Characterising the tear bacterial microbiome in young adults.

Conjunctival swabs (CS) are the major source of sampling for ocular microbiome studies, however collecting CS from the diseased eyes is difficult and painful. In this study, as an alternative to CS, a less invasive approach of tear collection was used to establish the bacterial microbiome in healthy eyes. Tear bacterial microbiome was generated from the DNA of tears (n = 24; male = 16 and female = 8) of healthy volunteers aged from 20 to 52 years. Sequencing of V3-V4 region of 16S rRNA gene was performed on the Illumina platform. Reads were processed in QIIME to assign the taxa. Statistical analysis of the tear microbiome was done in R to assess the alphadiversity and betadiversity indices. Tear microbiome was generated in all the 24 tear samples. Eight out of the top 10 predominant bacterial genera remained same in both tear and CS microbiomes, which include genera such as Corynebacterium, Staphylococcus, Streptococcus, Mycobacterium, Escherichia-Shigella, Lactobacillus, Bacillus and Acinetobacter. The similarity network analysis indicates that 144 out of 145 genera of tear cohort matched with conjunctival swabs. However, tear and CS microbiomes differed in the abundance of the predominant bacterial genera. The bacterial microbiome of tears in adults appears to be stable and is comparable with that of CS microbiome.

Fungi of the human eye: Culture to mycobiome.

The focus of the current review is multi-fold and compares the diversity and abundance of fungi on the ocular surface by the conventional culture-based method with the more sensitive, high throughput, culture-independent NGS method. The aim is to highlight the existence of a core ocular mycobiome and explore the transition of the ocular fungal microbiota from the normal eye to the diseased eye. PubMed, Google Scholar and Medline were used to search for publications and reviews related to cultivable fungi and the mycobiome of the normal and diseased eye. The conventional cultivable approach and the NGS approach confirm that the eye has its own mycobiome and several confounding factors (age, gender, ethnicity etc.) influence the mycobiome. Further, dysbiosis in the mycobiome appears to be associated with ocular diseases and thus impacts the health of the human eye. Considering that the mycobiome of the eye is influenced by several confounding factors and also varies with respect to the disease status of the eye there is a need to extensively explore the mycobiome under different physiological conditions, different ethnicities, geographical regions etc. Such studies would unravel the diversity and abundance of the mycobiomes and contribute to our understanding of ocular health. Research focused on ocular mycobiomes may eventually help to build a targeted and individualized treatment.

DNMT1 and DNMT3B gene variants and their association with endometriosis in South Indian women.

BACKGROUND: Endometriosis is a multifactorial estrogen dependent gynecological disease characterized by implantation of functional endometrial tissue at ectopic positions. Though this disease is benign, it is associated with an increased risk of malignant transformation. Epigenetic disruptions like aberrant DNA methylation, resulting changes in gene expression capacity, are important in tumor progression and malignant cellular transformation. Therefore, variation in genes involved in DNA methylation might lead to disease susceptibility. PURPOSE: To investigate the association between DNA methyl transferases (DNMT1 and DNMT3B) single nucleotide polymorphisms (SNPs) and the risk of endometriosis in South Indian women. METHODS: In the present study, we examined the genotypic and allele distribution of DNMT1 (rs10423341C/A, rs2228611G/Aandrs4804490C/A) and DNMT3B (rs1569686G/T) among the endometriosis patients (n = 150) and controls (n = 150). The genotypes were analyzed by polymerase chain reaction (PCR) and sequencing methods. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were surveyed by Haploview Software. RESULT: Significant increase in the frequencies of DNMT1 rs10423341 (P = 0.04601), rs2228611 (P = 0.00175) and DNMT3B rs1569686 (P = 0.033) genotypes and alleles was observed in patients compared to controls. In addition, the frequency of A/A/C (P = 0.0065) haplotype was significantly high in patients. But the DNMT1 (rs4804490) SNP did not show significant association with the disease. CONCLUSION: The DNMT1 and DNMT3B polymorphism may constitute an inheritable risk factor for endometriosis in South Indian women. To the best of our knowledge there is no reported study on the association of polymorphisms in DNMT1 and DNMT3B with endometriosis risk.

