Characterization of Bioactive Metabolites and Antioxidant Activities in Solid and Liquid Fractions of Fresh Duckweed (Wolffia globosa) Subjected to Different Cell Wall Rupture Methods.

Fresh Wolffia globosa, the smallest flowering plant well-known for its favorable nutrient composition and rich content of bioactive compounds, was subjected to boiling, freeze-thawing, and mechanical crushing to reduce its excessive (95-96%) moisture level and consequent drying time. The resultant three wolffia matrixes were filtered through a plankton net to fractionate into the residue and the filtrate. The proximate composition, bioactive metabolites, antioxidant activity, and characterization of bioactive metabolites by LC-ESI-QTOF-MS/MS and Fourier transform infrared spectroscopy were made from oven-dried residues and filtrates. Among residues, crude protein (29.84%), crude lipid (5.77%), total carotenoids (TCC; 722.8 µg/g), and vitamin C (70.02 mg/100 g) were the highest (p < 0.05) for freeze-thawing against higher ash (7.99%), total phenolic content (TPC; 191.47 mg GAE g-1 dry weight), total flavonoid content (TFC; 91.54 mg QE g-1 dry weight), DPPH activity (47.46%), and ferric reducing antioxidant power (FRAP) activity (570.19 µmol FeSO4 equiv/mg) for the crushed counterpart and Chl-b in residues from boiling. No significant variation was evident in the total tannin content (TTC). Among filtrates, higher total phenolic content (773.29 mg GAE g-1 dry weight), TFC (392.77 mg QE g-1 dry weight), TTC (22.51 mg TAE g-1), and antioxidant activity as DPPH activity (66.46%) and FRAP (891.62 µmol FeSO4 equiv/mg) were evident for boiling, while that from crushing exhibited the highest TCC (1997.38 µg/g DM). LC-ESI-QTOF-MS/MS analysis identified 72 phenolic compounds with the maximum in residue (33) and filtrate (33) from freeze-thawing, followed by crushing (18 and 19) and boiling (14 and 13) in order, respectively. The results indicated that the predrying cell rupturing method significantly impacted quantitative, as well as qualitative compositions of residues and filtrates from fresh wolffia.

Emergence of multidrug resistant, ctx negative seventh pandemic Vibrio cholerae O1 El Tor sequence type (ST) 69 in coastal water of Kerala, India.

Seventh pandemic Vibrio choleare O1 El Tor strain is responsible for the on-going pandemic outbreak of cholera globally. This strain evolved from non-pathogenic V. cholerae by acquiring seventh pandemic gene (VC 2346), pandemic Islands (VSP1 and VSP2), pathogenicity islands (VP1 and VP2) and CTX prophage region. The cholera toxin production is mainly attributed to the presence of ctx gene in these strains. However, several variants of this strain emerged as hybrid strains or atypical strains. The present study aimed to assess the aquatic environment of Cochin, India, over a period of 5 years for the emergence of multidrug resistant V. cholerae and its similarity with seventh pandemic strain. The continuous surveillance and monitoring resulted in the isolation of ctx negative, O1 positive V. cholerae isolate (VC6) from coastal water, Cochin, Kerala. The isolate possessed the biotype specific O1 El Tor tcpA gene and lacked other biotype specific ctx, zot, ace and rst genes. Whole genome analysis revealed the isolate belongs to pandemic sequence type (ST) 69 with the possession of pandemic VC2346 gene, pathogenic island VPI1, VPI2, and pandemic island VSP1 and VSP2. The isolate possessed several insertion sequences and the SXT/R391 family related Integrative Conjugative Elements (ICEs). In addition to this, the isolate genome carried virulence genes such as VgrG, mshA, ompT, toxR, ompU, rtxA, als, VasX, makA, and hlyA and antimicrobial resistance genes such as gyrA, dfrA1, strB, parE, sul2, parC, strA, VC1786ICE9-floR, and catB9. Moreover, the phylogenetic analysis suggests that the isolate genome is more closely related to seventh pandemic V. cholerae O1 N16961 strain. This study reports the first incidence of environmental ctx negative seventh pandemic V. choleare O1 El Tor isolate, globally and its presence in the aquatic system likely to induce toxicity in terms of public health point of view. The presence of this isolate in the aquatic environment warns the strict implementation of the epidemiological surveillance on the occurrence of emerging strains and the execution of flagship program for the judicious use of antibiotics in the aquatic ecosystem.

