Point-of-care impedimetric aptasensor to detect the luteinizing hormone.

Luteinizing hormone (LH) is a useful biomarker for identifying ovulation events in the cows to predict the time of ovulation to achieve a high success rate of conception following artificial insemination. Although antibody-based radioimmunoassay and enzyme-linked immunosorbent assay are being used for LH measurement, these techniques are expensive, time-consuming, and require expertise and sophisticated laboratory facilities. So, there is a need for a field-applicable, affordable, easy-to-use method for LH detection. For developing such a specific, quantitative, and inexpensive system, an aptamer-based smartphone-enabled aptasensor has been investigated. The aptamer was used instead of the antibody as a biorecognition element due to its comparative stability at ambient temperature, ease of synthesis, and cost-effectiveness. Electrochemical impedance spectroscopy has been used to obtain label-free detection of LH within 20 min in ~ 20 µL sample volume. The screen-printed gold electrode is compatible with a smartphone-enabled miniaturized device (Sensit Smart; Palmsens BV, The Netherlands) and was fabricated with the aptamer to detect LH in biological fluids (limit of detection 0.80 and 0.61 ng/mL in buffer and undiluted/unprocessed serum, respectively, with the dynamic range of detection of 0.01 to 50 ng/mL). All the data were obtained in the 10 kHz to 0.10 Hz frequency range at a bias potential of 0.30 V with an alternating potential of 10 mV. The clinical relevance of the sensor was evaluated in 10 serum samples collected from dairy animals which established a high correlation with standard LH-ELISA (κ > 0.87). The aptasensor can be stored at room temperature for 30 days without any significant loss in electrochemical sensing ability.

Gonadal transcriptome analysis of the common carp, Cyprinus carpio: Identification of differentially expressed genes and SSRs.

Common carp (Cyprinus carpio) is a world-wide freshwater fish of eutrophic waters. C. carpio, have various reproductive traits, including early sexual maturity, that may make them excellent, large, realistic, aquaculture model species. In the present work, de novo assembly of gonadal (testicular and ovarian) transcriptomes from juvenile common carp was performed to identify genes involved in gonadal development. A total of 81,757 and 43,257 transcripts with average lengths of 769 and 856 bp, were obtained from the immature testicular and ovarian transcriptomes, respectively. About 84,367 unigenes were constructed after removing redundancy involving representation of transcripts in both gonadal transcriptomes. Gene ontology (39,171 unigenes), clusters of orthologous group's analysis (6651 unigenes) and Kyoto encyclopedia of genes, and genomes automatic annotation server analysis (4783 unigenes) were performed to identify potential genes along with their functions. Furthermore, 18,342 (testis) and 8693 (ovary) simple sequence repeats were identified. About 298 differentially expressed genes were identified, of which 171 and 127 genes were up-regulated in testis and ovary, respectively. Quantitative real-time reverse transcription PCR was performed to validate differential expression of selected genes in testis and ovary. Nearly 809 genes related to reproduction were identified, sex-wise expression pattern of genes related to steroid synthesis, endocrine regulation, germ cell maintenance and others factors related to gonadal differentiation was observed, and expression analysis of nanos, ad4bp/sf-1, and gdf9 was performed. The present study identified certain important genes/factors involved in the gonadal development of C. carpio which may provide insights into the understanding of sex-differentiation and gonadal development processes.

Cloning, localization and differential expression of Neuropeptide-Y during early brain development and gonadal recrudescence in the catfish, Clarias gariepinus.

Neuropeptide-Y (NPY) has diverse physiological functions which are extensively studied in vertebrates. However, regulatory role of NPY in relation to brain ontogeny and recrudescence with reference to reproduction is less understood in fish. Present report for the first time evaluated the significance of NPY by transient esiRNA silencing and also analyzed its expression during brain development and gonadal recrudescence in the catfish, Clarias gariepinus. As a first step, full-length cDNA of NPY was cloned from adult catfish brain, which shared high homology with its counterparts from other teleosts upon phylogenetic analysis. Tissue distribution revealed dominant expression of NPY in brain and testis. NPY expression increased during brain development wherein the levels were higher in 100 and 150days post hatch females than the respective age-matched males. Seasonal cycle analysis showed high expression of NPY in brain during pre-spawning phase in comparison with other reproductive phases. Localization studies exhibited the presence of NPY, abundantly, in the regions of preoptic area, hypothalamus and pituitary. Transient silencing of NPY-esiRNA directly into the brain significantly decreased NPY expression in both the male and female brain of catfish which further resulted in significant decrease of transcripts of tryptophan hydroxylase 2, catfish gonadotropin-releasing hormone (cfGnRH), tyrosine hydroxylase and 3ß-hydroxysteroid dehydrogenase in brain and luteinizing hormone-ß/gonadotropin-II (lh-ß/GTH-II) in pituitary exhibiting its influence on gonadal axis. In addition, significant decrease of several ovary-related transcripts was observed in NPY-esiRNA silenced female catfish, indicating the plausible role of NPY in ovary through cfGnRH-GTH axis.

sox19 regulates ovarian steroidogenesis in common carp.

