Determination of the Most Efficient Household Technique for the Reduction of Pesticide Residues from Raw Fish Muscles.

Substantial quantities of pesticides are routinely applied to enhance agricultural crop production. Pesticides used in this way continuously accumulate in the environment and in foods. Harvested crops contain pesticide residues at various concentrations, with potential harmful impacts on human health. Hence, it is of value to identify techniques for the effective decontamination of tainted foods. However, cleaning with water or household agents, e.g., acetic acid and sodium bicarbonate, are recognized treatments for the efficient degradation of pesticides from vegetables and fruits. There is an apparent void of information about the decontamination treatments for raw fishes using household agents that are affordable for all classes of consumers. Hence, the present study was performed to determine the most efficient household technique for reducing pesticide residue levels from precooked raw fish to ensure the utmost food safety. Fish muscles of four species of fishes, viz., Clarias gariepinus, Channa striatus, Anabas testudineus and Trichogaster trichopterus, were treated with six treatments: washing with running tap water (T1), dipping in normal water (T2), dipping in 2% salt solution (T3), dipping in 2% vinegar (T4), dipping in 0.1% sodium bicarbonate solution (T5) as well as dipping in 0.1% sodium bicarbonate solution + 2% vinegar + 2% salt solution + lemon juice (T6), as fish muscle is the major consumable portion of fish. The current study demonstrated that the removal percentage of lindane, heptachlor, aldrin, endosulfan, dieldrin, endrin, DDT, methoxychlor and cypermethrin residues against the treated household treatments, in downward order, were soaking in 0.1% sodium bicarbonate solution + 2% vinegar + 2% salt solution + lemon juice solution (T6) > soaking in 2% vinegar (T4) solution > soaking in 0.1% sodium bicarbonate (T5) solution > soaking in 2% salt (T3) solution > washing with running tap water (T1) > soaking in stable normal water (T2). The treatment of raw fish muscle samples by soaking them in 0.1% sodium bicarbonate solution + 2% vinegar + 2% salt solution + lemon juice was found to be the most efficient household treatment, performing significant reductions (%) in pesticide concentration: 72−80% (p < 0.05) in Channa striata, 71−79% (p < 0.05) in Clarias gariepinus, 74−80% (p < 0.05) in Anabas testudineus as well as 78−81% (p < 0.05) in Trichogaster trichopterus before cooking.

Determination of Median Lethal Concentration (LC50) for Endosulfan, Heptachlor and Dieldrin Pesticides to African Catfish, Clarias gariepinus and Their Impact on Its Behavioral Patterns and Histopathological Responses.

Pesticides such as endosulfan, heptachlor and dieldrin persist in aquatic environments as a result of their resistance to biodegradation. However, there is no adequate information about the toxicity of endosulfan, heptachlor and dieldrin to the aquatic organism, African catfish (Clarias gariepinus)-a high valued widely distributed commercially interesting species. The current experiment was performed with the aim to determine the median lethal concentration (LC50) of endosulfan, heptachlor and dieldrin to African catfish (Clarias gariepinus); their behavioral abnormalities and histopathological alterations in several vital organs. A total of 324 juvenile fish were exposed for 96 h to six concentrations of endosulfan and dieldrin at 0, 0.001, 0.002, 0.004, 0.008 and 0.016 ppm, and to heptachlor at concentrations of 0, 0.02, 0.04, 0.08, 0.16 and 0.32 ppm for dose-response tests. The study demonstrated that the species is highly susceptible to those contaminants showing a number of behavioral abnormalities and histopathological changes in gill, liver and muscle. The 96-h LC50 value of endosulfan, dieldrin and heptachlor for the African catfish was found as 0.004 (0.001-0.01) mg/L, 0.006 mg/L and 0.056 (0.006-0.144) mg/L, respectively. Abnormal behaviors such as erratic jerky swimming, frequent surfacing movement with gulping of air, secretion of mucus on the body and gills were observed in response to the increasing exposure concentrations. Histopathological alterations of liver, gill and muscle tissues were demonstrated as vacuolization in hepatocytes, congestion of red blood cells (RBCs) in hepatic portal vein; deformed secondary lamellae and disintegrated myotomes with disintegrated epidermis, respectively. These findings are important to monitor and responsibly manage pesticide use in and around C. gariepinus aquacultural areas.

Zooplankton species composition and diversity in the seagrass habitat of Lawas, Sarawak, Malaysia.

