Effects of dietary α-linolenic acid/linoleic acid ratio on growth performance, tissue fatty acid profile, serum metabolites and Δ6 fad and elovl5 gene expression in silver barb (Puntius gonionotus).

BACKGROUND: Silver barb (Puntius gonionotus) is a medium-sized carp that is promising for freshwater aquaculture in Asia. This study's aim was to investigate the ideal dietary α-linolenic acid (ALA): linoleic acid (LA) ratio for maximizing long-chain polyunsaturated fatty acid (LC-PUFA) synthesis and their deposition in the muscle of silver barb, as that of fish oil based control diet. RESULT: Fish (with an initial body weight of 11.07 ± 0.12 g) were fed for 60 days with five experimental iso-proteinous, iso-lipidic, and iso-caloric diets, supplemented with linseed oil and peanut oil at varying levels to obtain ALA:LA ratios of 0.35, 0.51, 0.91, 2.04, 2.66. A control diet was prepared by supplementing fish oil. The dietary ALA:LA ratio did not influence the growth performance of fish. With increased dietary ALA:LA ratios, LA content decreased and ALA content increased in the muscle and liver of silver barb. The n-3 LC-PUFA level in muscle and liver was not influenced by feeding different ratios of ALA:LA, whereas n-6 LC-PUFA was decreased in the muscle and increased in the liver with increased dietary ALA:LA ratios. Increasing dietary ALA:LA ratio increased the Δ6fad and elovl5mRNA expression in the liver, muscle, brain, and intestinal tissues of silver barbs. CONCLUSION: Silver barb possess the ability to elongate and desaturate ALA and LA to their end products EPA and DHA. The highest level expression of Δ6 fad and elovl5 mRNA at the dietary ALA:LA ratio of 2.66 suggests greater affinity of these enzymes towards ALA than LA in silver barb. © 2019 Society of Chemical Industry.

Erythrocyte cAMP in Determining Frequency of Acute Pain Episodes in Sickle Cell Disease Patients from Odisha State, India.

Vaso-occlusive crisis (VOC) occurs more frequently during stress in sickle cell disease patients. Epinephrine released during stress increases adhesion of sickled red blood cells (RBCs) to endothelium and to leukocytes, a process mediated through erythrocyte cyclic adenosine monophosphate (cAMP). Increased adhesion of sickled RBCs retards blood flow through the capillaries and promotes vaso-occlusion. Therefore, we examined the association of RBC-cAMP levels with frequency of acute pain episodes in sickle cell disease subjects. Using a case control study design, we measured RBC-cAMP levels, fetal hemoglobin (Hb F), α-thalassemia (α-thal) and other hematological parameters at baseline (sham treated) and after stimulation with epinephrine. The cases consisted of sickle cell disease subjects with three or more acute pain episodes in the last 12 months, and those without a single acute pain episode in the last 12 months were considered as controls. Significantly higher cAMP values were found in cases than the controls, in both sham treated (p < 0.001) and epinephrine treated RBCs (p < 0.001) by Wilcoxon Rank Sum test. However, significant association of cAMP values was observed both on univariate [odds ratio (OR): 4.8, 95% confidence interval (95% CI): 1.51-15.19, p < 0.008) and multivariate logistic regression analyses only in epinephrine treated (OR: 5.07, 95% CI: 1.53-16.82, p < 0.008) but not in sham-treated RBCs. In the covariates, Hb F consistently showed protective effects in univariate as well as in multivariate analyses. Frequent acute pain episodes are associated with higher cAMP levels than those with less frequent pain episodes, only after stimulation with epinephrine but not with baseline level.

Gonadotropin receptors of Labeo rohita: Cloning and characterization of full-length cDNAs and their expression analysis during annual reproductive cycle.

Follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), secreted from pituitary, stimulate gonadal function by binding to their cognate receptors FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR). Rohu (Labeo rohita) is a commercially important seasonal breeder freshwater fish species, but till date, the regulation of expression of gonadotropins and their receptors gene during different phases of annual reproductive cycle has not been investigated. We envisaged the critical role of these molecules during seasonal gonadal development in this carp species. We cloned full- length cDNAs of fshra and lhcgrba from rohu testis using RACE (Rapid amplification of cDNA ends) and analyzed their expression along with fsh and lh by quantitative real time PCR (qRT-PCR) assay at various gonadal developmental stages of the annual reproductive cycle. Full-length rohu fshra and lhcgrba cDNA encodes 670 and 716 amino acids respectively, and in adult fish, they were widely expressed in brain, pituitary, gonad, liver, kidney, head kidney, heart, muscle, gill, fin, eye and intestine. In male, both fsh and fshra transcripts showed high level of expression during spermatogenesis, however, in female, expression level was found to be higher in the fully grown oocyte stages. The expression of rohu lh and lhcgrba mRNA increased with increment of gonadosomatic index and showed highest level during spermiation stage in male and fully matured oocyte stage in female. These results together may suggest the involvement of fshra and lhcgrba in regulating function of seasonal gonadal development in rohu.

