Arsenic speciation analysis in human urine for long term epidemiological studies: The Multi-Ethnic Study of Atherosclerosis (MESA).

Arsenic is a ubiquitous toxic metalloid causing serious health problems. Speciation analysis of arsenic in human urine provides valuable insights for large-scale epidemiological studies and informs on sources of exposure as well as human metabolism. The Multi-Ethnic Study of Atherosclerosis (MESA) is a valuable cohort for assessing chronic low-moderate arsenic exposure and health effects in an ethnically diverse US population. We present a state-of-the-art arsenic speciation analysis methodology and its application to 7677 MESA spot urine samples based on high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry. This method is fast, robust and detects a total of 11 individual As species at method detection limits of 0.02-0.03 µg arsenic/L urine for each individual species. Our analytical approach features excellent mean method accuracy (98%) and precision (5%) for the main arsenic species in urine (arsenobetaine, methylarsonic acid, dimethylarsinic acid, and total inorganic As); intra- (3-6%) and inter-day coefficients of variability (5-6%); column recovery (96 ± 7%); and spike recovery (97 ± 6%). The main arsenic species were detectable in ≥95% of urine samples due to the implementation of an oxidation step. Each individual minor arsenic species was detectable in ≤25% of all urines, although at least one of them was detected in almost half the participants. We identified two minor urinary arsenic species as dimethylarsinoylacetic acid and dimethylarsinoylpropionic acid, potential metabolites of seafood-related arsenicals. We observed differences in individual As species excretion by race/ethnicity, with Asian-American participants featuring 3-4 times higher concentrations compared to other participants. We also found differences by site, body mass index, smoking status, rice intake, and water arsenic levels, potentially indicating different exposures or related to individual bio-metabolism. The proposed approach is suitable for epidemiological studies and the collected data will constitute the base for future research on potential health effects of chronic low-level arsenic exposure.

High-Resolution Mass Spectrometry for Human Exposomics: Expanding Chemical Space Coverage.

In the modern "omics" era, measurement of the human exposome is a critical missing link between genetic drivers and disease outcomes. High-resolution mass spectrometry (HRMS), routinely used in proteomics and metabolomics, has emerged as a leading technology to broadly profile chemical exposure agents and related biomolecules for accurate mass measurement, high sensitivity, rapid data acquisition, and increased resolution of chemical space. Non-targeted approaches are increasingly accessible, supporting a shift from conventional hypothesis-driven, quantitation-centric targeted analyses toward data-driven, hypothesis-generating chemical exposome-wide profiling. However, HRMS-based exposomics encounters unique challenges. New analytical and computational infrastructures are needed to expand the analysis coverage through streamlined, scalable, and harmonized workflows and data pipelines that permit longitudinal chemical exposome tracking, retrospective validation, and multi-omics integration for meaningful health-oriented inferences. In this article, we survey the literature on state-of-the-art HRMS-based technologies, review current analytical workflows and informatic pipelines, and provide an up-to-date reference on exposomic approaches for chemists, toxicologists, epidemiologists, care providers, and stakeholders in health sciences and medicine. We propose efforts to benchmark fit-for-purpose platforms for expanding coverage of chemical space, including gas/liquid chromatography-HRMS (GC-HRMS and LC-HRMS), and discuss opportunities, challenges, and strategies to advance the burgeoning field of the exposome.

Development and validation of general plasma screening method for performance enhancing drugs in racehorses utilizing liquid chromatography-high-resolution mass spectrometry (LC-HRMS).