Microbes of the human eye: Microbiome, antimicrobial resistance and biofilm formation.

BACKGROUND: The review focuses on the bacteria associated with the human eye using the dual approach of detecting cultivable bacteria and the total microbiome using next generation sequencing. The purpose of this review was to highlight the connection between antimicrobial resistance and biofilm formation in ocular bacteria. METHODS: Pubmed was used as the source to catalogue culturable bacteria and ocular microbiomes associated with the normal eyes and those with ocular diseases, to ascertain the emergence of anti-microbial resistance with special reference to biofilm formation. RESULTS: This review highlights the genetic strategies used by microorganisms to evade the lethal effects of anti-microbial agents by tracing the connections between candidate genes and biofilm formation. CONCLUSION: The eye has its own microbiome which needs to be extensively studied under different physiological conditions; data on eye microbiomes of people from different ethnicities, geographical regions etc. are also needed to understand how these microbiomes affect ocular health.

Alterations in the conjunctival surface bacterial microbiome in bacterial keratitis patients.

Microbial keratitis is an infectious disease of the eye, in which the cornea is inflamed. Under severe conditions, keratitis can lead to significant loss of vision and enucleation of the eye. Ocular trauma is the major risk factor causing keratitis and microorganisms viz., bacteria, fungi, viruses are the causative agents. The current study characterized the conjunctival bacterial microbiomes of healthy individuals and individuals with bacterial keratitis (BK) and assessed whether ocular microbiome dysbiosis is prevalent in BK patients. Ocular bacterial microbiomes were generated from the conjunctival swabs of healthy controls (HC-SW) and conjunctival swabs (BK-SW) and corneal scrapings (BK-CR) of BK patients using V3-V4 amplicon sequencing and data analysed using QIIME and R software. The Alpha diversity indices, diversity and abundance of different phyla and genera, heat map analysis, NMDS plots and inferred functional pathway analysis clearly discriminated the bacterial microbiomes of conjunctival swabs of healthy controls from that of BK patients. Preponderance of negative interactions in the hub genera were observed in BK-CR and BK-SW compared to HC-SW. In addition, a consistent increase in the abundance of pathogenic bacteria, as inferred from published literature, was observed in the conjunctiva of BK patients compared to HC and this may be related to causing or exacerbating ocular surface inflammation. This is the first study demonstrating dysbiosis in the ocular bacterial microbiome of conjunctiva of bacterial keratitis patients compared to the eye of healthy controls. The bacterial microbiome associated with the corneal scrapings of keratitis individuals is also described for the first time.

Association of genetic variations in phosphatase and tensin homolog (PTEN) gene with polycystic ovary syndrome in South Indian women: a case control study.

PURPOSE: The purpose of the study was to investigate the association between gene phosphate and tensin homolog (PTEN) single nucleotide polymorphisms (SNPs) and risk of developing polycystic ovary syndrome (PCOS) in South Indian women. PTEN is one of the most important tumor suppressor genes that regulate cell proliferation, migration, and death. It is also involved in the maintenance of genome stability. PCOS is one of the most common endocrine disorders among women of reproductive age. It is a heterogeneous syndrome characterized by abnormal reproductive cycles, irregular ovulation, hormonal imbalance, hyperandrogenism, acne and hirsutism. RESEARCH QUESTION: What is the association status of PTEN SNPs with PCOS? METHODS: A total of 240 subjects were recruited in this case-control study comprising 110 patients with PCOS and 130 individuals without PCOS. All the subjects were of South Indian origin. The genotyping of PTEN SNPs (rs1903858 A/G, rs185262832G/A and rs10490920T/C) was carried out on DNA from subjects by polymerase chain reaction (PCR) and sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were surveyed by Haploview Software. RESULTS: Our results showed significant increase in the frequencies of rs1903858 A/G (P = 0.0016), rs185262832 G/A (P = 0.0122) and rs10490920 T/C (P = 0.0234) genotypes and alleles in cases compared to controls. CONCLUSION: The PTEN (rs1903858A/G, rs185262832G/A and rs10490920T/C) gene polymorphisms may constitute an inheritable risk factor for PCOS in South Indian women.