Complexities in viral replication strategies as a potential explanation for prevalence of asymptomatic carriers in Covid-19 infections: analytical observation on SARS-Cov2 genome characteristics.

Analytical observations (in silico) indicate molecular features of SARS-Cov2 genome that potentially explains the high prevalence of asymptomatic cases in Covid-19 pandemic. We observed that the virus maintains a low preference for 'GGG' codon for glycine (3%) in its genome. We also observed multiple putative introns of 26-44 nucleotide (nt) length in the genomic region between the coding regions of Nsp10 and RPol in the viral ORF1ab, like several other beta-coronaviruses of similar infectivity levels. It appears that the virus employs a dual strategy to ensure unhindered replication within the host. One of the strategies employ a (- )1 frameshift translation event through programmed ribosomal slippage at the ribosomal slippage site in the ORF1ab. The alternate strategy relies on intron excision to generate a read through frame. The presence of 'GGG' in this conserved ribosomal slippage site ensures adequate tRNA in cytoplasm to match the codon, implying no additional frameshift translation due to ribosomal stalling. With fewer replication events, viral load remains low and resulting in asymptomatic cases. We suggest that this strategy is the primary reason for the prevalence of asymptomatic cases in the disease, enabling the virus to spread rapidly.

Molecular Mechanisms of Natural Carotenoid-based Pigmentation of Queen Loach, Botia dario (Hamilton, 1822) Under Captive Condition.

The genetic basis and expression patterns of key genes are important aspects of study to understand the colouration. This trait differs between wild and domesticated fish which is a matter of research. Botia dario is an indigenous fish, having ornamental and aesthetic value, which shows faded appearance in terms of colour in domesticated condition than wild. In the present study the carotenoid-fed B. dario were examined through incorporation of marigold petal meal in the diets at the rate of 5, 10 and 15% w/w along with wild fish. The carotenoid content of tissues that is skin, muscle and intestine along with intensity of colouration increased in a dose dependant manner of carotenoid in the diet. Important carotenoid-based colouration genes that is csf1r, BCDO2, SR-B1, MLN64, STAR5, GSTA2 and PLIN2 were characterized in the fish, to find out their role in fish pigmentation. The significant difference (p < 0.05) in the expression of these genes in different tissues, when compared among carotenoid-fed domesticated and wild fish, revealed the mechanism responsible for faded colouration and also revealed the means to enhance colour in the fish.

Diagnosis of mitogenome for robust phylogeny: A case of Cypriniformes fish group.

Phylogenetic tree using mitochondrial genes and nuclear genes have long been used for augmenting biological classification and understanding evolutionary processes in different lineage of life. But a basic question still exists for finding the most suitable gene for constructing robust phylogenetic tree. Much of the controversy appears due to monophyletic, paraphyletic and polyphyletic clade making deviations from original taxonomy. In the present study we report the first complete mitochondrial genome (mitogenome) of queen loach, generated through next-generation sequencing methods. The assembled mitogenome is a 16,492 bp circular DNA, comprising of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. Further in this study we also investigated the suitability of different mitochondrial region for phylogenetic analysis in Cyprinidae and loach group. For this genetic tree were constructed on COI, COII, COIII, 16S rRNA, 12S rRNA, Cyt b, ATPase 6, D-loop, ND1, ND2, ND3, ND4, ND5, and ND6 along with complete mitogenome. The complete mitogenome based phylogenetic tree got inclusive support from available classical taxonomy for these groups. On individual gene basis Cyt b, 12S rRNA, ND2 and ND3 also produced perfect clade at family and subfamily level. For rest of the genes polyphyly were observed for the fishes belonging to same family or subfamily which makes their use questionable for phylogenetic tree construction.

Silencing and augmentation of IAG hormone transcripts in adult Macrobrachiumrosenbergii males affects morphotype transformation.