The screening of drugs in plasma and urine often requires initial extraction (such as liquid-liquid extraction and solid-phase extraction) before the samples are submitted to instrumental analyses. These extraction procedures are often laborious and time-consuming. In this manuscript, a high-throughput automated assay based on liquid chromatography-high-resolution mass spectrometry (LC-HRMS) suitable for use as an initial testing procedure covering multiple classes of compounds prohibited in horse racing is described. The assay requires a 600-µL plasma aliquot, which is subjected to solid phase extraction (SPE) using OASIS HLB 96-well SPE with Biotage Extrahera system, evaporation, and reconstitution in a 96-well collection plate. LC-HRMS analyses were carried out on a Thermo Q-Exactive Mass spectrometer coupled with Thermo UHPLC system equipped with Thermo Accela ALS 2.4.0 autosampler linked to ACE Excel column. Drug targets were detected by retention time and accurate mass, with a mass tolerance window of 5 ppm in positive and negative ionization mode. The screening method was validated for over 300 drug targets in a 13-min run. Validation data including sensitivity, specificity, extraction recovery, and precision are presented. As the method employs full-scan mass spectrometry, unlimited number of drug targets can theoretically be incorporated into this method.

Oral administration of recombinant outer membrane protein A-based nanovaccine affords protection against Aeromonas hydrophila in zebrafish.

Aeromonas hydrophila, an opportunistic warm water pathogen, has always been a threat to aquaculture, leading to substantial economic losses. Vaccination of the cultured fish would effectively prevent Aeromoniasis, and recent advancements in nanotechnology show promise for efficacious vaccines. Oral delivery would be the most practical and convenient method of vaccine delivery in a grow-out pond. This study studied the immunogenicity and protective efficacy of a nanoparticle-loaded outer membrane protein A from A. hydrophila in the zebrafish model. The protein was over-expressed, purified, and encapsulated using poly lactic-co-glycolic acid (PLGA) nanoparticles via the double emulsion method. The PLGA nanoparticles loaded with recombinant OmpA (rOmpA) exhibited a size of 295 ± 15.1 nm, an encapsulation efficiency of 72.52%, and a polydispersity index of 0.292 ± 0.07. Scanning electron microscopy confirmed the spherical and isolated nature of the PLGA-rOmpA nanoparticles. The protective efficacy in A. hydrophila-infected zebrafish after oral administration of the nanovaccine resulted in relative percentage survival of 77.7. Gene expression studies showed significant upregulation of immune genes in the vaccinated fish. The results demonstrate the usefulness of oral administration of nanovaccine-loaded rOmpA as a potential vaccine since it induced a robust immune response and conferred adequate protection against A. hydrophila in zebrafish, Danio rerio.

Polylactic-Co-glycolic Acid Polymer-Based Nano-Encapsulation Using Recombinant Maltoporin of Aeromonas hydrophila as Potential Vaccine Candidate.

Aquaculture production has been incurring economic losses due to infectious diseases by opportunistic pathogens like Aeromonas hydrophila, a bacterial agent that commonly affects warm water aquacultured fish. Developing an effective vaccine with an appropriate delivery system can elicit an immune response that would be a useful disease management strategy through prevention. The most practical method of administration would be the oral delivery of vaccine developed through nano-biotechnology. In this study, the gene encoding an outer membrane protein, maltoporin, of A. hydrophila, was identified, sequenced, and studied using bioinformatics tools to examine its potential as a vaccine candidate. Using a double emulsion method, the molecule was cloned, over-expressed, and encapsulated in a biodegradable polymer polylactic-co-glycolic acid (PLGA). The immunogenicity of maltoporin was identified through in silico analysis and thus taken up for nanovaccine preparation. The encapsulation efficiency of maltoporin was 63%, with an in vitro release of 55% protein in 48 h. The particle size and morphology of the encapsulated protein exhibited properties that could induce stability and function as an effective carrier system to deliver the antigen to the site and trigger immune response. Results show promise that the PLGA-mediated delivery system could be a potential carrier in developing a fish vaccine via oral administration. They provide insight for developing nanovaccine, since sustained in vitro release and biocompatibility were observed. There is further scope to study the immune response and examine the protective immunity induced by the nanoparticle-encapsulated maltoporin by oral delivery to fish.

