Thymic size correlates with cord blood zinc levels in low-birth-weight newborns.

UNLABELLED: Thymus is essential for immunity as it provides environment for T cell differentiation and maturation. There is limited information on various factors which determine thymic size at birth. We studied the influence of cord blood zinc and copper levels and maternal and neonatal nutritional status on thymic size in term low-birth-weight (LBW) newborns. A prospective observational study on 44 term LBW (<2,500 g) newborns (cases) and 71 gestational age-matched newborns weighing ≥2,500 g (controls). Sonographically determined thymic index was correlated to cord blood zinc and copper levels and maternal and neonatal nutritional status. Thymic index measured 3.74 ± 1.57 cm(3) in LBW newborns compared to 4.90 ± 2.33 cm(3) in normal-birth-weight newborns. Thymic index was significantly correlated to cord blood zinc levels but not to cord blood copper levels and had linear relationship to the maternal body mass index and midarm circumference and neonatal anthropometric parameters. CONCLUSION: Thymic index is linearly related to cord blood zinc levels and maternal and neonatal nutritional status. Compared to thymic size in the Western newborns, the thymus is less than half in size in Indian newborns of normal birth weight. Reduced thymic size in Indian newborns in general and LBW infants in particular may have consequences for their immune competence and the risk of infections. Improving nutrition of pregnant women, particularly zinc nutriture might favorably influence thymic size in their offspring.

Assessment of microcystin concentration in carp and catfish: a case study from Lakshmikund pond, Varanasi, India.

The present study was conducted to analyse microcystin concentrations in Lakshmikund pond, Varanasi, India, as well as in carp and catfish of the pond. The concentrations of microcystin were found well above the WHO guidelines (1 µg/L) both for the dissolved and particulate fractions of bloom samples. The microcystin concentrations in different organs of carp and catfish were in the following sequence; liver > gut > kidney > gall bladder > gills > muscles and gut > liver > kidney > gall bladder > gills > muscles, respectively. The bioaccumulation of microcystin in carp and catfish was negatively correlated with body weight, and showed species specificity. The higher bioaccumulation of microcystin in muscles of catfish (>tenfold) over carp indicates a possible threat to human beings on consumption of catfish. Therefore, to avoid animal and human intoxication, routine analyses of microcystin in pond water as well as fishes are strongly recommended.

Monitoring approaches for a toxic cyanobacterial bloom.

Cyanobacterial blooms, dominated by Microcystis sp. and associated microcystin variants, have been implicated in illnesses of humans and animals. Little is known regarding the formation of blooms and the presence of cyanotoxin variants in water bodies. Furthermore, the role played by ecological parameters, in regulating Microcystis blooms is complicate and diverse. Local authorities responsible for water management are often faced with the challenging task of dealing with cyanobacterial blooms. Therefore, the development of suitable monitoring approaches to characterize cyanobacterial blooms is an important goal. Currently, various biological, biochemical and physicochemical methods/approaches are being used to monitor cyanobacterial blooms and detect microcystins in freshwater bodies. Because these methods can vary as to the information they provide, no single approach seemed to be sufficient to accurately monitor blooms. For example, immunosensors are more suited for monitoring the presence of toxins in clear water bodies while molecular methods are more suited to detect potentially toxic strains. Thus, monitoring approaches should be tailored for specific water bodies using methods based on economic feasibility, speed, sensitivity and field applicability. This review critically evaluates monitoring approaches that are applicable to cyanobacterial blooms, especially those that focus on the presence of Microcystis, in freshwater bodies. Further, they were characterized and ranked according to their cost, speed, sensitivity and selectivity. Suggested improvements were offered as well as future research endeavors to accommodate anticipated environmental changes.

Desiccation induced changes in osmolytes production and the antioxidative defence in the cyanobacterium Anabaena sp. PCC 7120.

