controls (26.22). The threshold level of 30.28% was obtained as cut-off value to discriminate infertile men from fertile controls. Sperm count, forward motility, and normal morphology found to be negatively associated with DFI in overall study subjects. Infertile men with severe oligozoospermia had higher mean DFI (40.01 ± 11.31) than infertile men with oligozoospermia (35.11 ± 10.05) and normal sperm count (33.99 ± 9.96). Moreover 64% of infertile men have DFI > 30 against 6% of fertile controls (P < .0001). Higher sperm DNA fragmentation may be the underlying cause for poor semen quality in idiopathic infertile men and the threshold value of 30.28% is a clear discriminator to distinguish infertile men from fertile men of Indian population. Thus, DFI is a good prognostic marker as cases with higher sperm DFI may have poor success rate even after assisted conception and may experience recurrent pregnancy loss (RPL) and should be counseled accordingly.

Sperm DNA integrity assays: diagnostic and prognostic challenges and implications in management of infertility.

Sperm is not a simple carrier of paternal genetic information but its role extends clearly beyond fertilization. Integrity of sperm genome is an essential pre-requisite for birth of healthy offspring and evaluation of sperm should entail DNA integrity analysis. DNA integrity analysis is a better diagnostic and prognostic marker of sperm reproductive potential. Conventional semen analysis emphasizes on sperm concentration, viability, motility and morphology and has been proven to be a poor indicator of reproductive potential and pregnancy outcome. To overcome the drawbacks associated with conventional semen analysis more useful fertility tests and molecular biomarkers have been explored. Among the different tests which have evolved for assessing the sperm reproductive potential, tests for sperm DNA quality are most promising. Sperm DNA damage has been closely associated with numerous indicators of reproductive health including fertilization, embryo quality, implantation, spontaneous abortion, congenital malformations and childhood diseases. It therefore has great potential as a prognostic test for both in vitro and in vivo conception. This review presents an updated account of tests that have better diagnostic and prognostic implications in the evaluation of sperm DNA damage. The basic principles, outline of methodology, advantage, disadvantage, clinical significance of each technique and implications of these tests have been discussed. The logistics of each test with respect to available resources and equipment in an andrology laboratory, the feasibility of performing these tests in routine diagnostic workup of infertile men and the opportunities and challenges provided by DNA testing in male fertility determination are also presented.

Idiopathic recurrent pregnancy loss: role of paternal factors; a pilot study.

BACKGROUND: This case-control study was designed with the aim of evaluating the role of sperm, oxidative stress and DNA damage in idiopathic recurrent pregnancy loss (iRPL). This pilot study is the first study done on the Indian population which reports the association between DFI, TAC and ROS in couples experiencing iRSA. METHODS: Twenty infertile men with a history of iRPL and 20 fertile controls (having fathered a child a year earlier) were included in the study which was performed in Laboratory for Molecular Reproduction and Genetics, India, from March 2010 to July 2011. The female partners of the participants were normal on gynaecological examination and had normal endocrine and blood profiles. Conventional semen analysis was performed (concentration, motility, morphology; WHO criteria, 2010) within 1 hour of sample collection. Levels of reactive oxygen species (ROS) were assessed by luminol-dependant chemiluminescence. The total antioxidant capacity (TAC) was quantified by ELISA. The Sperm chromatin structure assay (SCSA) was performed by flow cytometry to determine DNA fragmentation Index (DFI). Statistical analysis was performed using SPSS version 15 and parameters were compared by Mann-Whitney test. Pearson correlation test was used to find the correlation between parameters and a p-value <0.05 was considered significant. Receiver operating characteristics (ROC) curve analysis was applied to find out the cut-off value of DNA fragmentation index. RESULTS: No significant differences in age, seminal volume, liquefaction time, pH and sperm concentration were observed between the male partner of iRPL cases and the controls, but sperm morphology and motility were significantly (p <0.05) lower in the male partner of cases with idiopathic recurrent spontaneous abortion (RSA). The mean ROS levels observed were 47427.00 relative light unit (RLU)/min/20 million sperm in the male partners as compared to 13644.57 RLU/ min/20 million sperm in the controls (normal <15000 RLU/min/20 million). The mean TAC levels in the controls (6.95 mM trolox) were significantly (p <0.05) higher as compared to the male partners of women with IRPL (2.98 mM trolox). The average mean DFI of male partners were found to be 23.37±9.9 and the mean DFI of controls was 13.89±5.40. The mean DFI was significantly (p <0.05) higher when compared to the controls. The range of DFI in male partners was 8.50-44.07. However, in the controls the range was 7.70-23.50. CONCLUSION: Sperm DNA integrity is critical for normal embryonic development and birth of healthy offspring. Oxidative stress due to the imbalance between raised free radical levels and low total antioxidant capacity is one of the critical causes of DNA damage. Thus assay of oxidative stress and sperm genomic integrity is essential in couples with iRSA following natural and spontaneous conception.

