Argulosis is one of the most unrestrained economically significant freshwater fish ectoparasitic diseases. Proper selection or normalization of the best reference gene governs the accuracy of results ...of gene expression studies using real-time PCR. Earlier studies in rohu carp (Labeo rohita) have used reference genes without proper validation. Here, seven candidate reference genes viz., acidic ribosomal protein (ARP0), glyceraldehyde 3-phosphate dehydrogenase, RNA polymerase II (RPo), elongation factor1α (EF1α), α- tubulin (AT), ribosomal protein L 10, and β-actin were evaluated using four algorithms (geNorm, BestKeeper, NormFinder and ∆Ct) followed by a comprehensive gene expression analysis using skin tissue of rohu at varied time points of experimental Argulus siamensis infection. ARP0 and EF1α were found to be the most stable whereas RPo and AT were considered as least stable genes based on basal expression level and variation in expression levels. Validation of candidate reference genes was undertaken by looking into the expression of six immune-related genes using the two most stable and two least stable genes as housekeeping genes in Argulus-infected rohu skin at different time points of infection. An increased expression of immune genes indicated the role of inflammation and the immune modulation process at the site of attachment of parasites in governing infection.
Fish possess numerous enzymatic antioxidant systems as part of their innate immunity. These systems have been poorly studied in
(rohu). The present study characterized and investigated the role of ...antioxidant genes in the defence mechanisms against two types of stressors, including infection and ammonia stress. Four key genes associated with antioxidant activity-catalase, glutathione peroxidase, glutathione S-transferase, and CuZn superoxide dismutase were successfully cloned and sequenced. These genes were found to be expressed in different tissues and developmental stages of rohu. The expression levels of these antioxidant genes in the liver and anterior kidney tissues of rohu juveniles were modulated in response to bacterial infection (
), parasite infection (
), poly I:C stimulation and ammonia stress. Additionally, the recombinant proteins derived from these genes exhibited significant antioxidant and antibacterial activities. These proteins also demonstrated a protective effect against
infection in rohu and had an immunomodulatory role. Furthermore, indirect ELISA assay systems were developed to measure these protein levels in healthy as well as
and ammonia-induced rohu serum. Overall, this study characterized and emphasised the importance of the antioxidant mechanism in rohu's defence against oxidative damage and microbial diseases.
•Argulosis, a disease caused by Argulus spp. of ectoparasites in scaly fish, is a global concern for aquaculture industry.•The resistance of the parasite to anti-parasitic drugs and the quantum of ...loss has been felt world-wide.•The current scenario of management and the development in vaccination are discussed herewith.
The knowledge of mucosa-associated molecular events that occur during infections is scarce despite the well-established importance of mucus in fish immunity. Using qRT-PCR, we analyzed the immune ...gene expression patterns in mucus of Labeo rohita experimentally infected with an ectoparasite Argulus siamensis. Mucus samples were collected at 0 h, 12 h, 24 h, 3 d, 7 d, 15 d, and 30 d post challenge of L. rohita with metanauplii of A. siamensis. All interleukins studied herein (IL 6, IL 15, and IL 1β) showed significant upregulation of expression levels in mucus of A. siamensis-infected fish compared to control samples. Further, the expression levels of molecules involved in pathogen recognition, toll like receptor 22, and pathogen presentation, β2 microglobulin, were found to be significantly upregulated in experimental samples until 7 d post challenge compared to control samples. The upregulated expression of lysozyme G at all time points post infection indicated the early activation of acute phase responses in mucus of infected L. rohita. Moreover, the expression levels of natural killer cell enhancing factor B were found to be higher in infected fish than they were in the control fish. The early upregulation of the immune genes observed herein reinforces the role of mucus as the first line of defense against pathogenic assault; furthermore, it expands our understanding of mucosal-immune responses to A. siamensis infection, which can aid development of immunological interventions.
Primary pleomorphic liposarcoma of the chest wall is a rare entity. Most of the reported cases of the chest wall liposarcomas are low grade, well differentiated with mediastinal extension. Majority ...of these develop from a subcutaneous lipoma. Complete surgical resection is the preferred treatment modality. The benefit of adjuvant radiotherapy or chemoradiotherapy for these tumors is a subject of debate. We report, an unusual case of high grade primary pleomorphic subtype of liposarcoma in a 50-year-old male presenting with a painful swelling in the anterior thoracic wall extending into the pleura but without any mediastinal involvement or distant metastasis.
•Complete CDs of NKEF-B of rohu was cloned and structurally characterized.•Its expression profile was studied in bacterial/viral/parasitic infection models.•Recombinant NKEF-B showed antioxidant and ...antibacterial activities.•An indirect ELISA was developed to measure LrNKEF-B concentration in serum.