Subsaxibacter sediminis sp. nov., isolated from Arctic glacial sediment and emended description of the genus Subsaxibacter.

A Gram-stain-negative, yellowish-orange pigmented, rod-shaped, motile bacterium, designated strain ARC111T, was isolated from sediment of Arctic permafrost at Midtre Lovénbreen glacier, Svalbard. 16S rRNA gene based identification of strain ARC111T demonstrated highest sequence similarities to Subsaxibacter broadyi P7T (97.8 %) and Subsaxibacter arcticus JCM30334T (97.5 %) and ≤95.2 % with all other members of the family Flavobacteriaceae. Phylogenetic analysis revealed the distinct positioning of strain ARC111T within the genus Subsaxibacter. The G+C content of ARC111T was 37.8±0.5 mol% while DNA-DNA hybridization depicted 35.6 % relatedness with S. arcticus JCM30334T. Strain ARC111T had C15 : 0iso, C16 : 0iso 3-OH, C15 : 1iso G, C15 : 0anteiso, C16 : 1iso H and C17 : 0iso 3-OH as major (>5 % of the total) cellular fatty acids and MK-6 was the predominant respiratory quinone. The polar lipid profile of strain ARC111T consisted of phosphatidylethanolamine, aminolipid and an unidentified lipid. Strain ARC111T harboured sym-homospermidine as the major polyamine. Characteristic differences obtained using polyphasic analysis of strain ARC111T and its closest relatives suggested that strain ARC111T is a novel species of genus Subsaxibacter, for which the name Subsaxibacter sediminis sp. nov. has been proposed. The type strain is ARC111T (=MCC 3191T=KCTC 42965T=LMG 29783T=GDMCC 1.1201T).

Vitamin D receptor gene polymorphisms and risk of polycystic ovary syndrome in South Indian women.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of reproductive age women. Emerging evidence suggests that Vitamin D Receptor (VDR) might be a causal factor for characteristics associated with PCOS such as obesity and type 2 diabetes. Present study investigated association between VDR gene BsmI A/G (rs1544410), ApaI A/C (rs7975232) and TaqI T/C (rs731236) single nucleotide polymorphisms and PCOS risk in South Indian women. Genotyping of VDR gene SNPs was carried out in PCOS patients (n = 95) and controls (n = 130) by PCR-RFLP method and confirmed by sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were assessed by Haploview software. Results showed significantly increased frequencies of BsmI G/G (p = .0197), ApaI C/C (p = .048), TaqI C/C (p = .044) genotypes and BsmI G (p = .0181), ApaI C (p = .0092), TaqI C (p = .0066) alleles in patients compared to controls. In addition, the frequency of the 'BsmI G, ApaI C, TaqI C' haplotype was also significantly elevated in patients (p = .0087). In conclusion, the VDR gene BsmI A/G ApaI A/C TaqI T/C and haplotype may constitute an inheritable risk factor for PCOS in South Indian women.

Dysbiosis in the Gut Bacterial Microbiome of Patients with Uveitis, an Inflammatory Disease of the Eye.

Uveitis (UVT), an inflammatory disease of the eye significantly contributes to vision impairment and blindness. Uveitis is associated with systemic infectious and autoimmune diseases, but in most cases, the aetiology remains unidentified. Dysbiosis in the gut microbiome has been implicated in autoimmune diseases, inflammatory diseases, cancers and mental disorders. In a mice model of autoimmune UVT, it was observed that manipulating the gut microbiome reduces the inflammation and disease severity. Further, alterations in the bacterial gut microbiome and their metabolites were reported in UVT patients from a Chinese cohort. Hence, it is worth comparing the bacterial gut microbiome of UVT patients with that of healthy controls (HC) to ascertain whether dysbiosis of the gut microbiome has implications in UVT. Our analyses showed reduced diversity of several anti-inflammatory organisms including Faecalibacterium, Bacteroides, Lachnospira, Ruminococcus and members of Lachnospiraceae and Ruminococcaceae families, and enrichment of Prevotella (proinflammatory) and Streptococcus (pathogenic) OTUs in UVT microbiomes compared to HC. In addition, decrease in probiotic and antibacterial organisms was observed in UVT compared to HC microbiomes. Heatmap and PCoA plots also indicated significant variations in the microbiomes of UVT versus HC. This is the first study demonstrating dysbiosis in the gut bacterial communities of UVT patients in an Indian cohort and suggests a role of the gut microbiome in the pathophysiology of UVT.