Attenuation of oxidative stress & DNA damage in varicocelectomy: implications in infertility management.

Sperm DNA integrity is of vital importance for foetal development and birth of healthy offspring. Oxidative stress and consequent DNA damage are the major cause of decline in semen quality in men with varicocele. A preliminary study was conducted on 11 men with clinical varicocele who also had high levels of reactive oxygen species (ROS), to assess DNA damage in sperms and ROS levels before and after varicocelectomy. Varicocelectomy resulted in rapid (1 month) significant (P<0.001) decline in free radical levels and slow (3-6 months) significant decline in DNA damage levels. Thus men undergoing varicocelectomy should try concieving only 6 months following surgery.

Mitochondrial DNA Mutations in etiopathogenesis of male infertility.

OBJECTIVE: To understand role of mitochondrial (mt) mutations in genes regulating oxidative phosphorylation (OXPHOS) in pathogenesis of male infertility. Infertility affects approximately 15% of couples trying to conceive. Infertility is frequently attributed to defects of sperm motility and number. Mitochondrion and mitochondrial DNA (mtDNA) play an important role in variety of physiological process. They control the oxidative energy supply and thus are central to growth, development and differentiation. Mitochondrial function is controlled by a fine-tuned crosstalk between mtDNA and nuclear DNA (nDNA). As mitochondria supply energy by OXPHOS, any mutation in mtDNA disrupts adenosine triphosphate (ATP) production and thus result in an impaired spermatogenesis and impaired flagellar movement. As sperm midpiece has few mtDNA copies, thus enhanced number of mutant mtDNA results in early phenotypic defect which manifest as spermatogenic arrest or asthenozoospermia. Oxidative stress and mtDNA mutations are positively correlated and mutations in mitochondrial genome (mt genome) are implicated in the lowered fertilising capacity of the sperm and affects the reproductive potential of an individual. MATERIALS AND METHODS: A thorough review of articles in the last 15 years was cited with reference to the below-mentioned keywords. The articles considered discuss the role of mt genome in the normal functioning of sperm and the factors associated with mt mutations and impact of these mutations on the reproductive potential. RESULTS: Sperm motility is a very important factor for the fertilisation of ova. The energy requirements of sperm are therefore very critical for sperm. Mutations in the mitochondrial genes as COX II, ATPase 6 and 8 play an important role and disrupts ATP production affecting the spermatogenesis and sperm motility. Therefore, the aberrations in mt genome are an important etiopatholgy of male infertility. CONCLUSION: In the context of male infertility, mt mutations, generation of reactive oxygen species and lowered antioxidant capacity are interlinked and constitute a unified pathogenic molecular mechanism. In the era of assisted reproduction technique (ART), it is very important to distinguish between mutations in nuclear and mitochondrial genomes in sperm, as mtDNA mutations are better diagnostic and prognostic markers in infertile men opting for ART.