Lysophosphatidylcholines are associated with P-tau181 levels in early stages of Alzheimer's Disease.

Background: We profiled circulating plasma metabolites to identify systemic biochemical changes in clinical and biomarker-assisted diagnosis of Alzheimer's disease (AD). Methods: We used an untargeted approach with liquid chromatography coupled to high-resolution mass spectrometry to measure small molecule plasma metabolites from 150 clinically diagnosed AD patients and 567 age-matched healthy elderly of Caribbean Hispanic ancestry. Plasma biomarkers of AD were measured including P-tau181, Aß40, Aß42, total-tau, neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP). Association of individual and co-abundant modules of metabolites were tested with clinical diagnosis of AD, as well as biologically-defined AD pathological process based on P-tau181 and other biomarker levels. Results: Over 6000 metabolomic features were measured with high accuracy. First principal component (PC) of lysophosphatidylcholines (lysoPC) that bind to or interact with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (AHA) was associated with decreased risk of AD (OR = 0.91 [0.89-0.96], p = 2e-04). Association was restricted to individuals without an APOE ε4 allele (OR = 0.89 [0.84-0.94], p = 8.7e-05). Among individuals carrying at least one APOE ε4 allele, PC4 of lysoPCs moderately increased risk of AD (OR = 1.37 [1.16-1.6], p = 1e-04). Essential amino acids including tyrosine metabolism pathways were enriched among metabolites associated with P-tau181 levels and heparan and keratan sulfate degradation pathways were associated with Aß42/Aß40 ratio. Conclusions: Unbiased metabolic profiling can identify critical metabolites and pathways associated with ß-amyloid and phosphotau pathology. We also observed an APOE-ε4 dependent association of lysoPCs with AD and biologically based diagnostic criteria may aid in the identification of unique pathogenic mechanisms.

In vitro determination of probiotic efficacy of Bacillus subtilis TLDK301120C24 isolated from tilapia against warm water fish pathogens and in vivo validation using gnotobiotic zebrafish model.

Eco-friendly alternatives such as probiotics are needed to prevent economically relevant infectious diseases for a successful disease-free harvest in aquaculture. The use of antibiotics has been the favored practice, but its empirical and indiscriminate use has led to antibiotic resistance in the aquatic environment and residues in the food fish. With this rationale, a probiotic was isolated from tilapia, a commercially important cultured fish worldwide. The characteristics of the probiotic were checked against common bacterial pathogens affecting aquaculture. In vitro tests demonstrated the inhibitory effects of the isolated probiotic on the growth of Aeromonas hydrophila, Edwardsiella tarda, Vibrio anguillarum, and V. alginolyticus. The candidate probiotic, referred to as TLDK301120C24, was identified as Bacillus subtilis by a battery of biochemical tests and genotypic confirmation by 16S rDNA sequencing. The in vitro results revealed the ability of the probiotic to withstand the gut conditions that included pH range of 3-9, salt concentration of 0.5-6%, and bile salt concentration of up to 6%. The isolate could hydrolyze starch (12-14 mm clearance zone), protein (20-22 mm clearance zone), and cellulose (22-24 mm clearance zone). Further, the inhibitory ability of the probiotic against aquatic pathogens was determined in vivo using gnotobiotic zebrafish by employing a novel approach that involved tagging the probiotic with a red fluorescent protein and the pathogens with a green fluorescent protein, respectively. The colonizing ability of probiotics and its inhibitory effects against the pathogens were evaluated by fluorescence microscopy, PCR, and estimation of viable counts in LBA + Amp plates. Finally, the competitive inhibition and exclusion of fish pathogens A. hydrophila and E. tarda by B. subtilis was confirmed semi-quantitatively, through challenge experiments. This study shows the potential of B. subtilis as a probiotic and its excellent ability to inhibit major fish pathogens in vivo and in vitro. It also shows promise as a potent substitute for antibiotics.