Seagrass habitats are considered to be some of the most biodiverse ecosystems on the planet and safeguard some ecologically and economically important fauna, amongst which are some globally threatened species, including dugong. Malaysian seagrass ecosystems are not widespread, but their existence supports some significant marine fauna. A rigorous zooplankton study was conducted from May 2016 to February 2017, in the seagrass habitat of Lawas, Sarawak, Malaysia, to examine their temporal composition and diversity, together with their ecological influences. A total of 45 zooplankton species from 13 significant groups were recorded in the seagrass habitat. The population density of zooplankton ranged between 2,482 ind/m³ and 22,670 ind/m³ over three different seasons. A single zooplankton copepod was found to be dominant (47.40%), while bivalves were the second largest (31.8%) group in terms of total abundance. It was also noticed that the average relative abundance (0.62) and important species index (62.08) of copepods were higher than for other groups that exist in the seagrass meadow, whereas copepod Parvocalanus crassirostris showed both the highest average relative abundance (0.41) and the highest important species index (41.15). The diversity (H') and richness index of the intermediate season were found to be highest due to favourable physico-chemical conditions. Within the referred seasonal cluster, the wet and dry seasons were almost similar in terms of species abundance, while the intermediate season was distinct, with high species diversity backed by ANOSIM analysis results. Copepod and bivalves formed one group with a common similarity level of 0.80. The CCA (Canonical Correspondence Analysis) model established that abiotic factors, especially turbidity, NO2, rainfall, dissolved oxygen and pH were significantly correlated with abundance of individual groups of zooplankton. Zooplankton assemblage and abundance in Lawas were found to be very rich in multiple seasons, indicating that the productivity of uninterrupted seagrass habitat might be high and the system rich in biodiversity.

A comparative transcriptome approach for identification of molecular changes in Aphanomyces invadans infected Channa striatus.

Snakehead murrel, Channa striatus is an economically important aquatic species in Asia and are widely cultured and captured because of its nutritious and medicinal values. Their growth is predominantly affected by epizootic ulcerative syndrome (EUS) which is primarily caused by an oomycete fungus, Aphanomyces invadans. However, the molecular mechanism of immune response in murrel against this infection is still not clear. In this study, transcriptome technique was used to understand the molecular changes involved in C. striatus during A. invadans infection. RNA from the control (CF) and infected fish (IF) groups were sequenced using Illumina Hi-seq sequencing technology. For control group, 28,952,608 clean reads were generated and de novo assembly was performed to produce 60,753 contigs. For fungus infected group, 25,470,920 clean reads were obtained and assembled to produce 58,654 contigs. Differential gene expression analysis revealed that a total of 146 genes were up-regulated and 486 genes were down regulated. Most of the differentially expressed genes were involved in innate immune mechanism such as pathogen recognition, signalling and antimicrobial mechanisms. Interestingly, few adaptive immune genes, especially immunoglobulins were also significantly up regulated during fungal infection. Also, the results were validated by qRT-PCR analysis. These results indicated the involvement of various immune genes involved in both innate and adaptive immune mechanism during fungal infection in C. striatus which provide new insights into murrel immune mechanisms against A. invadans.

Corrigendum: Efficient decellularization of whole porcine kidneys improves reseeded cell behavior (2016 Biomed. Mater. 11 025003).

The manuscript 'Efficient decellularization of whole porcine kidneys improves reseeded cell behavior' (Poornejad et al 2016 Biomedical Materials 11: 025003) describes our efforts to improve the process for recellularization of porcine kidneys. We obtained what we believed to be an immortalized cell line of human renal cortical tubular epithelium (RCTE) cells from the Feinberg School of Medicine, Northwestern University to conduct our reseeding experiments. The RCTE cells that were provided to us were later discovered to actually be Madin-Darby Canine Kidney (MDCK) epithelial cells. A published erratum pertaining to this issue has been published (Caralt et al 2017 American Journal of Transplantation 17: 1429). Despite being of canine origin, MDCK cells are a distal tubule epithelial cell line that behave similarly to human RCTE cells. The conclusions regarding reseeding as reported in our paper are still sound.

Immunohistochemical and molecular analysis of spitzoid neoplasms with pulverocyte subclones.

