Microalgae in the Southwest Coast of India

Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology

Seawater carbonate chemistry, cell numbers and growth rate during experiments with dinoflagellates, 2007

The effects of dissolved inorganic carbon (DIC) on the growth of 3 red-tide dinoflagellates (Ceratium lineatum, Heterocapsa triquetra and Prorocentrum minimum) were studied at pH 8.0 and at higher pH levels, depending upon the pH tolerance of the individual species. The higher pH levels chosen for experiments were 8.55 for C. lineatum and 9.2 for the other 2 species. At pH 8.0, which approximates the pH found in the open sea, the maximum growth in all species was maintained until the total DIC concentration was reduced below ~0.4 and 0.2 mM for C. lineatum and the other 2 species, respectively. Growth compensation points (concentration of inorganic carbon needed for maintenance of cells) were reached at ~0.18 and 0.05 mM DIC for C. lineatum and the other 2 species, respectively. At higher pH levels, maximum growth rates were lower compared to growth at pH 8, even at very high DIC concentrations, indicating a direct pH effect on growth. Moreover, the concentration of bio-available inorganic carbon (CO2 + HCO3-) required for maintenance as well as the half-saturation constants were increased considerably at high pH compared to pH 8.0. Experiments with pH-drift were carried out at initial concentrations of 2.4 and 1.2 mM DIC to test whether pH or DIC was the main limiting factor at a natural range of DIC. Independent of the initial DIC concentrations, growth rates were similar in both incubations until pH had increased considerably. The results of this study demonstrated that growth of the 3 species was mainly limited by pH, while inorganic carbon limitation played a minor role only at very high pH levels and low initial DIC concentrations.

Seawater carbonate chemistry and cell numbers during experiments with dinoflagellates, 2007

The effects of dissolved inorganic carbon (DIC) on the growth of 3 red-tide dinoflagellates (Ceratium lineatum, Heterocapsa triquetra and Prorocentrum minimum) were studied at pH 8.0 and at higher pH levels, depending upon the pH tolerance of the individual species. The higher pH levels chosen for experiments were 8.55 for C. lineatum and 9.2 for the other 2 species. At pH 8.0, which approximates the pH found in the open sea, the maximum growth in all species was maintained until the total DIC concentration was reduced below ~0.4 and 0.2 mM for C. lineatum and the other 2 species, respectively. Growth compensation points (concentration of inorganic carbon needed for maintenance of cells) were reached at ~0.18 and 0.05 mM DIC for C. lineatum and the other 2 species, respectively. At higher pH levels, maximum growth rates were lower compared to growth at pH 8, even at very high DIC concentrations, indicating a direct pH effect on growth. Moreover, the concentration of bio-available inorganic carbon (CO2 + HCO3-) required for maintenance as well as the half-saturation constants were increased considerably at high pH compared to pH 8.0. Experiments with pH-drift were carried out at initial concentrations of 2.4 and 1.2 mM DIC to test whether pH or DIC was the main limiting factor at a natural range of DIC. Independent of the initial DIC concentrations, growth rates were similar in both incubations until pH had increased considerably. The results of this study demonstrated that growth of the 3 species was mainly limited by pH, while inorganic carbon limitation played a minor role only at very high pH levels and low initial DIC concentrations.

Ecologia do fitoplâncton impactado por cultivo de tilápia do Nilo, oreochromis niloticus, em tanques-rede no açude Ministro João Alves, Parelhas, Rio Grande do Norte