Nanovaccines to Combat Aeromonas hydrophila Infections in Warm-Water Aquaculture: Opportunities and Challenges.

The application of nanotechnology in aquaculture for developing efficient vaccines has shown great potential in recent years. Nanovaccination, which involves encapsulating antigens of fish pathogens in various polymeric materials and nanoparticles, can afford protection to the antigens and a sustained release of the molecule. Oral administration of nanoparticles would be a convenient and cost-effective method for delivering vaccines in aquaculture while eliminating the need for stressful, labour-intensive injectables. The small size of nanoparticles allows them to overcome the degradative digestive enzymes and help deliver antigens to the target site of the fish more effectively. This targeted-delivery approach would help trigger cellular and humoral immune responses more efficiently, thereby enhancing the protective efficacy of vaccines. This is particularly relevant for combating diseases caused by pathogens like Aeromonas hydrophila, a major fish pathogen responsible for significant morbidity and mortality in the aquaculture sector. While the use of nanoparticle-based vaccines in aquaculture has shown promise, concerns exist about the potential toxicity associated with certain types of nanoparticles. Some nanoparticles have been found to exhibit varying degrees of toxicity, and their safety profiles need to be thoroughly assessed before widespread application. The introduction of nanovaccines has opened new vistas for improving aquaculture healthcare, but must be evaluated for potential toxicity before aquaculture applications. Details of nanovaccines and their mode of action, with a focus on protecting fish from infections and outbreaks caused by the ubiquitous opportunistic pathogen A. hydrophila, are reviewed here.

Lysophosphatidylcholines are associated with P-tau181 levels in early stages of Alzheimer's Disease.

Background: We investigated systemic biochemical changes in Alzheimer's disease (AD) by investigating the relationship between circulating plasma metabolites and both clinical and biomarker-assisted diagnosis of AD. Methods: We used an untargeted approach with liquid chromatography coupled to high-resolution mass spectrometry to measure exogenous and endogenous small molecule metabolites in plasma from 150 individuals clinically diagnosed with AD and 567 age-matched elderly without dementia of Caribbean Hispanic ancestry. Plasma biomarkers of AD were also measured including P-tau181, Aß40, Aß42, total tau, neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP). Association of individual and co-expressed modules of metabolites were tested with the clinical diagnosis of AD, as well as biologically-defined AD pathological process based on P-tau181 and other biomarker levels. Results: Over 4000 metabolomic features were measured with high accuracy. First principal component (PC) of lysophosphatidylcholines (lysoPC) that bind to or interact with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (AHA) was associated with decreased risk of AD (OR=0.91 [0.89-0.96], p=2e-04). Restricted to individuals without an APOE ε4 allele (OR=0.89 [0.84-0.94], p= 8.7e-05), the association remained. Among individuals carrying at least one APOE ε4 allele, PC4 of lysoPCs moderately increased risk of AD (OR=1.37 [1.16-1.6], p=1e-04). Essential amino acids including tyrosine metabolism pathways were enriched among metabolites associated with P-tau181 levels and heparan and keratan sulfate degradation pathways were associated with Aß42/Aß40 ratio reflecting different pathways enriched in early and middle stages of disease. Conclusions: Our findings indicate that unbiased metabolic profiling can identify critical metabolites and pathways associated with ß-amyloid and phosphotau pathology. We also observed an APOE ε4 dependent association of lysoPCs with AD and that biologically-based diagnostic criteria may aid in the identification of unique pathogenic mechanisms.

Evaluation of Probiotic Efficacy of Bacillus subtilis RODK28110C3 Against Pathogenic Aeromonas hydrophila and Edwardsiella tarda Using In Vitro Studies and In Vivo Gnotobiotic Zebrafish Gut Model System.