BACKGROUND: Clonal naevi are characterized by a focal proliferation of pigmented melanocytes in an otherwise banal naevus. These subclones are often composed of aggregates of larger, epithelioid melanocytes with nuclear atypia and dusty-grey cytoplasmic pigmentation, which are referred to as 'pulverocytes', and this finding may lead to a misdiagnosis of malignant melanoma (MM). AIM: To characterize the significance of subclones of dusty-grey pigmented epithelioid melanocytes within spitzoid neoplasms. METHODS: We studied the histological and molecular features of a series of 20 spitzoid neoplasms with pulverocyte subclones encountered in our practice, including both atypical Spitz tumours (ASTs) and invasive MMs. RESULTS: Pulverocytes were predominantly dermal, and the percentage of subclones ranged from 2% to 40%, with a median of 10% in ASTs and 25% in lesions we classified as MM. In cases with > 10% subclones, there was an increased odds of fluorescence in situ hybridization positivity (OR = 12; 95% CI 1.2-293.4; P = 0.03) and an increased odds of homozygous 9p21 deletion (OR = 3.6; 95 CI 0.28-89.82; P = 0.33), although the latter did not reach statistical significance. CONCLUSIONS: We consider spitzoid lesions with a small subclone population to be a variant of a clonal naevus with indolent behaviour, whereas lesions with larger pulverocyte populations are more likely to have chromosomal copy number aberrations and in some cases may represent malignant transformation.

Biosensing estrogenic endocrine disruptors in human blood and urine: A RAPID cell-free protein synthesis approach.

Many diseases and disorders are linked to exposure to endocrine disrupting chemicals (EDCs) that mimic the function of natural estrogen hormones. Here we present a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting chemicals that interact with the human estrogen receptor ß (hERß). This biosensor consists of an allosteric fusion protein, which is expressed using cell-free protein synthesis technology and is directly assayed by a colorimetric response. The resultant biosensor successfully detected known EDCs of hERß (BPA, E2, and DPN) at similar or better detection range than an analogous cell-based biosensor, but in a fraction of time. We also engineered cell-free protein synthesis reactions with RNAse inhibitors to increase production yields in the presence of human blood and urine. The RAPID biosensor successfully detects EDCs in these human samples in the presence of RNAse inhibitors. Engineered cell-free protein synthesis facilitates the use of protein biosensors in complex sample matrices without cumbersome protein purification.

Improvement of feed pellet characteristics by dietary pre-gelatinized starch and their subsequent effects on growth and physiology in tilapia.

A 9-week study was conducted to compare dietary corn starch (CS) or tapioca starch (TS), with or without being pre-gelatinized (PG), on the growth, feeding efficiencies, plasma and muscle biochemistry, intestinal short chain fatty acids (SCFA), and liver glycogen of triplicate groups of 20 red hybrid tilapia (Orecohromis sp.). Various pellet characteristics were evaluated, along with their surface and cross sectional microstructure. The PG diets had significantly higher water stability, bulk density, and protein solubility, along with a smoother surface. Tilapia fed the TS diet had lower growth than had all other tilapia, but were significantly improved when diet was pre-gelatinized. In the PG treatments, intestinal SCFA significantly decreased while plasma glucose, cholesterol and triglycerides, as well as liver glycogen, significantly increased. Fish fed the CS diet had significantly more long chain polyunsaturated fatty acid than had those fed by other treatments. Pre-gelatinization may improve fish productivity and offer greater flexibility during aquafeed production.

A mini review on immune role of chemokines and its receptors in snakehead murrel Channa striatus.

Chemokines are ubiquitous cytokine molecules involved in migration of cells during inflammation and normal physiological processes. Though the study on chemokines in mammalian species like humans have been extensively studied, characterization of chemokines in teleost fishes is still in the early stage. The present review provides an overview of chemokines and its receptors in a teleost fish, Channa striatus. C. striatus is an air breathing freshwater carnivore, which has enormous economic importance. This species is affected by an oomycete fungus, Aphanomyces invadans and a Gram negative bacteria Aeromonas hydrophila is known to cause secondary infection. These pathogens impose immune changes in the host organism, which in turn mounts several immune responses. Of these, the role of cytokines in the immune response is immense, due to their involvement in several activities of inflammation such as cell trafficking to the site of inflammation and antigen presentation. Given that importance, chemokines in fishes do have significant role in the immunological and other physiological functions of the organism, hence there is a need to understand the characteristics, activities and performace of these small molecules in details.

Oral steroids alone or followed by intranasal steroids versus watchful waiting in the management of otitis media with effusion.

OBJECTIVE: To evaluate the effects of oral steroids alone or followed by intranasal steroids versus watchful waiting on the resolution of otitis media with effusion in children aged 2-11 years. METHODS: A total of 290 children with bilateral otitis media with effusion were assigned to 3 groups: group A was treated with oral steroids followed by intranasal steroids, group B was treated with oral steroids alone and group C was managed with watchful waiting. Patients were evaluated with audiometry and tympanometry. RESULTS: The complete resolution rates of otitis media with effusion were higher in groups A and B than in group C at six weeks. There were no significant differences in otitis media with effusion resolution rates between the groups at three, six and nine months. CONCLUSION: Oral steroids lead only to a quick resolution of otitis media with effusion, with no long-term benefits. There was no benefit of using intranasal steroids in the management of otitis media with effusion.