The dams are limnic ecosystems of great importance for its multiple uses, among them, water supply for the public and to culture of artisanal fish are most relevant. The aim of the present study is to evaluate the physical-chemical characteristics and the phytoplankton community in two chosen sites (Point 1 littoral zone of point source; Point 2 pelagic zone of non-point source) of the Minister João Alves dam, which is also known as Boqueirão de Parelhas/RN. This represents the spatial distribution of the phytoplankton species in order to understand any possible alterations of the water quality and the phytoplankton composition in relation to the water quality originating from the impact of the tilapia, Oreochromis niloticus, culture. The study period also encompasses temporal variations exhibited in two seasons of an annual cycle, one during the dry season (Oct, Nov and Dec of 2008 and Jan of 2009), and the other rainy season (Mar, Apr, May and June of 2008) to extend the observation. The physicalchemical parameters, such as pH, temperature, electrical conductivity, concentration of dissolved oxygen were measured in situ and the values of the inorganic nutrients (nitrate, ammonium and orto-phosfato) and chlorophyll in the laboratory. The quali-quantitative analyses of the phytoplankton had been carried through sedimentation technique and the enumeration of the random of 400 cells, colonies and filaments counted using Sedgwick-Rafter counting chamber. The results of pH varied widely from the acidic to alkaline range with the minimum of 5.8 (± 0.8) and the maximum of 9.2 (± 0.7-0.8), at point 1 and 2. The dissolved oxygen content was higher in the rainy period than that in the dry period. The maximum electrical conductivity was of 1409 μScm-1 in point 1 and 431 minim of μScm-1, in point 2. There was a considerable alteration in the levels of inorganic nutrients such as nitrate-nitrogen, ammoniacal nitrogen and orthophosphate during the two cycles of study period. Phytoplankton assemblages presented a picture of alternate dominance among species Cyanobacteria, Bacillariophyceae and Chlorophyceae. The trophic state index diagnosed to the category of mesotrophic, which is based on the values of chlorophyll, total phosphorus and Secchi-disc measurements. The wind driven turbulence of the water column and the fresh inflow of water (flushing and dilution) during rainy season acted as constraint and did-not allow an exaggerated growth of the species of cyanobacteria. On the basis of the present we conclude that the culture of tilapias in cage-culture fails to produce pollution load that could compromise the quality of the water of the dam, probably be due to small dimension of the culture in relation to the size, volume of the water and the reservoir capacity support its own environment

Estudo comparativo da variação temporal da comunidade fitoplanctônica do açude Cruzeta inserido no bioma caatinga, Rio Grande do Norte

The Cruzeta impoundment, situated in the city of Cruzeta, hinterland of the state of the Rio Grande do Norte state has significant importance to the municipality as it represents the only source of supplying water to the region. It was hypothesized that the regional consequence of the global warming and the warming of surface water could substantially contribute the significant growth of the aquatic macrophytes in the years 2008-2009. The growth of these vegetation believed to be improved the degree of water transparency and as a consequence of the improved growth of phytoplankton species and chlorophyll biomass. At the same time the aquatic macrophyte could interact and compete potentially for the dissolved inorganic nutrients resources and the phytoplankton community. This study presents a comparison of years 2004-2005 when it did not have the expressive presence of the aquatic macrophyte community or restricted to the littoral region. In contrast, the years 2008-2009 showed a significant growth of the aquatic macrophyte in the Cruzeta/RN impoundments. The present study is an attempt to elucidate the significant presence of the aquatic macrophyte, Eichhornia crassipes, Ceratophyllum submersum, Nymphea sp and Pistia sp, and its interference on the ecology of phytoplankton. The samplings had been carried out from September of 2008 to April of 2009 and consistently between 10:00 h and 12:00 h with the aid of Van Dorn bottle and the plankton net of mesh size 20 Qm. The collections were made in three depths ie., surface, mid-column and bottom. The Physical-Chemical parameters such as pH, temperature, electrical conductivity and dissolved oxygen had been analyzed in situ. The samples for analysis of nutrients and chlorophyll were kept under refrigeration for posterior analysis in the laboratory. Phytoplankton samples were preserved in Lugol-iodine and kept for sedimentation for quali-quantitative analysis of phytoplankton. Enumeration of cells, colonies and filaments was done with the aid of Sedgwick-Rafter counting chamber and expressed as numbers/ml. Chlorophyll a was analyzed as a functional component of phytoplankton biomass and extracted with cold 90% acetone. The results indicate that the chlorophyll concentration varied between 5,65-8,08 Qg.L-1 for the dry period and 5,09-6,23 Qg.L-1 for the rainy period and showed considerable reduction when compared to the values to the 2004-2005 study period. The temperature was always presented higher in relation to the 2004-2005 study. Phytoplankton species showed a relative abundance of the Cyanophyceae for both the period of dry and rainy. The predominance species are filamentous Leptolymbya geophila Borzi (Planctolyngbya sp), Anabaena plankctônica Brunnthaler, Oscillatória limosa Ag. and Cylindrospermopsis raciborskii (Wolosz). The concentration of the nutrients such as nitrate and orto-phosfato had always presented higher values during the rainy period and the ammoniacal nitrogen retained moderate values in the dry period and a slight increase in rainy season. The main conclusions are the reduction of the concentration of chlorophyll, diversity of phytoplankton, and the increase in temperature and transparency of the water during the period of the study