The indiscriminate use of antibiotics in aquaculture has led to the emergence of resistance; hence, eco-friendly, host-specific alternatives to mitigate bacterial infections have become imminent. In this study, bacteria that could possibly serve as probiotics were isolated and evaluated for their efficacy with in vitro experiments and in vivo zebrafish gut model. One isolate from each of the 23 rohu fish (Labeo rohita) was shortlisted after preliminary screening of several isolates and tested for their ability to inhibit two important warm water bacterial fish pathogens, Aeromonas hydrophila, and Edwardsiella tarda. An isolate (RODK28110C3) that showed broad-spectrum inhibitory activity against a battery of different isolates of the two fish pathogens included in this study and maintained in our repository was selected for further characterization. The culture was identified phenotypically as Bacillus subtilis and confirmed by 16S rDNA sequencing. The isolate was able to hydrolyze fish feed constituents that include starch, protein, and cellulose. Further in vitro tests ensured that the potential isolate with probiotic attributes could tolerate different gut conditions, which included a range of pH, salinity, and varying concentrations of bile salt. Exposure of 4 days post fertilization zebrafish embryos to the RFP-tagged isolate confirmed the colonization of B. subtilis in the gut of the zebrafish embryo, which is an important attribute of a probiotic. The isolate was able to inhibit both A. hydrophila and E. tarda in gnotobiotic zebrafish embryo in triplicate. The study demonstrates the probiotic characteristics of the B. subtilis isolated from L. rohita and its ability to inhibit A. hydrophila and E. tarda using in vitro conditions and in the zebrafish gut and could serve as an effective alternative to antibiotics in aquaculture.

The mobile gene cassette carrying tetracycline resistance genes in Aeromonas veronii strain Ah5S-24 isolated from catfish pond sediments shows similarity with a cassette found in other environmental and foodborne bacteria.

Aeromonas veronii is a Gram-negative bacterium ubiquitously found in aquatic environments. It is a foodborne pathogen that causes diarrhea in humans and hemorrhagic septicemia in fish. In the present study, we used whole-genome sequencing (WGS) to evaluate the presence of antimicrobial resistance (AMR) and virulence genes found in A. veronii Ah5S-24 isolated from catfish pond sediments in South-East, United States. We found cphA4, dfrA3, mcr-7.1, valF, bla FOX-7, and bla OXA-12 resistance genes encoded in the chromosome of A. veronii Ah5S-24. We also found the tetracycline tet(E) and tetR genes placed next to the IS5/IS1182 transposase, integrase, and hypothetical proteins that formed as a genetic structure or transposon designated as IS5/IS1182/hp/tet(E)/tetR/hp. BLAST analysis showed that a similar mobile gene cassette (MGC) existed in chromosomes of other bacteria species such as Vibrio parahaemolyticus isolated from retail fish at markets, Aeromonas caviae from human stool and Aeromonas media from a sewage bioreactor. In addition, the IS5/IS1182/hp/tet(E)/tetR/hp cassette was also found in the plasmid of Vibrio alginolyticus isolated from shrimp. As for virulence genes, we found the tap type IV pili (tapA and tapY), polar flagellae (flgA and flgN), lateral flagellae (ifgA and IfgL), and fimbriae (pefC and pefD) genes responsible for motility and adherence. We also found the hemolysin genes (hylII, hylA, and TSH), aerA toxin, biofilm formation, and quorum sensing (LuxS, mshA, and mshQ) genes. However, there were no MGCs encoding virulence genes found in A. veronii AhS5-24. Thus, our findings show that MGCs could play a vital role in the spread of AMR genes between chromosomes and plasmids among bacteria in aquatic environments. Overall, our findings are suggesting that MGCs encoding AMR genes could play a vital role in the spread of resistance acquired from high usage of antimicrobials in aquaculture to animals and humans.

Characterization of virulence and antimicrobial resistance genes of Aeromonas media strain SD/21-15 from marine sediments in comparison with other Aeromonas spp.