SRC family kinases in hamster spermatozoa: evidence for the presence of LCK.

Sperm capacitation is a prerequisite for successful fertilization. Increase in tyrosine phosphorylation is considered the hallmark of capacitation and attempts to understand its regulation are ongoing. In this regard, we attempted to study the role of SRC family kinases (SFKs) in the hamster sperm functions. Interestingly, we found the presence of the lymphocyte-specific protein tyrosine kinase, LCK, in mammalian spermatozoa and further characterized it in terms of its localization and function. LCK was found in spermatozoa of several species, and its transcript was identified in the hamster testis. Autophosphorylation of LCK at the Y394 residue increased as capacitation progressed, indicating an upregulation of LCK activity during capacitation. Inhibition of LCK (and perhaps the other SFKs) with the use of a specific inhibitor showed a significant decrease in protein tyrosine phosphorylation of several proteins, implying LCK/SFKs as key tyrosine kinase(s) regulating tyrosine phosphorylation during hamster sperm capacitation. Dihydrolipoamide dehydrogenase was identified as a substrate for LCK/SFK. LCK/SFKs inhibition significantly reduced the percentage fertilization (in vitro) but had no effect on sperm motility, hyperactivation and acrosome reaction. In summary, this is the first report on the presence of LCK, an SFK of hematopoietic lineage in spermatozoa besides being the first study on the role of SFKs in the spermatozoa of Syrian hamsters.

Effect of different cryo-devices on in vitro maturation and development of vitrified-warmed immature buffalo oocytes.

The aim of the study was to identify a cryo-device that would be best suited for the vitrification of buffalo immature cumulus-oocyte complexes (COCs) as judged by viability and meiotic competence of the vitrified-warmed oocytes and their development ability following in vitro fertilization (IVF). The expression of oocyte secreting factors and their receptors (GDF9, BMP15, BMPR2, TGFBR1) and apoptosis related genes (BCL2, BAX, P53, C-MYC) were compared in vitrified-warmed oocytes after in vitro maturation. COCs from the ovaries of slaughtered buffaloes were vitrified in a combination of dimethyl sulfoxide, ethylene glycol, and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). The fresh COCs were exposed to vitrification and warming solutions as in other vitrification methods without plunging in to liquid nitrogen (EC). The viability of vitrified-warmed COCs, 2 h post warming in HS and CT was similar to fresh and EC groups but significantly higher than CS and OPS methods. The proportions of oocytes with first polar body after 24 h in vitro maturation were significantly higher in HS and CT methods than in CS, OPS and CL methods. The development ability of these vitrified-warmed oocytes to blastocyst stage following IVF in all vitrified groups was significantly lower than control and EC groups. Among the vitrified groups, the blastocyst rate in HS, CT and CL groups was significantly higher than in OPS and CS groups. It was also observed that the expression levels of GDF9, BMP15, BMPR2, TGFBR1, BCL2, BAX, P53 and C-MYC genes in vitrified-warmed COCs in CT, HS and CL groups were similar to control. The results indicated that HS, CT and CL are more suitable cryo-devices for vitrification of buffalo immature oocytes.

Manganese-superoxide dismutase (Mn-SOD) overexpression is a common event in colorectal cancers with mitochondrial microsatellite instability.