Diagnosis of Mucormycosis Using Frozen Section, Histopathology, Culture, and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Techniques: A Comparative Study.

Mucormycosis usually occurs in immunocompromised patients or those with uncontrolled diabetes. Along the third wave of SARS-CoV-2, an associated angioinvasive opportunistic infection with Mucor, a life-threatening fungal infection, was rampant and emerging. With an increase in the usage of steroids in the COVID scenario, the rate of mucormycosis did take a rapid and alarming increase in King Edward Memorial Hospital, Pune, India. Any delay in the diagnosis and management of the disease was life-threatening. The most conventional methods to diagnose mucormycosis are microbiological culture and histopathology of the tissue. The microbiological culture method plays an important role in the diagnosis of mucormycosis. However, the technique is labour-intensive, taking seven to eight days. Histopathology leads to false-negative reports if the tissue is not biopsied from representative sites. On the other hand, molecular methods are rapid, reliable, and applicable to different body samples, such as tissue, paraffin-embedded tissue blocks, plasma, and urine. We aimed to use a reverse transcriptase polymerase chain reaction (RT-PCR) method to detect Mucor in plasma samples. Due to a lack of availability of fresh samples, nucleic acid was extracted from the tissue sections of 69 cases diagnosed as Mucor by histopathology. These samples were subjected to RT-PCR using the MucorGenius kit (Pathonostics, Maastricht, Netherlands). A total of 57 tissue samples were sent for culture, and 49% of our cases were positive by culture and equally by RT-PCR. There was 80% sensitivity and 76% specificity between culture and PCR techniques. However, the use of blood/plasma for RT-PCR for early diagnosis of mucormycosis will be the method of choice.

Uncommon presentation of chromoblastomycosis.

Chromoblastomycosis is an uncommon subcutaneous mycosis seen in the tropical and subtropical regions that is caused by traumatic inoculation of a specific group of dematiaceous fungi through the skin. It is a therapeutic challenge due to its recalcitrant nature. Although there currently is no evidence-based treatment protocol, itraconazole and terbinafine are considered drugs of first choice. We present the case of a 25-year-old dairy farmer with slowly growing verrucous lesions on both cheeks of 20 years' duration. This case highlights the usefulness of amphotericin B in cases of chromoblastomycosis that do not respond to first-choice drugs.

Influence of dietary lipid levels on growth, nutrient utilization, tissue fatty acid composition and desaturase gene expression in silver barb (Puntius gonionotous) fingerlings.

Silver barb (Puntius gonionotus) is considered as a promising medium-sized carp species for freshwater aquaculture in Asia. This study in silver barb was carried out to evaluate the effects of increasing dietary levels of lipid on growth, nutrient utilization, whole-body composition, tissue fatty acid composition and Δ6 fatty acyl desaturase (Δ6 fad) gene expression. Fish (11.3 ±â€¯0.23 g of initial body weight) was fed for 60 days with five experimental diets: FO-0 (control feed); FO-30; FO-60; FO-90 and FO-120 containing 0, 30, 60, 90 and 120 g fish oil kg-1 diet, respectively. Among the diets, the highest specific growth rate (SGR), protein efficiency ratio (PER) and whole-body lipid content, and the lowest feed conversion ratio (FCR) were recorded with FO-120 diet. The saturated fatty acids (SFA) level in the muscle was significantly (P < .05) increased with the enhanced FO supplementation, whereas monounsaturated fatty acids (MUFA) level decreased. Increased level of fish oil in the diet also enhanced the n-3 PUFA and n-3 LC-PUFA (long-chain polyunsaturated fatty acid) in the muscle and liver. The expression of Δ6 fad gene was downregulated, whereas the serum biochemical constituents were either remain unchanged or enhanced with increased FO supplementation in the diets of silver barb.