Cells of Anabaena sp. PCC 7120, a low desiccation tolerant cyanobacterium, was subjected to prolonged desiccation and effect of loss of water was examined on production of osmolytes, and antioxidant response as well as on overall viability in terms of photosynthetic activity. During dehydration (22 h), the organism maintained about 98.5 % loss of cellular water, yet cells remained viable as about 30 % of photosynthetic O2-evolution activity resumed upon hydrating (1 h) such cells. In desiccated state, cyanobacterial cells accumulated osmolytes within 1 h though their contents decreased thereafter. The highest levels of trehalose (179 nmol mg(-1) protein), sucrose (805 nmol mg(-1) protein) and proline (23.2 nmol mg(-1) protein) were attained within 1 h. Chlorophyll a and carotenoid contents also increased within 1 h but phycocyanin level showed opposite trend. The oxygen-evolving activity declined in desiccated cyanobacterial biomass while rehydration led to instant recovery, indicating that cells protect the photosynthetic machinery against desiccation. Notwithstanding, activities of antioxidant enzymes (catalase, peroxidase and superoxide dismutase) attained their peaks after 3 h of desiccation, though within 10 min of rehydration, their levels returned back close to basal activities of the cultured cells. We propose that onset of osmolyte production in conjunction with upshift of antioxidant enzymes apparently protects the cyanobacterial cells from desiccation stress.

Proline biosynthesizing enzymes (glutamate 5-kinase and pyrroline-5-carboxylate reductase) from a model cyanobacterium for desiccation tolerance.

Drought is the most important abiotic stress, challenging sustainable agriculture globally. For desiccation being the multigenic trait, a combination of identified genes from the appropriate organism may render crop tolerant to the water stress. Among the compatible solutes, proline plays multifaceted role in counteracting such stress. The genes encoding proline biosynthesizing enzymes, glutamate 5-kinase (G5K), and pyrroline-5-carboxylate reductase (P5CR) from the low-desiccation-tolerant cyanobacterium Anabaena sp. PCC 7120, were cloned and overexpressed in Escherichia coli BL21(DE3) individually. The recombinant E. coli cells harboring G5K, failed to exhibit enhanced desiccation tolerance relative to those with P5CR that showed increased growth/survival over the wild type. This may be ascribed to the overexpression of the reductase gene. Multiple sequence alignment showed P5CR to be conserved in all the organisms. We hypothesize that P5CR gene from high-desiccation-tolerant cyanobacteria may be adopted as the candidate for making transgenic N2-fixing cyanobacterium for paddy fields and/or crop development in future.

In silico and in vitro evaluation of extract derived from Dunaliella salina, a halotolerant microalga for its antifungal and antibacterial activity.

In the present study little explored halotolerant wall-less green alga Dunaliella salina was found to be a potent source of antibacterial and antifungal biomolecules. Both the target pathogens, bacteria (Escherischia coli, Klebsiella pneumoniae, and Acinetobacter baumannii) and fungi (Candida albicans, C. tropicalis, and Cryptococus sp.) were WHO prioritized. The bioassay guided approach led us to evaluate antibacterial and antifungal lead molecule(s) from an array of compounds using spectroscopic and in silico studies. The methanol derived crude extract was purified via thin layer chromatography (TLC) using solvent system methanol: chloroform (1:19). Maximum antimicrobial activity was observed in fractions D5, D6 and D7, the components of which were then recognized using high resolution-liquid chromatography/mass spectroscopy (Orbitrap) (HR-LC/MS). The screened compounds were then docked with target enzymes sterol-14-alpha demethylase and OmpF porin protein. The energy scores revealed that amongst all, lariciresinol-4-O-glucoside showed better binding affinity, in silico, using the Schrödinger Maestro 2018-1 platform. The 3-dimensional crystal structures of both the proteins were retrieved from the protein data bank (PDB), and showed binding energies of -14.35 kcal/mol, and -11.0 kcal/mol against respective drug targets. The molecular dynamics (MD) simulations were performed for 100 ns, using Desmond package, Schrödinger to evaluate the conformational stability and alteration of protein-ligand complexes during the simulation. Thus, our findings confirmed that lariciresinol-4-O-glucoside, a lignan derivative and known strong antioxidant, may be used as an important "lead" molecule to be developed as antibacterial and antifungal drugs in the future.Communicated by Ramaswamy H. Sarma.

Variation in the LRR region of Pi54 protein alters its interaction with the AvrPi54 protein revealed by in silico analysis.