Aeromonas media is a Gram-negative bacterium ubiquitously found in aquatic environments. It is a foodborne pathogen associated with diarrhea in humans and skin ulceration in fish. In this study, we used whole genome sequencing to profile all antimicrobial resistance (AMR) and virulence genes found in A. media strain SD/21-15 isolated from marine sediments in Denmark. To gain a better understanding of virulence and AMR genes found in several A. media strains, we included 24 whole genomes retrieved from the public databanks whose isolates originate from different host species and environmental samples from Asia, Europe, and North America. We also compared the virulence genes of strain SD/21-15 with A. hydrophila, A. veronii, and A. salmonicida reference strains. We detected Msh pili, tap IV pili, and lateral flagella genes responsible for expression of motility and adherence proteins in all isolates. We also found hylA, hylIII, and TSH hemolysin genes in all isolates responsible for virulence in all isolates while the aerA gene was not detected in all A. media isolates but was present in A. hydrophila, A. veronii, and A. salmonicida reference strains. In addition, we detected LuxS and mshA-Q responsible for quorum sensing and biofilm formation as well as the ferric uptake regulator (Fur), heme and siderophore genes responsible for iron acquisition in all A. media isolates. As for the secretory systems, we found all genes that form the T2SS in all isolates while only the vgrG1, vrgG3, hcp, and ats genes that form parts of the T6SS were detected in some isolates. Presence of bla MOX-9 and bla OXA-427 ß-lactamases as well as crp and mcr genes in all isolates is suggestive that these genes were intrinsically encoded in the genomes of all A. media isolates. Finally, the presence of various transposases, integrases, recombinases, virulence, and AMR genes in the plasmids examined in this study is suggestive that A. media has the potential to transfer virulence and AMR genes to other bacteria. Overall, we anticipate these data will pave way for further studies on virulence mechanisms and the role of A. media in the spread of AMR genes.

Aeromonas species isolated from aquatic organisms, insects, chicken, and humans in India show similar antimicrobial resistance profiles.

Aeromonas species are Gram-negative bacteria that infect various living organisms and are ubiquitously found in different aquatic environments. In this study, we used whole genome sequencing (WGS) to identify and compare the antimicrobial resistance (AMR) genes, integrons, transposases and plasmids found in Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii isolated from Indian major carp (Catla catla), Indian carp (Labeo rohita), catfish (Clarias batrachus) and Nile tilapia (Oreochromis niloticus) sampled in India. To gain a wider comparison, we included 11 whole genome sequences of Aeromonas spp. from different host species in India deposited in the National Center for Biotechnology Information (NCBI). Our findings show that all 15 Aeromonas sequences examined had multiple AMR genes of which the Ambler classes B, C and D ß-lactamase genes were the most dominant. The high similarity of AMR genes in the Aeromonas sequences obtained from different host species point to interspecies transmission of AMR genes. Our findings also show that all Aeromonas sequences examined encoded several multidrug efflux-pump proteins. As for genes linked to mobile genetic elements (MBE), only the class I integrase was detected from two fish isolates, while all transposases detected belonged to the insertion sequence (IS) family. Only seven of the 15 Aeromonas sequences examined had plasmids and none of the plasmids encoded AMR genes. In summary, our findings show that Aeromonas spp. isolated from different host species in India carry multiple AMR genes. Thus, we advocate that the control of AMR caused by Aeromonas spp. in India should be based on a One Health approach.

Efficiency, sensitivity and specificity of a quantitative real-time PCR assay for Tilapia Lake virus (TiLV).