Molecular characterization, tissue distribution and differential nutritional regulation of putative Elovl5 elongase in silver barb (Puntius gonionotus).

Fatty acid desaturase (Fads) and elongase (Elovl) are two important enzymes involved in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) in fishes. Silver barb (Puntius gonionotus) is considered a promising medium carp species for freshwater aquaculture in Asia. Earlier, Δ6 fads cDNA was characterized to understand the molecular mechanism of LC-PUFA biosynthesis in this species. In the present study elovl5-like elongase full-length cDNA was cloned and characterized by RACE (rapid amplification of cDNA ends), and analyzed its basal expression in various organs/tissues by quantitative real time PCR (qRT-PCR) assay. Differential expression of elovl5 cDNA in various tissues following replacement of fish oil (FO) with linseed oil (LO) in the diet also studied. The full length cDNA sequence of silver barb elovl5-like elongase consisted of 1853bp, including an open reading frame (ORF) of 876bp encoding a precursor protein of 291 amino acids possessing the entire characteristic features of Elovl protein. The elovl5 transcript was constitutively expressed in all the tissues examined: muscle, liver, brain, kidney, skin, intestine, gill and eye. The expression of elovl5 gene was upregulated, whereas the serum biochemical constituents were either remain unchanged or lowered following replacement of FO with LO in the diets. We report for the first time, an in silico 3D structure model of elongase protein from silver barb. All these findings may contribute to a better understanding the potential regulatory mechanisms involved and may be useful for enhancing endogenous LC-PUFA production in silver barb.

Dietary fish oil replacement by linseed oil: Effect on growth, nutrient utilization, tissue fatty acid composition and desaturase gene expression in silver barb (Puntius gonionotus) fingerlings.

Silver barb (Puntius gonionotus) is considered a promising medium carp species for freshwater aquaculture in Asia. This study in silver barb was carried out to evaluate the effects of total or partial substitution of dietary fish oil (FO) with linseed oil (LO) on growth, nutrient utilization, whole-body composition, muscle and liver fatty acid composition. Fish (12.1±0.4g of initial body weight) were fed for 60days with five experimental iso-proteinous, iso-lipidic and iso-caloric diets in which FO (control diet) was replaced by 33.3%, 50%, 66.7% and 100% LO. Final weight, weight gain, percent weight gain, SGR decreased linearly (p<0.001) with increasing LO levels in the diets. Dietary LO substitution levels did not significantly (p>0.05) affect the feed conversion ratio (FCR), protein efficiency ratio (PER) and whole body proximate composition. Furthermore, enhanced level of LO increased α-linolenic acid (ALA; 18:3n3) and linoleic acid (LA; 18:2n6) and decreased eicosapentaenoic acid (EPA; 20:5n3) and docosahexaenoic acid (DHA; 22:6n3) in muscle and liver. To understand the molecular mechanism of long chain-polyunsaturated fatty acid (LC-PUFA) biosynthesis, we cloned and characterized the fatty acyl Δ6 desaturase (Δ6 fad) cDNA and investigated its expression in various organs/tissues following replacement of FO with LO in the diet. The full-length Δ6 fad cDNA was 2056bp encoding 444 amino acids and was widely expressed in various organs/tissues. Replacement of FO with LO increased the expression of Δ6 fad mRNA in liver, muscle and intestine but no significant difference was found in the brain.

Molecular characterization of two kiss genes and their expression in rohu (Labeo rohita) during annual reproductive cycle.

Kisspeptin is an important regulator of reproduction in mammals and presumably non-mammalian species. In the Indian subcontinent, rohu (Labeo rohita) is a commercially important seasonal breeder freshwater fish species, but till date, the expression of kisspeptin gene during different phases of annual reproductive cycle has not been investigated. To address this, we cloned and characterized kiss1 and kiss2 full-length cDNA by RACE (rapid amplification of cDNA ends), and analyzed their expressions in brain-pituitary-gonad (BPG) axis by quantitative real time PCR (qRT-PCR) assay at various gonadal developmental stages of the annual reproductive cycle. Full-length rohu kiss1 and kiss2 cDNA encodes 116 and 125 amino acids respectively, and in the adult fish, they were widely expressed in brain, pituitary, gonad, liver, muscle, kidney, intestine and eye. In male, kiss1 mRNA in brain and testis showed the highest level of expression during meiosis division of the gonad. The kiss2 mRNA revealed the highest expression during recrudescence stages in the brain, spermiation stages in pituitary and post-spawning stages in testes. In females, significantly (p<0.05) a higher level of kiss1 transcript was expressed in brain and ovary, in the full grown oocyte stages, whereas during pre-vitellogenic and vitellogenic stages in pituitary. The kiss2 gene expression was almost similar at various gonadal developmental stages in the brain and ovary, but, highest expression was detected in full grown oocyte stages in the pituitary. These results together may suggest the involvement of two kiss genes in the control of seasonal gonadal development in rohu.