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae is a destructive disease of rice and responsible for causing extensive damage to the crop. Pi54, a dominant blast resistance gene cloned from rice line Tetep, imparts a broad spectrum resistance against various M. oryzae isolates. Many of its alleles have been explored from wild Oryza species and landraces whose sequences are available in the public domain. Its cognate effector gene AvrPi54 has also been cloned from M. oryzae. Complying with the Flor's gene-for-gene system, Pi54 protein interacts with AvrPi54 protein following fungal invasion leading to the resistance responses in rice cell that prevents the disease development. In the present study Pi54 alleles from 72 rice lines were used to understand the interaction of Pi54 (R) proteins with AvrPi54 (Avr) protein. The physiochemical properties of these proteins varied due to the nucleotide level polymorphism. The ab initio tertiary structures of these R- and Avr- proteins were generated and subjected to the in silico interaction. In this interaction, the residues in the LRR region of R- proteins were shown to interact with the Avr protein. These R proteins were found to have variable strengths of binding due to the differential spatial arrangements of their amino acid residues. Additionally, molecular dynamic simulations were performed for the protein pairs that showed stronger interaction than Pi54tetep (original Pi54 from Tetep) protein. We found these proteins were forming h-bond during simulation which indicated an effective binding. The root mean square deviation values and potential energy values were stable during simulation which validated the docking results. From the interaction studies and the molecular dynamics simulations, we concluded that the AvrPi54 protein interacts directly with the resistant Pi54 proteins through the LRR region of Pi54 proteins. Some of the Pi54 proteins from the landraces namely Casebatta, Tadukan, Varun dhan, Govind, Acharmita, HPR-2083, Budda, Jatto, MTU-4870, Dobeja-1, CN-1789, Indira sona, Kulanji pille and Motebangarkaddi cultivars show stronger binding with the AvrPi54 protein, thus these alleles can be effectively used for the rice blast resistance breeding program in future.

Role of Ca2+ as protectant under heat stress by regulation of photosynthesis and membrane saturation in Anabaena PCC 7120.

The present study was aimed at understanding the effects of heat stress on selected physiological and biochemical parameters of a model cyanobacterium, Anabaena PCC 7120 in addition to amelioration strategy using exogenous Ca2+. A comparison of the cells exposed to heat stress (0-24 h) in the presence or absence of Ca2+ clearly showed reduction in colony-forming ability and increase in reactive oxygen species (ROS) leading to loss in the viability of cells of Ca2+-deficient cultures. There was higher level of saturation in membrane lipids of the cells supplemented with Ca2+ along with higher accumulation of proline. Similarly, higher quantum yield (7.8-fold) in Ca2+-supplemented cultures indicated role of Ca2+ in regulation of photosynthesis. Relative electron transport rate (rETR) decreased in both the sets with the difference in the rate of decrease (slow) in Ca2+-supplemented cultures. The Ca2+-supplemented sets also maintained high levels of open reaction centers of PS II in comparison to Ca2+-deprived cells. Increase in transcripts of both subunits ((rbcL and rbcS) of RubisCO from Ca2+-supplemented Anabaena cultures pointed out the role of Ca2+ in sustenance of photosynthesis of cells via CO2 fixation, thus, playing an important role in maintaining metabolic status of the heat-stressed cyanobacterium.

Mass cultivation of Dunaliella salina in a flat plate photobioreactor and its effective harvesting.

Mass cultivation of Dunaliella salina was standardized in a flat plate photobioreactor followed by a vertical flat plate photobioreactor. Maximum biomass productivity (14.95 ±â€¯0.43 mgL-1d-1 dry cell weight) was achieved in the latter at inoculum concentration of OD680nm = 0.1, 100 µmolm-2s-1 light illumination and 1.0 L min-1 aeration. Semicontinuous operation with varying KNO3 and NaHCO3 concentrations resulted highest biomass productivity (17.85 ±â€¯0.55 mgL-1d-1) at 0.50 mM NaHCO3 and 15 mM KNO3. However, maximum lipid (16.36 ±â€¯1.18% dry cell weight) was achieved at 0.75 mM NaHCO3 and 10 mM KNO3. Flocculation studies employing potash alum, FeCl3·6H2O or pH showed harvesting efficiencies exceeding 90% in 0.75 mM potash alum or FeCl3·6H2O or pH 11, but they yielded low concentration factor (<5) and were detrimental (Fv/Fm < 0.50). A combination of 0.50 mM FeCl3·6H2O and pH 9 was found as most suitable flocculating strategy with maximum concentration factor (>14) and least damaging (Fv/Fm > 0.54).