Cytotoxicity of two novel formulations of calcium phosphate cements: A comparative in vitro study

The purpose was to evaluate the cytotoxicity of two novel formulations (alpha and beta) of calcium phosphate cements. Positive control, represented by a commercial hydroxyapatite cement, and negative control were included for comparative purposes. A continuous lineage of fibroblastic cells was used, and the effect of the tested materials on both cell proliferation and viability was assessed by counting cell number on hemocytometer and by the trypan blue exclusion test, respectively. Study design attempted to simulate clinical use by allowing direct and indirect contact of cells and cements. Results were analyzed by the Kruskal-Wallis test and indicated that the beta formulation was extremely cytotoxic (P < 0.001), because this material induced the greatest reduction on cell proliferation and viability. The alpha formulation behaved similarly to the positive control regarding its effect on cell proliferation and viability. Thus, it is concluded that alpha formulation has promise for further evaluation of its behavior in vivo.

Ecologia do fitoplâncton em áreas de cultivo de Tilápia do Nilo, Oreochromis niloticus, em tanques-rede no açude Ministro João Alves, Parelhas, Rio Grande do Norte

The dams are limnic ecosystems of great importance for its multiple uses, among them, water supply for the public and to culture of artisanal fish are most relevant. The aim of the present study is to evaluate the physical-chemical characteristics and the phytoplankton community in two chosen sites (Point 1 littoral zone of point source; Point 2 pelagic zone of non-point source) of the Minister João Alves dam, which is also known as Boqueirão de Parelhas/RN. This represents the spatial distribution of the phytoplankton species in order to understand any possible alterations of the water quality and the phytoplankton composition in relation to the water quality originating from the impact of the tilapia, Oreochromis niloticus, culture. The study period also encompasses temporal variations exhibited in two seasons of an annual cycle, one during the dry season (Oct, Nov and Dec of 2008 and Jan of 2009), and the other rainy season (Mar, Apr, May and June of 2008) to extend the observation. The physicalchemical parameters, such as pH, temperature, electrical conductivity, concentration of dissolved oxygen were measured in situ and the values of the inorganic nutrients (nitrate, ammonium and orto-phosfato) and chlorophyll in the laboratory. The quali-quantitative analyses of the phytoplankton had been carried through sedimentation technique and the enumeration of the random of 400 cells, colonies and filaments counted using Sedgwick-Rafter counting chamber. The results of pH varied widely from the acidic to alkaline range with the minimum of 5.8 (± 0.8) and the maximum of 9.2 (± 0.7-0.8), at point 1 and 2. The dissolved oxygen content was higher in the rainy period than that in the dry period. The maximum electrical conductivity was of 1409 μScm-1 in point 1 and 431 minim of μScm-1, in point 2. There was a considerable alteration in the levels of inorganic nutrients such as nitrate-nitrogen, ammoniacal nitrogen and orthophosphate during the two cycles of study period. Phytoplankton assemblages presented a picture of alternate dominance among species Cyanobacteria, Bacillariophyceae and Chlorophyceae. The trophic state index diagnosed to the category of mesotrophic, which is based on the values of chlorophyll, total phosphorus and Secchi-disc measurements. The wind driven turbulence of the water column and the fresh inflow of water (flushing and dilution) during rainy season acted as constraint and did-not allow an exaggerated growth of the species of cyanobacteria. On the basis of the present we conclude that the culture of tilapias in cage-culture fails to produce pollution load that could compromise the quality of the water of the dam, probably be due to small dimension of the culture in relation to the size, volume of the water and the reservoir capacity support its own environment