Mitochondrial displacement loop (D-loop) is a hot spot for mitochondrial DNA (mtDNA) alterations that effects cellular reactive oxygen species (ROS) generation. Manganese-superoxide dismutase (Mn-SOD) is a major antioxidant enzyme that protects cells from ROS-mediated damage. In the present study, we investigated the relationship between sequence alterations of mitochondrial D-loop and Mn-SOD expression in colorectal cancer (CRC). Genotyping of entire mitochondrial D-loop (1124 bp) was carried out on mtDNA of analogous tumor and normal tissues from 35 CRC patients of south Indian origin by PCR-sequencing analysis. Tumor-specific large-scale mtDNA deletions and Mn-SOD expression was analyzed by PCR and Western blot analysis, respectively. We identified 87 polymorphisms in the D-loop region of tumor and/or control tissues. Polymorphisms were predominantly located in hypervariable region I (67.9 %) than in II (32.1 %) of D-loop. Significantly increased mtDNA microsatellite instability (mtMSI) [310'C' insertion (P = 0.00001) and T16189C (P = 0.0007)] and elevated Mn-SOD expression was observed in tumor tissues compared with controls. Interestingly, mtMSI was significantly high in tumors with Mn-SOD overexpression. Tumor-specific large-scale mtDNA deletions were not observed in CRC tissues. In conclusion, mtMSI and Mn-SOD overexpression are a common event in CRC. The analysis of mtMSI and/or Mn-SOD expression might help to identify patients at high risk for disease outcome, thereby helping to refine therapeutic decisions in CRC.

Description of Thalassospira lohafexi sp. nov., isolated from Southern Ocean, Antarctica.

A gram-negative, aerobic, obligatory halophilic, curved-to-spiral rod-shaped, uni- or bi-polar flagellated motile bacterium 139Z-12(T) was isolated from water samples collected from Southern Ocean surrounding Antarctica as part of the Indo-German iron fertilization experiment "LOHAFEX." The bacterium was positive for catalase, oxidase, and gelatinase, with C18:1ω7c (20.1 %), C16:0 (7.3 %), C16:1ω7c (28.9 %), and C12:0 (19.0 %) as the predominant fatty acids, phosphatidylglycerol, and phosphatidylethanolamine as major lipids and Q-10 as the major respiratory quinone. DNA G + C content (mol%) of strain 139Z-12(T) was 53.0. Both BLAST and EzTaxon identified strain 139Z-12(T) as affiliated species of the genus Thalassospira with 16S rRNA gene sequence similarities ranging from 97.9 to 99.9 %. Based on above characteristics, strain 139Z-12(T) was assigned to the genus Thalassospira. Since strain 139Z-12(T) shared a DNA-DNA similarity of <70.0 % with members of the genus Thalassospira, 139Z-12(T) was identified as a novel species of the genus and named as Thalassospira lohafexi sp. nov. In addition to this, strain 139Z-12(T), based on REP-PCR, delineated at a similarity coefficient of <0.67 from other species of the genus Thalassospira and also exhibited a MLSA distance of <95.5 %, a value less than the interspecies distance determined previously for the genus Thalassospira, further supporting the species status. In addition, species rank was further confirmed by other polyphasic characteristics. The type strain of Thalassospira lohafexi sp. nov. is 139Z-12(T) (KCTC 32347(T) = LMG 27449(T) = MCC 2233(T) = NBRC 110402(T)).

Tolerance of an Antarctic Bacterium to Multiple Environmental Stressors.

A population of cold-tolerant Antarctic bacteria was screened for their ability to tolerate other environmental stress factors. Besides low temperature, they were predominantly found to be tolerant to alkali. Attempt was also made to postulate a genetic basis of their multistress-tolerance. Transposon mutagenesis of an isolate Pseudomonas syringae Lz4W was performed, and mutants with delayed growth at low temperature were further screened for sensitivity to some other stress factors. A number of multistress-sensitive mutants were isolated. The mutated gene in one of the mutants sensitive to low temperature, acid and alkali was found to encode citrate synthase. Possible role of citrate synthase in conferring multistress-tolerance was postulated.

BRCA1 alterations are associated with endometriosis, but BRCA2 alterations show no detectable endometriosis risk: a study in Indian population.