Role of calcium in the mitigation of heat stress in the cyanobacterium Anabaena PCC 7120.

The effects of exogenously added CaCl2 (0.25mM) on photopigments, photosynthetic O2-evolution, antioxidative enzyme activity, membrane damage, expression of two heat shock genes (groEL and groES) and apoptotic features in Anabaena 7120 under heat stress (45°C) for up to 24h were investigated. Heat stress lowered the level of photopigments; however, Ca2+--supplemented cultures showed a low level reduction in Chl a but induced accumulation of carotenoids and phycocyanin under heat stress. Photosynthetic O2-evolving capacity was maintained at a higher level in cells from Ca2+-supplemented medium. Among the antioxidative enzymes, superoxide dismutase activity was unaffected by the presence or absence of Ca2+ in contrast to increases in catalase, ascorbate peroxidase and glutathione reductase activities in cells grown in Ca2+-supplemented medium. Lower levels of lipid peroxidation were recorded in Anabaena cells grown in Ca2+-supplemented medium in comparison to cells from Ca2+--deprived medium. Target cells grown in Ca2+-deprived medium developed apoptotic features in the early stages of heat shock, while Ca2+ application seemed to interfere with apoptosis because only a few cells showed such features after 24 h of heat exposure, indicating a role for Ca2+ in maintaining cell viability under heat stress. There was also continuous up regulation of two important heat shock genes (groEL and groES) in Ca2+-supplemented cultures, exposed to heat shock, again indicating a role for Ca2+ in stress management.

Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes.

Roles of nutrients and other environmental variables in development of cyanobacterial bloom and its toxicity are complex and not well understood. We have monitored the photoautotrophic growth, total microcystin concentration, and microcystins synthetase gene (mcyA) expression in lab-grown strains of Microcystis NIES 843 (reference strain), KW (Wangsong Reservoir, South Korea), and Durgakund (Varanasi, India) under different nutrient regimes (nitrogen, phosphorus, and boron). Higher level of nitrogen and boron resulted in increased growth (avg. 5 and 6.5 Chl a mg/L, resp.), total microcystin concentrations (avg. 1.185 and 7.153 mg/L, resp.), and mcyA transcript but its expression was not directly correlated with total microcystin concentrations in the target strains. Interestingly, Durgakund strain had much lower microcystin content and lacked microcystin-YR variant over NIES 843 and KW. It is inferred that microcystin concentration and its variants are strain specific. We have also examined the heterotrophic bacteria associated with cyanobacterial bloom in Durgakund Pond and Wangsong Reservoir which were found to be enriched in Alpha-, Beta-, and Gammaproteobacteria and that could influence the bloom dynamics.

Status, alert system, and prediction of cyanobacterial bloom in South Korea.

Bloom-forming freshwater cyanobacterial genera pose a major ecological problem due to their ability to produce toxins and other bioactive compounds, which can have important implications in illnesses of humans and livestock. Cyanobacteria such as Microcystis, Anabaena, Oscillatoria, Phormidium, and Aphanizomenon species producing microcystins and anatoxin-a have been predominantly documented from most South Korean lakes and reservoirs. With the increase in frequency of such blooms, various monitoring approaches, treatment processes, and prediction models have been developed in due course. In this paper we review the field studies and current knowledge on toxin producing cyanobacterial species and ecological variables that regulate toxin production and bloom formation in major rivers (Han, Geum, Nakdong, and Yeongsan) and reservoirs in South Korea. In addition, development of new, fast, and high-throughput techniques for effective monitoring is also discussed with cyanobacterial bloom advisory practices, current management strategies, and their implications in South Korean freshwater bodies.

Temporal variations in microcystin-producing cells and microcystin concentrations in two fresh water ponds.