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase-coated magnetic beads: A key step toward codon emancipation.

Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate-based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase-coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real-time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1401-1407, 2017.

Cell-Free Protein Synthesis Approach to Biosensing hTRß-Specific Endocrine Disruptors.

Here we introduce a Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The RAPID platform can be adapted for field use, allowing rapid evaluation of endocrine disrupting chemicals (EDCs) presence or absence in environmental samples, and can also be applied for drug screening. The biosensor is based on an engineered, allosterically activated fusion protein, which contains the ligand binding domain from a target NHR (human thyroid receptor ß in this work). In vitro expression of this protein using cell-free protein synthesis (CFPS) technology in the presence of an EDC leads to activation of a reporter enzyme, reported through a straightforward colorimetric assay output. In this work, we demonstrate the potential of this biosensor platform to be used in a portable "just-add-sample" format for near real-time detection. We also demonstrate the robust nature of the cell-free protein synthesis component in the presence of a variety of environmental and human samples, including sewage, blood, and urine. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.

Morphometric relations in the grey eel catfish Plotosus canius in the coastal waters of Port Dickson, Peninsular Malaysia.

Samples of grey eel catfish Plotosus canius were collected from the coastal waters of Port Dickson, Malaysia from January to December, 2012. A total of 341 specimens (172 males and 169 females) were used to estimate the length-weight relationship parameters. Mean population size of females were 0.72 cm taller than the males, however difference was not significant (t-test, P > 0.05). The overall relationship equations between total length (TL) and body weight (BW) were established for males as Log TW = 2.71 Log TL - 1.85 (R2 = 0.95) and for females as Log TW = 2.88 Log TL-2.10 (R2 = 0.95). The estimated relative growth co-efficient (b) values were 2.71 for males and 2.88 for females. It is revealed that growth pattern of the species showed negative allometry. In both males and females, relationship between TL and SL gave highest regression coefficient (0.99). While relationship between TL and EL gave lowest regression coefficient in both males and females (0.87 and 0.81 respectively). The findings from this study contributed first information on morphometric relations of the fish from Malaysian coastal waters and could be useful for sustainable management options of P. canius in Malaysia.

Escherichia coli-based cell-free extract development for protein-based cancer therapeutic production.

Cell-free protein synthesis has been around for decades but it has never been close to becoming a robust tool for the production of biotherapeutic agents. In this review, we focus on how Escherichia coli-based cell-free protein synthesis can be modified in various ways to produce challenging, complex anticancer biotherapeutics. Here we report progress in extract preparation and its relation to cell-free cancer research. The future prospects of cell-free technology and its potential in various areas of cancer therapeutics production are also highlighted.

Phylogenetics and population genetics of Plotosus canius (Siluriformes: Plotosidae) from Malaysian coastal waters.

Plotosus canius (Hamilton, 1822) is a significant marine species in Malaysia from nutritional and commercial perspectives. Despite numerous fundamental research on biological characteristics of P. canius, there are various concerns on the level of population differentiation, genomic structure, and the level of genetic variability among their populations due to deficiency of genetic-based studies. Deficiency on basic contexts such as stock identification, phylogenetic relationship and population genetic structure would negatively impact their sustainable conservation. Hence, this study was conducted to characterize the genetic structure of P. canius for the first time through the application of mitochondrial Cytochrome Oxidase I (COI) gene, cross amplification of Tandanus tandanus microsatellites, and a total of 117 collected specimens across five selected populations of Malaysia. The experimental results of the mitochondrial analysis revealed that the haplotype diversity and nucleotide diversity varied from 0.395-0.771 and 0.033-0.65 respectively. Moreover, the statistical analysis of microsatellites addressed a considerable heterozygote insufficiency in all populations, with average observed heterozygosity (Ho ) value of 0.2168, which was lower than the standard heterozygosity in marine populations (Ho = 0.79). This alongside the high Fis values estimation, high pairwise differentiation among populations and low within population variations are supposed to be associated with small sample size, and inbreeding system. Besides, the significant finding of this study was the sharing of common haplotype KR086940, which reflects a historical genetic connectivity between Peninsular Malaysia and Borneo populations due to the geological history of Southeast Asia during Pleistocene era. Demographic analyses showed that all populations were in an equilibrium state with no significant evidence of population expansion. To put it briefly, the current study has managed to provide an initial genomic database toward understanding of the genetic characterization, phylogenetic, molecular diversification and population structure in P. canius, and should be necessary highlighted for appropriate management and conservation of species. Further studies must be carried out involving more geographical and sampling sites, larger population size per site, and utilization of species specific microsatellites loci.