PURPOSE: To investigate the role of genetic variations and expression alterations of BRCA1 and BRCA2 genes in the pathophysiology of endometriosis. METHODS: A genetic association study was conducted in 573 endometriosis cases and 490 controls of Indian origin. We genotyped 13 selected promoter SNPs of BRCA1 gene and 2 selected promoter SNPs of BRCA2 gene by PCR-sequencing analysis. In addition, to better understand genetic contributions to the pathophysiology of endometriosis, the expression pattern of BRCA1 & 2 was analyzed in the eutopic endometria of endometriosis cases and controls by western-blot and immunohistochemical analysis. RESULTS: Our results revealed significant association between BRCA1 rs71361504 (-/GTT) SNP and endometriosis risk in Indian women (P < 0.0001), while the remaining SNPs of both BRCA1 & 2 genes showed no difference between cases and controls. Western-blot and immunohistochemical analysis revealed significantly decreased BRCA1 expression levels in eutopic endometria of patients compared with controls (P < 0.05). Furthermore, nuclear BRCA1 was frequently lost compared with cytoplasmic BRCA1 in eutopic endometria of patients. Expression of BRCA2 did not differ between patients and controls. CONCLUSIONS: BRCA1 rs71361504 SNP may modify the endometriosis risk in Indian women. In addition, decreased expression of BRCA1 may play an important role in the pathophysiology of endometriosis. The analysis of BRCA1 genetic variants and/or expression might help to identify patients at high risk for disease outcome.

Regulation of gene expression at low temperature: role of cold-inducible promoters.

Psychrophilic micro-organisms are the most dominant flora in cold habitats. Their unique ability to survive and multiply at low temperatures (<5 °C) is based on their ability to modulate the rigidity of the membrane, to transcribe, to translate and to catalyse biochemical reactions at low temperature. A number of genes are known to be upregulated during growth at low temperature and cold-inducible promoters are known to regulate the expression of genes at low temperature. In this review, we attempted to compile promoter sequences of genes that are cold-inducible so as to identify similarities and to compare the distinct features of each type of promoter when microbes are grown in the cold.

Mutations in the PTEN tumor gene and risk of endometriosis: a case-control study.

STUDY QUESTION: Are mutations in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene associated with endometriosis? SUMMARY ANSWER: Loss of heterozygosity (LOH) at the 10q23.3 locus, PTEN somatic mutations and changes in the levels and distribution of proteins in the PTEN-PI3K/Akt signal transduction pathway are associated with endometriosis. WHAT IS KNOWN ALREADY: Endometriosis has a strong genetic basis. Recent genome-wide association and linkage studies have reported a significant association of endometriosis with 7p15.2, 9p21 and 10q23-26 loci. PTEN, which maps to 10q23.3, acts as a tumor suppressor gene through the action of its phosphatase protein product, phosphatase and tensin homolog (PTEN). This phosphatase is involved in the regulation of the cell cycle, and mutations of PTEN are a step in the development of many cancers. STUDY DESIGN, SIZE, DURATION: A total of 1252 subjects of Indian origin (endometriosis patients = 752; controls = 500) were recruited to participate in this case-control study. Recruitment took place from 2001 to 2009 at Institute of Reproductive Medicine (IRM), Kolkata, India; Infertility Institute and Research Centre (IIRC), Secundrabad, India and Vasavi Medical and Research Centre, Hyderabad, India. PARTICIPANTS/MATERIALS, SETTING, METHODS: LOH on 10q, 9p and 7p was analyzed in analogous ectopic-eutopic endometria along with blood samples from 32 advanced stage endometriosis patients by PCR-GeneScan analysis. Genotyping of PTEN was carried out on genomic DNA of analogous ectopic-eutopic endometria (n = 32) as well as blood samples from 720 patients and 500 controls by PCR-sequencing analysis to explore somatic and germ-line mutations, respectively. The levels and distribution of PTEN, p-Akt, p-Bad and p27 were analyzed in the eutopic endometria of patients (n = 5) and controls (n = 5) using western-blot and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: PCR-GeneScan analysis revealed a higher LOH frequency at 10q23.3 (84.4%) compared with other loci analyzed, hence we focused our attention on PTEN. PCR-sequencing analysis revealed seven novel somatic mutations and 23 germ-line polymorphisms in patients. Among somatic mutations, a frame-shift insertion at 10:89692992-89692993 (in the functionally important N-terminal phosphatase domain of PTEN) occurred in 11 of the 32 ectopic endometria. Western-blot and immunohistochemical analysis revealed decreased PTEN and increased p-Akt and p-Bad levels in eutopic endometria of patients compared with controls (all comparisons, P < 0.0001). Furthermore, PTEN loss was more frequent in the nucleus than in the cytoplasm. Expression of p27 did not differ between patients and controls. LIMITATIONS, REASONS FOR CAUTION: Protein analysis was performed in eutopic endometrial samples from only a small number of patients and controls. In future investigations, a larger sample size should be used and the role of the other genes involved in the PTEN-PI3K/Akt signal transduction pathway should be analyzed. WIDER IMPLICATIONS OF THE FINDINGS: Our findings revealed a possible involvement of the PTEN-PI3K/Akt-Bad axis in the pathogenesis of endometriosis, which may facilitate the discovery of suitable pathway inhibitors for disease treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Science & Engineering Research Board (SERB), India (Lr No: SR/FT/LS-188/2009) to BM. The authors have no competing interests to declare.