The relationship between microcystin production, microcystin-producing cyanobacteria, including Microcystis spp., and various biological and physicochemical parameters in Sankuldhara and Lakshmikund, situated in the same geographical area was studied over a period of 1.5 years. Seasonal variation in cyanobacterial 16S rRNA, Microcystis spp. 16S rRNA, mcyA and mcyB genes were quantitatively determined by real-time PCR. Microcystis was the dominant microcystin producer in both study sites constituting 67% and 97% of the total microcystin-producing cyanobacteria at Sankuldhara and Lakshmikund, respectively. Microcystin concentrations were 2.19-39.60 µg/L and 15.22-128.14 µg/L at Sankuldhara and Lakshmikund, respectively, as determined by LC-MS. Principal component analysis revealed a strong positive correlation between microcystin concentration and the copy number of mcyA and mcyB, chlorophyll a and cyanobacterial biomass at both sites. The higher microcystin concentrations in Lakshmikund pond were attributed to the high copy number of mcy genes present coupled with the pond's eutrophication status, as indicated by high total algal biomass, high chlorophyll a content, high nutrient load and low DO. Therefore, a significant difference in microcystin concentrations, correlating with these various biological and physicochemical parameters, confirms the importance of local environmental variables in the overall regulation of microcystins production.

Fresh Water Cyanobacteria Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 as an Anticancer Drug Resource.

An increasing number of cancer patients worldwide, especially in third world countries, have raised concern to explore natural drug resources, such as the less explored fresh water filamentous cyanobacteria. Six strains of cyanobacteria (Phormidium sp. CCC727, Geitlerinema sp. CCC728, Arthrospira sp. CCC729, Phormidium sp. CCC731, Phormidium sp. CCC730, and Leptolyngbya sp. CCC732) were isolated (paddy fields and ponds in the Banaras Hindu University, campus) and five strains screened for anticancer potential using human colon adenocarcinoma (HT29) and human kidney adenocarcinoma (A498) cancer cell lines. Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 were the most potent as determined by examination of morphological features and by inhibition of growth by graded concentrations of crude extracts and thin-layer chromatography (TLC) eluates. Cell cycle analysis and multiplex assays using cancer biomarkers also confirmed Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 as cancer drug resources. Apoptotic studies in the cells of A498 (cancer) and MCF-10A (normal human epithelial) exposed to crude extracts and TLC fractions revealed no significant impact on MCF-10A cells emphasizing its importance in the development of anticancer drug. Identification of biomolecules from these extracts are in progress.

Zinc supplementation for neonatal hyperbilirubinemia: a randomized controlled trial.

OBJECTIVE: To determine the efficacy of oral zinc for treatment of idiopathic neonatal hyperbilirubinemia in near-term and term (35-41 weeks) neonates. DESIGN: Randomized placebo-controlled trial. SETTING: Tertiary-care teaching hospital. PARTICIPANTS: Eighty newborns with idiopathic neonatal hyperbilirubinemia. INTERVENTION: Neonates were randomized to receive either oral zinc sulfate (10 mg/d) or placebo for 7 days. MAIN OUTCOME MEASURES: Primary: total serum bilirubin levels at 48 (±12) h, 96 (±12) h and 144 (±12) h after intervention. Secondary: duration of phototherapy, and serum zinc and copper levels. RESULTS: Baseline mean (SD) total serum bilirubin levels were 14.8 (3.8) and 14.4 (3.5) mg/dL in zinc and placebo groups, respectively. No significant differences were observed in total bilirubin levels between the two groups after the intervention. Mean (SD) total serum bilirubin levels in zinc and placebo groups were 13.9 (2.5) vs. 13.4 (1.9) mg/dL (mean difference 0.566; 95% CI -0.535, 1.668, P=0.038) at 48 h, 13.1 (2.7) vs. 12.8 (2.3) mg/dL (mean difference 0.234; 95% CI -1.011, 1.479, P =0.708) at 96 h and 8.0 (2.0) vs. 8.6 (1.2) mg/dL (mean difference -0.569, 95% CI -1.382, 0.242, P=0.166) at 144 h. Although the mean duration of phototherapy in the zinc group was less by 21.3 h (95% CI 11.6, 30.9, P=0.052), the difference was not significant. Post-intervention, serum zinc levels were significantly higher in the zinc-supplemented group while serum copper levels were comparable between the two groups. CONCLUSIONS: Oral zinc sulfate, in a dose of 10 mg/day, is not effective in the management of idiopathic neonatal hyperbilirubinemia.