Plasma cell vulvitis.

Plasma cell vulvitis is a very rare inflammatory disorder of vulva, characterized by a bright-red mucosal lesion of significant chronicity, which may be symptomatic. Very few case studies of this condition are reported in literature. We describe one such classical patient, who presented with slight dyspareunia. The diagnosis was confirmed on histopathological examination. It is important for clinicians to accurately diagnose this alarming condition in time.

Thermosensitive ion channel TRPV1 is endogenously expressed in the sperm of a fresh water teleost fish (Labeo rohita) and regulates sperm motility.

Sperm cells exhibit extremely high sensitivity in response to slight changes in temperature, osmotic pressure and/or presence of various chemical stimuli. In most cases throughout the evolution, these physico-chemical stimuli trigger Ca (2+)-signaling and subsequently alter structure, cellular function, motility and survival of the sperm cells. Few reports have recently demonstrated the presence of Transient Receptor Potential (TRP) channels in the sperm cells from higher eukaryotes, mainly from higher mammals. In this work, we have explored if the sperm cells from lower vertebrates can also have thermo-sensitive TRP channels. In this paper, we demonstrate the endogenous presence of one specific thermo-sensitive ion channel, namely Transient Receptor Potential Vanilloid family member sub type 1 (TRPV1) in the sperm cells collected from fresh water teleost fish, Labeo rohita. By using western blot analysis, fluorescence assisted cell sorting (FACS) and confocal microscopy; we confirm the presence of this non-selective cation channel. Activation of TRPV1 by an endogenous activator NADA significantly increases the quality as well as the duration of fish sperm movement. The sperm cell specific expression of TRPV1 matches well with our in silico sequence analysis. The results demonstrate that TRPV1 gene is conserved in various fishes, ranging from 1-3 in copy number, and it originated by fish-specific duplication events within the last 320 million years (MY). To the best of our knowledge, this is the first report demonstrating the presence of any thermo-sensitive TRP channels in the sperm cells of early vertebrates as well as of aquatic animals, which undergo external fertilization in fresh water. This observation may have implications in the aquaculture, breeding of several fresh water and marine fish species and cryopreservation of fish sperms.

Miliary tuberculosis with hemophagocytosis in a two months old infant.

A two months old male infant, presented with fever, hepatosplenomegaly and pancytopenia. On bone marrow aspirate, hemophagocytosis was noted. Trephine biopsy showed epithelioid granulomas. ZN stain AFB detected, which confirmed the diagnosis of miliary tuberculosis. This case highlights the rare association of hemophagocytic syndrome with tuberculosis in infancy.

Immunohistochemical analysis of Mallory bodies in Wilsonian and non-Wilsonian hepatic copper toxicosis.

Patients with Wilson's disease (WD), Indian childhood cirrhosis (ICC), and idiopathic copper toxicosis (ICT) develop severe liver disease morphologically characterized by ballooning of hepatocytes, inflammation, cytoskeletal alterations, and Mallory body (MB) formation, finally leading to mostly micronodular cirrhosis. The pathogenesis of MBs in copper toxicosis is still unresolved. Immunohistochemical analysis of MBs in different types of copper intoxication revealed that keratin, p62, and ubiquitin are integral components. Thus MBs associated with copper intoxication resemble those present in alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH). p62 is a multifunctional immediate early gene product that, on the one hand, is involved in stress-induced cell signaling (particularly that of oxidative stress) by acting as an adapter protein linking receptor-interacting protein (RIP) with the atypical protein kinase C. On the other hand, p62 binds with high affinity to polyubiquitin and ubiquitinated proteins. In conclusion, p62 accumulation in WD, ICC, and ICT and deposition in MBs indicates a central role of protein misfolding induced by oxidative stress in copper-induced liver toxicity. By sequestering potentially harmful misfolded ubiquitinated proteins as inert cytoplasmic inclusion bodies (e.g., as MBs), p62 may be a major player in an important cellular rescue mechanism in oxidative hepatocyte injury.