Long-term Spinal Ventral Root Reimplantation, But Not Bone Marrow Mononuclear Cell Treatment, Positively Influences Ultrastructural Synapse Recovery And Motor Axonal Regrowth.

We recently proposed a new surgical approach to treat ventral root avulsion, resulting in motoneuron protection. The present work combined such a surgical approach with bone marrow mononuclear cells (MC) therapy. Therefore, MC were added to the site of reimplantation. Female Lewis rats (seven weeks old) were subjected to unilateral ventral root avulsion (VRA) at L4, L5 and L6 levels and divided into the following groups (n = 5 for each group): Avulsion, sealant reimplanted roots and sealant reimplanted roots plus MC. After four weeks and 12 weeks post-surgery, the lumbar intumescences were processed by transmission electron microscopy, to analyze synaptic inputs to the repaired α motoneurons. Also, the ipsi and contralateral sciatic nerves were processed for axon counting and morphometry. The ultrastructural results indicated a significant preservation of inhibitory pre-synaptic boutons in the groups repaired with sealant alone and associated with MC therapy. Moreover, the average number of axons was higher in treated groups when compared to avulsion only. Complementary to the fiber counting, the morphometric analysis of axonal diameter and g ratio demonstrated that root reimplantation improved the motor component recovery. In conclusion, the data herein demonstrate that root reimplantation at the lesion site may be considered a therapeutic approach, following proximal lesions in the interface of central nervous system (CNS) and peripheral nervous system (PNS), and that MC therapy does not further improve the regenerative recovery, up to 12 weeks post lesion.

Mesenchymal Stem Cells Engrafted In A Fibrin Scaffold Stimulate Schwann Cell Reactivity And Axonal Regeneration Following Sciatic Nerve Tubulization.

The present study investigated the effectiveness of mesenchymal stem cells (MSCs) associated with a fibrin scaffold (FS) for the peripheral regenerative process after nerve tubulization. Adult female Lewis rats received a unilateral sciatic nerve transection followed by repair with a polycaprolactone (PCL)-based tubular prosthesis. Sixty days after injury, the regenerated nerves were studied by immunohistochemistry. Anti-p75NTR immunostaining was used to investigate the reactivity of the MSCs. Basal labeling, which was upregulated during the regenerative process, was detected in uninjured nerves and was significantly greater in the MSC-treated group. The presence of GFP-positive MSCs was detected in the nerves, indicating the long term survival of such cells. Moreover, there was co-localization between MSCs and BNDF immunoreactivity, showing a possible mechanism by which MSCs improve the reactivity of SCs. Myelinated axon counting and morphometric analyses showed that MSC engrafting led to a higher degree of fiber compaction combined with a trend of increased myelin sheath thickness, when compared with other groups. The functional result of MSC engrafting was that the animals showed higher motor function recovery at the seventh and eighth week after lesion. The findings herein show that MSC+FS therapy improves the nerve regeneration process by positively modulating the reactivity of SCs.