Cyanobacteria, Lyngbya aestuarii and Aphanothece bullosa as antifungal and antileishmanial drug resources.

OBJECTIVE: To investigate two cyanobacteria isolated from different origins i.e. Lyngbya aestuarii (L. aestuarii) from brackish water and Aphanothece bullosa (A. bullosa) from fresh water paddy fields for antifungal and antileishmanila activity taking Candida albicans and Leishmania donovain as targets. METHODS: Biomass of L. aestuarii and A. bullosa were harvested after 40 and 60 d respectively and lyophilized twice in methanol (100%) and redissolved in methanol (5%) for bioassay. Antifungal bioassay was done by agar well diffusion method while antileishmanial, by counting cell numbers and flageller motility observation of promastigotes and amastigotes from L. donovani. Fluconazole and 5% methanol were used as control. RESULTS: Both the cyanobacteria were found to be potent source of antifungal activity keeping fluconazole as positive control, however, methanolic crude extract (15 mg/mL) of A. bullosa was found more potent (larger inhibition zone) over that of methanolic crude extract of L. aestuarii. Similarly antileishmanial activity of crude extract (24.0 mg/mL) of A. bullosa was superior over that of methanolic crude extract of L. aestuarii (25.6 mg/mL). CONCLUSIONS: Antifungal and antileishmanial drugs are still limited in the market. Screening of microbes possessing antifungal and antileishmanial activity drug is of prime importance. Cyanobacteria are little explored in this context because most of the drugs in human therapy are derived from microorganisms, mainly bacterial, fungal and actinomycetes. Thus in the present study two cyanobacterial strains from different origins showed potent source of antifungal and antileishmanial biomolecules.

Recent trends in development of biosensors for detection of microcystin.

Increased cyanobacterial blooms, a source of cyanotoxins are linked with climate change and eutrophication in aquatic bodies, a major concern worldwide. Microcystins are potently hepatotoxic, nephrotoxic as well as carcinogenic. Thus microcystins are threat to tourism, agriculture and animal's health. However, there is a still lacuna in the knowledge of regulation of microcystins production. Presence of toxic and non-toxic cyanobacterial strains together and occurrence of various microcystin variants in aquatic bodies compounded the problem. Although several analytical techniques for microcystin detection such as bioassay, ELISA, HPLC and LC-MS etc. have been already prevalent, the development of biosensors offered rapid and accurate detection, high reproducibility and portability. Sequencing of Microcystis spp., opened the new vistas towards the development of biosensor at molecular and genetic level. This review incorporates the current trends in the development of biosensors for microcystin detection in the light of state-of-the-art techniques.

Dynamics of microcystin production and quantification of potentially toxigenic Microcystis sp. using real-time PCR.

Cyanobacterial blooms in eutrophied water body are generally composed of various genotypes with or without microcystin-producing genes (mcy gene cluster). Thus there is a need for quantification of potent toxin producing strains. The present study aimed at identifying microcystin variants and its producer strains in Durgakund pond, Varanasi, India, based on quantification of cpcBA-IGS and mcyA (condensation domain) genes using real-time PCR and LC-MS. Increase in microcystin concentrations was correlated with increase in mcyA copy number and the level of pigments (chlorophyll a, phycocyanin and carotenoids). Also, selected environmental factors (water temperature, light irradiance, rainfall, pH, N and P) and the concentration of microcystin variants (MC-LR, -RR and -YR) were also assessed in samples during May 2010 to April 2011 to establish the possible correlation among these parameters. Nutrients favored cyanobacterial bloom but it could not be correlated with the levels of microcystin variants and seemed to be geographically specific. Microcystis sp. dominant in the pond comprised potentially toxigenic cells. The ratio of potentially toxigenic Microcystis sp. to that of total Microcystis sp. ranged from 0% to 14%. Such studies paved the way to identify and quantify the most potent microcystin producer in the tropical aquatic body.