In teleost, ovarian steroidogenesis governed by the neuroendocrine system is also regulated by several transcription factors of gonadal origin. Investigating the synchronized interactions between the transcriptional and the hormonal factors is vital to comprehend the mechanisms that lead to gonadal differentiation. This study signifies the role of sry-related box (sox) 19 in ovarian steroidogenesis regulation of the common carp, Cyprinus carpio. Analysis of tissue distribution displayed higher sox19 expression in brain and ovary, and gonadal ontogeny showed higher expression of sox19 at 80 days post hatch (dph). Higher sox19 mRNA expression during spawning and increase of sox19 post human chorionic gonadotropin induction substantiate gonadotropin dependency. Estradiol-17ß treatment but not 17α-methyl-di-hydroxy-testosterone to 50 dph common carp for inducing mono-sex, elevated sox19 expression substantially. Sox19 protein was observed in granulosa cells of the follicular layer in common carp ovary. Higher sox19 expression was detected in isolated granulosa and theca cells, in vitro. Transient gene silencing with sox19-siRNA caused downregulation of various ovary-related genes including those specific to activator protein-1 factors, fibroblast growth factors, wnt-signaling, steroidogenic genes along with certain transcription factors. Serum 17α, 20ß-dihydroxy-4-pregnen-3-one and estradiol-17ß reduced significantly post sox19 silencing, in vivo. Concomitantly, a decrease in aromatase activity was detected post sox19-siRNA treatment, in vivo. This study demonstrates the impact of sox19 in the regulation of common carp ovarian growth and steroidogenesis.

Exposure of carbon nanotubes affects testis and brain of common carp.

Carbon nanotubes production has been rapidly increasing for many potential applications, however, the environmental impact of this nanomaterial needs to be comprehended. The present work focused on unraveling the effects of single-walled carbon nanotubes (SWCNT) in the common carp, Cyprinus carpio. The physicochemical properties of SWCNT were analyzed with X-ray diffraction, Fourier transforms infra-red, UV-Vis absorption, transmission electron microscopy (TEM), and Raman spectroscopy before testing for exposure impact. The effects of SWCNT, were investigated by exposing to two doses viz., 10 and 50 µg/L, for 7 days in adult common carp, in vivo. Expression of key steroidogenic and transcription factor genes related to testis and brain were downregulated after the treatment. The concomitant decreases in serum testosterone and 11-ketotestosterone levels revealed the impact of SWCNT after exposure. Further, SWCNT exposure induced antioxidant enzymes namely glutathione-S-transferases, superoxide dismutase, and catalase in both testis and brain. Concurrently, histological and TEM analysis of testis revealed structural disarray. In addition, SWCNT treatment, in testicular and brain primary cell cultures decreased cell viability with an increase of reactive oxygen species levels, leading to a significant elevation of apoptotic cells. In line with this, low mitochondrial membrane potential and DNA damage were also observed during post SWCNT treatment. Taken together, transient exposure of SWCNT causes toxic effects and alters testicular and brain function in the common carp. Thus, the discharge of carbon nanotubes poses a greater risk to the aquatic environment warranting regulatory measures.

Role of sox30 in regulating testicular steroidogenesis of common carp.

Expression of transcription factors is crucial for the regulation of steroidogenesis and gonadal development in fish. SRY-related box (SOX) proteins regulate gene expression of various events related to vertebrate reproduction. This study reports the role of sox30 and its influence on sox9a/b in regulating testicular steroidogenesis of the common carp, Cyprinus carpio. Tissue distribution showed predominant expression of sox30 in gonads, while gonadal ontogeny indicated significant dimorphic expression of sox30 from 120 days post hatch. Higher sox30 transcripts during the spawning season, an elevation of sox30 after human chorionic gonadotropin induction, and 11-ketotestosterone (11-KT) treatment authenticate gonadotropin dependency. Treatment of 17α-methyl-di-hydroxy-testosterone to juvenile common carp for mono-sex induction, vis-à-vis elevated sox30 expression. Sox30 protein was detected abundantly in spermatocytes and spermatid/sperm of carp testis. Transient silencing of sox30 using small interfering RNAs decreased sox9a/b expression, lead to downregulation of certain molecule/factor, transcription factor, germ/stem cell marker, and steroidogenesis-related enzyme genes. Serum testosterone and 11-KT decreased significantly upon transient silencing of sox30, in vivo. Concomitantly, a reduction in testicular microsomal 11-ß hydroxysteroid dehydrogenase activity was observed. These results demonstrate the influence of sox30 as well as sox9a/b in the regulation of testicular steroidogenesis in common carp.

Risk Factors for Scrub Typhus, Murine Typhus, and Spotted Fever Seropositivity in Urban Areas, Rural Plains, and Peri-Forest Hill Villages in South India: A Cross-Sectional Study.

Scrub typhus and spotted fever group rickettsioses are thought to be common causes of febrile illness in India, whereas they rarely test for murine typhus. This cross-sectional study explored the risk factors associated with scrub typhus, tick-borne spotted fever, and murine typhus seropositivity in three different geographical settings, urban, rural, and hill villages in Tamil Nadu, South India. We enrolled 1,353 participants living in 48 clusters. The study included a questionnaire survey and blood sampling. Blood was tested for Orientia tsutsugamushi (scrub typhus), Rickettsia typhi (murine typhus), and spotted fever group Rickettsia IgG using ELISA. The seroprevalence of scrub typhus, spotted fever, and murine typhus were 20.4%, 10.4%, and 5.4%, respectively. Scrub typhus had the highest prevalence in rural areas (28.1%), and spotted fever was most common in peri-forested areas (14.9%). Murine typhus was more common in rural (8.7%) than urban areas (5.4%) and absent in peri-forested hill areas. Agricultural workers had a higher relative risk for scrub typhus, especially in urban areas. For murine typhus, proximity to a waterbody and owning a dog were found to be major risk factors. The main risk factors for spotted fever were agricultural work and living in proximity to a forest. Urban, rural plains, and hill settings display distinct epidemiological pattern of Orientia and rickettsial infections. Although scrub typhus and spotted fever were associated with known risk factors in this study, the findings suggest a different ecology of murine typhus transmission compared with other studies conducted in Asia.