Efficient decellularization of whole porcine kidneys improves reseeded cell behavior.

Combining patient-specific cells with the appropriate scaffold to create functional kidneys is a promising technology to provide immunocompatible kidneys for the 100,000+ patients on the organ waiting list. For proper recellularization to occur, the scaffold must possess the critical microstructure and an intact vascular network. Detergent perfusion through the vasculature of a kidney is the preferred method of decellularization; however, harsh detergents could be damaging to the microstructure of the renal tissue and may undesirably solubilize the endogenous growth and signaling factors. In this study, automated decellularization of whole porcine kidneys was performed using an improved method that combined physical and chemical steps to efficiently remove cellular materials while producing minimal damage to the collagenous extracellular matrix (ECM). Freezing/thawing, incremental increases in flow rate under constant pressure, applying osmotic shock to the cellular membranes, and low concentrations of the detergent sodium dodecyl sulfate (SDS) were factors used to decrease SDS exposure time during the decellularization process from 36 to 5 h, which preserved the microstructure while still removing 99% of the DNA. The well-preserved glycosaminoglycans (GAGs) and collagen fibers enhanced cell-ECM interactions. Human renal cortical tubular epithelium (RCTE) cells grew more rapidly when cultured on the ECM obtained from the improved decellularization process and also demonstrated more in vivo-like gene expression patterns. The optimized, automated process that resulted from this work is now used routinely in our laboratory to rapidly decellularize porcine kidneys and could be adapted to other large organs (e.g. heart, liver, and lung).

Cell-free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just-add-water cell-free system.

Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. For example, difficult-to-express proteins may be cytotoxic or form inclusion bodies during expression, increasing the time, labor, and difficulty of purification and downstream characterization. One potential pathway to simplify the expression and screening of such therapeutics is to utilize cell-free protein synthesis. Cell-free systems offer a compelling alternative to in vivo production, due to their open and malleable reaction environments. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. This lyophilized system remains viable after being stored above freezing for up to one year. The beneficial features of these cell-free systems make them compelling candidates for future biotherapeutic screening and production.

Trypsin-modulating oostatic factor (TMOF) decreased the survival, growth and digestion enzymes of Macrobrachium rosenbergii.

Trypsin-modulating oostatic factor (TMOF) is an effective mosquito larvicide, but information on its potential toxicity to non-target organisms is limited. To investigate this, triplicate groups of 10 Macrobrachium rosenbergii were exposed to 0, 10, 50 or 100 mg/L nominal TMOF concentrations for 12 days. Tail moisture, crude protein, and hepatopancreatic glycogen/histopathology were unaffected, but increasing TMOF linearly decreased survival and growth. TMOF at the lowest concentration employed significantly decreased trypsin and chymotrypsin activities.

Impacts of gamete concentration, age and contact time on the fertilization success in the tropical species of white sea urchin, Salmaciella dussumieri.

The globoid sea urchin (Salmaciella dussumieri) occurs abundantly in the Indo-West Pacific from the Island of the West Indian Ocean, East Africa, Magdagascar, Red Sea, South-East Arabia, Ceylon, Bay of Bengal, North Australia, Philippine, China and South Japan and Australia, and also has significant biological, ecological, aquaculture, conservational and medicinal importance. An experiment was carried out to assess the influence of sperm dilution, egg concentration, sperm-egg exposure time, and gamete aging on fertilization success of S. dussumieri in a captive laboratory condition. It has been found that dilution, age and contact time of sperm to egg were successively the most dominant factors effecting the success of fertilization, but concentration of eggs did not significantly so over the range investigated. The sperms retained their competency for more than two hours only in relatively dense sperm concentrations (≥ 10-(4) dilution of 'dry' sperm), although they exhibited lower capability and potency with increasing dilutions and age. In the trials of the egg-sperm exposure time, >80% eggs were fertilized within 10 sec of contact time at lower dilutions (10(-3)-10(-2)) of concentrated sperm, while some longer times were required to get the higher rates of fertilization. On the contrary, eggs remained in good quality for up to a period of 3 h and no abnormality or any adverse effects in fertilization were observed. The findings obtained from the present experiment reveal that limited longevity of diluted sperm have an important effect on fertilization rate of sea urchin eggs during natural spawning seasons in the field. Hence, the globoid sea urchin (S. dussumieri) is under extensive selective pressures to breed synchronously with the purpose of producing high sperm concentrations and higher sperm-egg interactions to maximize the success of fertilization in the water column.