Rat Adipose Tissue-Derived Stem Cells Transplantation Attenuates Cardiac Dysfunction Post Infarction and Biopolymers Enhance Cell Retention

Background: Cardiac cell transplantation is compromised by low cell retention and poor graft viability. Here, the effects of co-injecting adipose tissue-derived stem cells (ASCs) with biopolymers on cell cardiac retention, ventricular morphometry and performance were evaluated in a rat model of myocardial infarction (MI). Methodology/Principal Findings: (99m)Tc-labeled ASCs (1 x 10(6) cells) isolated from isogenic Lewis rats were injected 24 hours post-MI using fibrin a, collagen (ASC/C), or culture medium (ASC/M) as vehicle, and cell body distribution was assessed 24 hours later by gamma-emission counting of harvested organs. ASC/F and ASC/C groups retained significantly more cells in the myocardium than ASC/M (13.8+/-2.0 and 26.8+/-2.4% vs. 4.8+/-0.7%, respectively). Then, morphometric and direct cardiac functional parameters were evaluated 4 weeks post-MI cell injection. Left ventricle (LV) perimeter and percentage of interstitial collagen in the spare myocardium were significantly attenuated in all ASC-treated groups compared to the non-treated (NT) and control groups (culture medium, fibrin, or collagen alone). Direct hemodynamic assessment under pharmacological stress showed that stroke volume (SV) and left ventricle end-diastolic pressure were preserved in ASC-treated groups regardless of the vehicle used to deliver ASCs. Stroke work (SW), a global index of cardiac function, improved in ASC/M while it normalized when biopolymers were co-injected with ASCs. A positive correlation was observed between cardiac ASCs retention and preservation of SV and improvement in SW post-MI under hemodynamic stress. Conclusions: We provided direct evidence that intramyocardial injection of ASCs mitigates the negative cardiac remodeling and preserves ventricular function post-MI in rats and these beneficial effects can be further enhanced by administrating co-injection of ASCs with biopolymers.

Cell Therapy Attenuates Cardiac Dysfunction Post Myocardial Infarction: Effect of Timing, Routes of Injection and a Fibrin Scaffold

Background: Cell therapy approaches for biologic cardiac repair hold great promises, although basic fundamental issues remain poorly understood. In the present study we examined the effects of timing and routes of administration of bone marrow cells (BMC) post-myocardial infarction (MI) and the efficacy of an injectable biopolymer scaffold to improve cardiac cell retention and function. Methodology/Principal Findings: (99m)Tc-labeled BMC (6x10(6) cells) were injected by 4 different routes in adult rats: intravenous (IV), left ventricular cavity (LV), left ventricular cavity with temporal aorta occlusion (LV(+)) to mimic coronary injection, and intramyocardial (IM). The injections were performed 1, 2, 3, or 7 days post-MI and cell retention was estimated by gamma-emission counting of the organs excised 24 hs after cell injection. IM injection improved cell retention and attenuated cardiac dysfunction, whereas IV, LV or LV* routes were somewhat inefficient (< 1%). Cardiac BMC retention was not influenced by timing except for the IM injection that showed greater cell retention at 7 (16%) vs. 1, 2 or 3 (average of 7%) days post-MI. Cardiac cell retention was further improved by an injectable fibrin scaffold at day 3 post-MI (17 vs. 7%), even though morphometric and function parameters evaluated 4 weeks later displayed similar improvements. Conclusions/Significance: These results show that cells injected post-MI display comparable tissue distribution profile regardless of the route of injection and that there is no time effect for cardiac cell accumulation for injections performed 1 to 3 days post-MI. As expected the IM injection is the most efficient for cardiac cell retention, it can be further improved by co-injection with a fibrin scaffold and it significantly attenuates cardiac dysfunction evaluated 4 weeks post myocardial infarction. These pharmacokinetic data obtained under similar experimental conditions are essential for further development of these novel approaches.