Morphotypic differentiation is the external manifestation of dominance hierarchy in Macrobrachium rosenbergii The intermediate morphotype orange claw (OC) male exhibits the highest growth rate and is subordinate in hierarchy to blue claw (BC) male while dominant on small male (SM). The present study was undertaken to examine the specific role of insulin-like androgenic gland (iag) hormone in morphotype differentiation of M. rosenbergii To achieve this, RNAi mediated knockdown as well as augmentation of iag transcripts were effected in ∼60 g OC males using plasmid-based constructs pcD-IAG-lh and pcD-IAGorf, respectively. The treatments were administered to animals maintained in isolation as well as in community. The knockdown plasmid construct that expresses iag-specific long hairpin RNA caused 16-fold reduction of iag transcripts in the SSN1 cell line in vitro When injected into OC males living in a community, 2.3-fold iag knockdown was recorded, while in isolated OC males it was 4.2-fold initially, but returned to normal subsequently. Compared with the respective controls, OC to BC transformations in the iag silenced animals were significantly lower in the community-reared group, while no difference was observed in the isolated animals. It is reported here for the first time that iag augmentation in OC males resulted in significantly higher OC to BC transformations, when animals were reared in community. This plasmid-based IAG knockdown approach could be developed into a low stress, feed or immersion treatment for controlling heterogeneous individual growth of M. rosenbergii males in aquaculture.

Analysis of variance, normal quantile-quantile correlation and effective expression support of pooled expression ratio of reference genes for defining expression stability.

Identification of a reference gene unaffected by the experimental conditions is obligatory for accurate measurement of gene expression through relative quantification. Most existing methods directly analyze variability in crossing point (Cp) values of reference genes and fail to account for template-independent factors that affect Cp values in their estimates. We describe the use of three simple statistical methods namely analysis of variance (ANOVA), normal quantile-quantile correlation (NQQC) and effective expression support (EES), on pooled expression ratios of reference genes in a panel to overcome this issue. The pooling of expression ratios across the genes in the panel nullify the sample specific effects uniformly affecting all genes that are falsely reflected as instability. Our methods also offer the flexibility to include sample specific PCR efficiencies in estimations, when available, for improved accuracy. Additionally, we describe a correction factor from the ANOVA method to correct the relative fold change of a target gene if no truly stable reference gene could be found in the analyzed panel. The analysis is described on a synthetic data set to simplify the explanation of the statistical treatment of data.

A GFP-based bacterial biosensor with chromosomally integrated sensing cassette for quantitative detection of Hg(II) in environment.

A mercury biosensor was constructed by integrating biosensor genetic elements into E. coli JM109 chromosome in a single copy number, using the attP/attB recombination mechanism of lambda phage. The genetic elements used include a regulatory protein gene (merR) along with operator/promoter (O/P) derived from the mercury resistance operon from pDU1358 plasmid of Serratia marcescens. The expression of reporter gene gfp is also controlled by merR/O/P. Integration of the construct into the chromosome was done to increase the stability and precision of the biosensor. This biosensor could detect Hg(II) ions in the concentration range of 100-1700 nmol/L, and manifest the result as the expression of GFP. The GFP expression was significantly different (P < or = 0.05) for each concentration of inducing Hg(II) ions in the detection range, which reduces the chances of misinterpretation of results. A model using regression method was also derived for the quantification of the concentration of Hg(II) in water samples.

High dietary protein combats the stress of Labeo rohita fingerlings exposed to heat shock.

The amelioration effect of high dietary protein against stress was evaluated in Labeo rohita fingerlings, exposed to heat shock. Two hundred and forty fingerlings (6.57 ± 0.04 g, average weight ± SE) were randomly distributed into 4 treatment groups, each with 4 replicates was fed with either of four diets containing different levels of protein (20, 30, 40 or 45%). Water temperatures of all the treatments were within the range of 25.5-26.5°C throughout the experimental period of 30 days. After 30 days of feeding, fish were given heat shock by exposing to 38°C for 2 h. Heat shock significantly decreased (P < 0.05) liver glycogen content in treatment groups fed with 20 and 30% dietary protein, whereas unaffected in the 40 and 45% protein-fed groups. Heat shock significantly increased (P < 0.05) serum glucose and cortisol level in all the treatments. The 40 and 45% dietary protein-fed groups registered significantly higher survival (%) after the heat shock compared with their lower-protein counterparts. Heat shock increased the glycolytic, gluconeogenic, protein metabolic and antioxidative enzymes to cope up with thermal stress. Our results indicate that high-protein diet (≥40%) combats the stress due to heat shock in Labeo rohita.