Natural killer enhancing factor (NKEF) of peroxiredoxin family is an important innate immune molecule with having anti-oxidant activity. Although this gene has already been studied in a few fish species, it is yet to be identified and functionally characterised in Indian major carps. In the present study, the complete NKEF-B cDNA of rohu, Labeo rohita was cloned that encoded a putative protein of 197 amino acids. The phylogenetic study showed that L. rohita NKEF-B (LrNKEF-B) is closely related to NKEF-B of Cyprinus carpio and Danio rerio species. Tissue-specific expression of LrNKEF-B gene revealed the highest transcript level in the liver tissue. In the ontogeny study, the highest level of the expression was observed in milt and at 18 h post-development. The expression pattern of this gene was also studied in various pathogen models viz., Gram-negative bacteria (Aeromonas hydrophila), ectoparasite (Argulus siamensis) and a dsRNA viral analogue (poly I:C) in the liver and anterior kidney tissues of L. rohita juveniles. During A. hydrophila infection, the increase in expression of transcripts was observed at 3 h post-infection in both liver (15-fold) and anterior kidney (8-fold). In A. siamensis infection, the expression gradually increased up to 3 d post-infection in the anterior kidney, whereas in liver 3-fold up-regulation was noticed at 12 h post-infection. Similarly, during poly I:C stimulation, up-regulation of NKEF-B transcript was observed in anterior kidney from 1 h to 24 h post-stimulation and down-regulated afterwards whereas, the transcript level increased gradually from 6 h to 15 d post-stimulation in liver tissue. In vitro exposure to concanavalin, A and formalin-killed A. hydrophila upregulated NKEF-B gene expression in anterior kidney and peripheral blood leukocytes of L. rohita, however, down-regulated the same in the splenic leukocytes. A recombinant protein of LrNKEF-B (rLrNKEF-B) of 22 kDa was produced and it showed anti-oxidant activity by protecting supercoiled DNA and reducing insulin disulfide bonds. The minimum bactericidal concentration of this recombinant protein was found to be 4.54 μM against A. hydrophila and Staphylococcus aureus. Interestingly, rLrNKEF-B showed relative percent survival of 72.6 % in A. hydrophila challenged L. rohita, and the survival was found to be associated with a high level of expression of different cytokines, anti-oxidant genes and perforin in the rLrNKEF-B treated L. rohita. An indirect ELISA assay for estimation of NKEF was developed in L. rohita, and the concentrations of NKEF-B increased with time periods post A. hydrophila challenge viz., 0 h (42.56 ng/mL), 12 h (174 ng/mL) and 48 h (370 ng/mL) in rohu serum. Our results suggest a crucial role of LrNKEF-B in innate immunity against biotic stress and oxidative damage and also having antibacterial activity.
Fish possess numerous enzymatic antioxidant systems as part of their innate immunity. These systems have been poorly studied in Labeo rohita (rohu). The present study characterized and investigated ...the role of antioxidant genes in the defence mechanisms against two types of stressors, including infection and ammonia stress. Four key genes associated with antioxidant activity–catalase, glutathione peroxidase, glutathione S-transferase, and CuZn superoxide dismutase were successfully cloned and sequenced. These genes were found to be expressed in different tissues and developmental stages of rohu. The expression levels of these antioxidant genes in the liver and anterior kidney tissues of rohu juveniles were modulated in response to bacterial infection (Aeromonas hydrophila), parasite infection (Argulus siamensis), poly I:C stimulation and ammonia stress. Additionally, the recombinant proteins derived from these genes exhibited significant antioxidant and antibacterial activities. These proteins also demonstrated a protective effect against A. hydrophila infection in rohu and had an immunomodulatory role. Furthermore, indirect ELISA assay systems were developed to measure these protein levels in healthy as well as A. hydrophila and ammonia-induced rohu serum. Overall, this study characterized and emphasised the importance of the antioxidant mechanism in rohu’s defence against oxidative damage and microbial diseases.
Recent advances on synthesis of C-glycosides Parida, Sonali Priyadarshini; Das, Tapaswini; Ahemad, Mohammed Ansar ...
Carbohydrate research,
August 2023, 2023-Aug, 2023-08-00, 20230801, Letnik:
530
Journal Article
Recenzirano
In recent years, C-glycosides have emerged as significant building blocks for many naturally occurring alkaloids and pharmaceutically active drug molecules. Therefore, significant efforts have been ...devoted to the construction of structurally important C-glycosidic linkages in carbohydrate compounds. Herein, we have summarized the recent developments of diverse synthesis of C-glycoside core between the time period from 2019 to 2022 focusing on different catalytic strategies, such as (i) transition-metal, and (ii) metal-free catalytic approaches. Further, the transition metal catalyzed C-glycosylations have been categorized into four sub classes: (a) metal based C-H activation, (b) cross-coupling reaction, (c) glycosyl radical intermediate-based process, and (d) Others.
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•Current advancements in the formation of various C-glycoside functionalities from the year 2019-2022.•Diverse transition metal catalyzed approaches, metal free approaches and photo-catalytic approaches.•Carbohydrate based drug synthesis from C-glycoside derivatives is described.•Stereoselective synthesis of a wide range of β-C-glycosides.
Argulus spp. are economically important fish ectoparasites. The development of antiparasitic drugs is thus important and real time PCR is an indispensable tool in drug development. The analytical ...potential of RT-PCR depends upon accurate normalisation by the use of stable reference genes. Here, we identified stable reference genes of Argulus siamensis for validation of efficacy of drugs and drug targets. Seven candidate genes were evaluated by evaluating their expression under different states of Argulus using the RefFinder tool. The four algorithms together generated a comprehensive ranking with elongation factor-1 alpha (EF-1α) being the most stable and 18S ribosomal protein (18S) the least stable gene. Taking EF-1α and 18S genes as references, the effectiveness of six anti-parasitic compounds against Argulus was evaluated by studying their effect on the expression pattern of few ion channel genes; this was to understand their mode of action, besides validating the reference genes. EF-1α was found to be the most stable gene in the validation. Collectively, this study is the first report to validate the optimal reference genes of A. siamensis for normalisation, and the potential of the ion channel genes for evaluating effective drug targets in parasite control.
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