Short communication: Evaluation of an on-farm test to estimate somatic cell count

The objective of this study was to compare the results of an on-farm test, named Somaticell, with results of electronic cell counting and for milk somatic cell count (SCC) among readers. The Somaticell test correctly determined the SCC in fresh quarter milk samples. Correlation between Somaticell and electronic enumeration of somatic cells was 0.92 and. coefficient 0.82. Using a threshold of 205,000 cells/mL, the sensitivity and specificity for determination of intramammary infections were 91.3 and 96.0%, respectively. The SCC was greater for milk samples from which major mastitis pathogens were recovered. Minor variation among readers was observed and most likely associated with the mixing procedure. However, the final analysis indicated that this variation was not significant and did not affect the amount of samples classified as having subclinical mastitis. The on-farm test evaluated in this study showed adequate capacity of determining SCC on quarter milk samples and may be considered as an alternative for on-farm detection of subclinical mastitis.

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

Purpose: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. Methods: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and H-3-thymidine incorporation in SMCs in culture. Results: Arterial HSPGs (680 mu g) reduced neointimal formation by 35% at 14 days after injury (P =.029), whereas 2000 mu g of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 mu g/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1% +/- 13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1% +/- 15.1%). In contrast, 100 mu g/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9% +/- 14.6%, controls 35.9% +/- 12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 mu g/mL compared with a value of 14 mu g/ml; for Enoxaparin (P

Improved method for counting virus and virus like particles

An improved method for counting virus and virus like particles by electron microscopy (EM) was developed. The procedure involves the determination of the absolute concentration of pure or semi-pure particles once deposited evenly on EM grids using either centrifugation or antibody capture techniques. The counting of particles was done with a Microfiche unit which enlarged approximately 50 x the image of particles on a developed negative film which had been taken at a relatively low magnification (2500 x) by EM. Initially, latex particles of a known concentration were counted using this approach, to prove the accuracy of the technique. The latex particles were deposited evenly on an EM grid using centrifugation (Modified Beckmen EM-90 Airfuge technique). Subsequently, recombinant Bluetongue virus (BTV) core-like particles (CLPs) captured by a Monoclonal antibody using a hovel sample loading method were counted by the Microfiche unit method and by a direct EM method. Comparison of the simplified counting method developed with a conventional method, showed good agreement. The method is simple, accurate, rapid, and reproducible when used with either pure particles or with particles from crude cell culture extracts.

Topical interleukin-1 receptor antagonist inhibits inflammatory cell infiltration into the cornea

Interleukin (IL)-1 alpha and beta are important modulators of many functions of corneal epithelial and stromal cells that occur following injury to the cornea, including the influx of bone marrow-derived inflammatory cells into the stroma attracted by chemokines released from the stroma and epithelium. In this study, we examined the effect of topical soluble IL-1 receptor antagonist on bone marrow-derived cell influx following corneal epithelial scrape injury in a mouse model. C57BL/6 mice underwent corneal epithelial scrape followed by application of IL-1 receptor antagonist (Amgen, Thousand Oaks, CA) at a concentration of 20 mg/ml or vehicle for 24 h prior to immunocytochemical detection of marker CD11b-positive cells into the stroma. In two experiments, topical IL-1 receptor antagonist had a marked effect in blocking cell influx. For example, in experiment 1, topical IL-1 receptor antagonist markedly reduced detectible CD11b-positive cells into the corneal stroma at 24 It after epithelial injury compared with the vehicle control (3.5 +/- 0.5 (standard error of the mean) cells/400x field and 13.9 +/- 1.2 cells/400x field, respectively, p < 0.01). A second experiment with a different observer performing cell counting had the same result. Thus, the data demonstrate conclusively that topical IL-1 receptor antagonist markedly down-regulates CD-11b-positive monocytic cell appearance in the corneal stroma. Topical IL-1 receptor antagonist could be an effective adjuvant for clinical treatment of corneal conditions in which unwanted inflammation has a role in the pathophysiology of the disorder. (c) 2008 Elsevier Ltd. All rights reserved.