Phylogenetic analyses of the genus Glaciecola: emended description of the genus Glaciecola, transfer of Glaciecola mesophila, G. agarilytica, G. aquimarina, G. arctica, G. chathamensis, G. polaris and G. psychrophila to the genus Paraglaciecola gen. nov. as Paraglaciecola mesophila comb. nov., P. agarilytica comb. nov., P. aquimarina comb. nov., P. arctica comb. nov., P. chathamensis comb. nov., P. polaris comb. nov. and P. psychrophila comb. nov., and description of Paraglaciecola oceanifecundans sp. nov., isolated from the Southern Ocean.

Phylogenetic analyses of the genus Glaciecola were performed using the sequences of the 16S rRNA gene and the GyrB protein to establish its taxonomic status. The results indicated a consistent clustering of the genus Glaciecola into two clades, with significant bootstrap values, with all the phylogenetic methods employed. Clade 1 was represented by seven species, Glaciecola agarilytica, G. aquimarina, G. arctica, G. chathamensis, G. mesophila, G. polaris and G. psychrophila, while clade 2 consisted of only three species, Glaciecola nitratireducens, G. pallidula and G. punicea. Evolutionary distances between species of clades 1 and 2, based on 16S rRNA gene and GyrB protein sequences, ranged from 93.0 to 95.0 % and 69.0 to 73.0 %, respectively. In addition, clades 1 and 2 possessed 18 unique signature nucleotides, at positions 132, 184 : 193, 185 : 192, 230, 616 : 624, 631, 632, 633, 738, 829, 1257, 1265, 1281, 1356 and 1366, in the 16S rRNA gene sequence and can be differentiated by the occurrence of a 15 nt signature motif 5'-CAAATCAGAATGTTG at positions 1354-1368 in members of clade 2. Robust clustering of the genus Glaciecola into two clades based on analysis of 16S rRNA gene and GyrB protein sequences, 16S rRNA gene sequence similarity of ≤95.0 % and the occurrence of signature nucleotides and signature motifs in the 16S rRNA gene suggested that the genus should be split into two genera. The genus Paraglaciecola gen. nov. is therefore created to accommodate the seven species of clade 1, while the name Glaciecola sensu stricto is retained to represent species of clade 2. The species of clade 1 are transferred to the genus Paraglaciecola as Paraglaciecola mesophila comb. nov. (type strain DSM 15026(T) = KMM 241(T)), P. agarilytica comb. nov. (type strain NO2(T) = KCTC 12755(T) = LMG 23762(T)), P. aquimarina comb. nov. (type strain GGW-M5(T) = KCTC 32108(T) = CCUG 62918(T)), P. arctica comb. nov. (type strain BSs20135(T) = CCTCC AB 209161(T) = KACC 14537(T)), P. chathamensis comb. nov. (type strain E3(T) = CGMCC 1.7001(T) = JCM 15139(T)), P. polaris comb. nov. (type strain ARK 150(T) = CIP 108324(T) = LMG 21857(T)) and P. psychrophila comb. nov. (type strain 170(T) = CGMCC1.6130(T) = JCM 13954(T)). The type species of the genus Paraglaciecola is Paraglaciecola mesophila. An emended description of the genus Glaciecola is provided. In addition, a novel strain, 162Z-12(T), was isolated from seawater collected as part of an iron fertilization experiment (LOHAFEX) conducted in the Southern Ocean in 2009 and was subjected to polyphasic taxonomic characterization. Cells of 162Z-12(T) were Gram-negative, aerobic, motile, ovoid to short rod-shaped, obligatorily halophilic and possessed all the characteristics of the genus Paraglaciecola. Strain 162Z-12(T) shared the highest 16S rRNA gene sequence similarity with the type strains of P. agarilytica (99.7 %), P. chathamensis (99.7 %), P. mesophila (98.5 %) and P. polaris (98.3 %). However, it exhibited DNA-DNA relatedness of less than 70.0 % with its nearest phylogenetic relatives, well below the threshold value for species delineation. Further, strain 162Z-12(T) differed from the nearest species in several phenotypic characteristics, in addition to the occurrence of unique nucleotides G, T, T and T at positions 1194, 1269, 1270 and 1271 of the 16S rRNA gene. Based on the cumulative differences it exhibited from its nearest phylogenetic neighbours, strain 162Z-12(T) was identified as a novel member of the genus Paraglaciecola and assigned to the novel species Paraglaciecola oceanifecundans sp. nov. The type strain of Paraglaciecola oceanifecundans is 162Z-12(T) ( = KCTC 32337(T) = LMG 27453(T)).

Non-invasive assessment of reproductive status and stress in captive Asian elephants in three south Indian zoos.

Asian elephants in captivity need immediate attention to be bred so as to meet the increasing demand for captive elephants and to overcome the dependence on supplementing the captive stock with wild animals. Unfortunately, captive breeding programs across the globe have met with limited success and therefore more effort is needed to improve breeding in captivity. Endocrine profiling of reproductive hormones (progestagens and androgens) and the stress hormone (glucocorticoids) could facilitate better management and breeding strategies. In the present study, we investigated reproductive and stress physiology of 12 captive Asian elephants for 10-27 months using a non-invasive method based on steroid analysis of 1700 elephant dung samples. Most of the elephants were cycling regularly. Males during musth showed increased fecal androgen metabolite concentrations and exhibited a slight increase in fecal glucocorticoid metabolite levels. Elephants used in public festivals and processions showed significantly increased in faecal glucocorticoid metabolite levels. The results indicate that captive elephants require periodic health care, better husbandry practices and scientific management for sustainable captive population.

Diversity and bioprospective potential (cold-active enzymes) of cultivable marine bacteria from the subarctic glacial Fjord, Kongsfjorden.

The diversity and abundance of culturable bacteria in Kongsfjorden water (15 stations) and sediments (12 stations) were studied. Viable numbers ranged between 105­106 CFU l−1 in water and 102­104 CFU g−1 in the sediments. A total of 291 and 43 bacterial isolates were retrieved from the water (KJF) and sediments (FS), respectively. Based on 16S rRNA gene sequence similarities, the KJF and FS isolates were grouped into 49 and 23 phylotypes, respectively. The KJF and FS phylotypes represented three phyla namely, Actinobacteria, Bacteroidetes, and Proteobacteria. At the genus level, Flavobacterium and Shewanella and at the species level, Pseudoaltermonas arctica and Colwellia psychrerythraea were dominant in the water and sediments, respectively. Most phylotypes were psychrotolerant with upper growth temperature limit of 25­37 °C and tolerated 0.3­2.5 M NaCl and pH values of 5.0­11.0. Majority of the phylotypes produced one or more of the extracellular hydrolytic enzymes amylase, lipase, caseinase, urease, gelatinase, and DNase at 4 and 18 °C, while none were chitinolytic. Few of the FS phylotypes exhibited extracellular activity only at 4 or 18 °C. Nine FS and 21 KJF isolates were pigmented. The predominant cellular fatty acids were unsaturated, branched, and modified fatty acids, which are unique to cold-adapted bacteria.