Tilapia lake virus (TiLV) is an emerging viral pathogen of tilapiines worldwide in wild and farmed tilapia. TiLV is an orthomyxo-like, negative sense segmented RNA virus, belonging to genus Tilapinevirus, family Amnoonviridae. Here we developed a quantitative real-time PCR (qRT-PCR) assay testing primer sets targeting the 10 segments of TiLV. Sensitivity, specificity, efficiency and reproducibility of these assays were examined. Detection sensitivity was equivalent to 2 TCID50/ml when tested on supernatants from cell culture-grown TiLV. Specificity tests showed that all primer sets amplified their respective TiLV segments, and standard curves showed linear correlation of R2 > 0.998 and amplification efficiencies between 93 % and 98 %. Intra- and inter-assay coefficients of variation (CV %) were in the range of 0.0 %- 2.6 % and 0.0 %- 5.9 %, respectively. Sensitivity tests showed that primer sets targeting segments 1, 2, 3 and 4 had the highest detection sensitivities (100.301 TCID50/ml). The qRT-PCR used for detection of viral genome in TiLV infected organs gave virus titers equivalent to 3.80 log10, 3.94 log10 and 3.52 log10 TCID50/ml for brain, kidney and liver tissues, respectively as calculated on the basis of Ct values. These findings suggest that primer optimization for qPCR should not only focus on attaining high amplification efficiency but also sensitivity comparison of primer sets targeting different viral segments in order to develop a method with the highest sensitivity.

Garvicin KS, a Broad-Spectrum Bacteriocin Protects Zebrafish Larvae against Lactococcus garvieae Infection.

Bacteriocins are emerging as a viable alternative to antibiotics due to their ability to inhibit growth or kill antibiotic resistant pathogens. Herein, we evaluated the ability of the bacteriocin Garvicin KS (GarKS) produced by Lactococcus garvieae KS1546 isolated from cow milk to inhibit the growth of fish and foodborne bacterial pathogens. We found that GarKS inhibited the growth of five fish L. garvieae strains isolated from infected trout and eels. Among fish pathogens, GarKS inhibited the growth of Streptococcus agalactiae serotypes Ia and Ib, and Aeromonas hydrophila but did not inhibit the growth of Edwardsiella tarda. In addition, it inhibited the growth of A. salmonicida strain 6421 but not A. salmonicida strain 6422 and Yersinia ruckeri. There was no inhibition of three foodborne bacterial species, namely Salmonella enterica, Klebsiella pneumoniae, and Escherichia coli. In vitro cytotoxicity tests using different GarKS concentrations showed that the highest concentration of 33 µg/mL exhibited low cytotoxicity, while concentrations ≤3.3 µg/mL had no cytotoxicity on CHSE-214 and RTG-2 cells. In vivo tests showed that zebrafish larvae treated with 33 µg/mL and 3.3 µg/mL GarKS prior to challenge had 53% and 48% survival, respectively, while concentrations ≤0.33 µg/mL were nonprotective. Altogether, these data show that GarKS has a broad inhibitory spectrum against Gram positive and negative bacteria and that it has potential applications as a therapeutic agent for a wide range of bacterial pathogens. Thus, future studies should include clinical trials to test the efficacy of GarKS against various bacterial pathogens in farmed fish.

Aeromonas species obtained from different farmed aquatic species in India and Taiwan show high phenotypic relatedness despite species diversity.

OBJECTIVES: Aeromonads cause severe diseases in farmed aquatic organisms. Herein, we examined 28 isolates causing disease in farmed aquatic organisms from India (n = 24) and Taiwan (n = 4) to gain insight of their genotypic and phenotypic properties. RESULTS: API 20NE biochemical phenotyping showed ≥ 90% similarity classifying all isolates as Aeromonas hydrophila. 16S rRNA genotyping showed ≥ 98% homology among all isolates with A. sobria (NR119044.1ATCC), A. veronii (MK990549.1), A. caviae (NR029252.1) and A. hydrophila (MG984625.1ATCC) and other reference strains. In contrast, gyrB showed a higher intraspecies diversity (≥ 96%) than 16S rRNA delineating the 28 isolates into three groups. Group-I consisted of seven Indian isolates clustered with A. sobria (MK484163.1ATCC), group-II comprised of five Indian and two Taiwanese isolates clustered with A. veronii AF417626.1ATCC while group-III had 11 Indian and three Taiwanese isolates grouped with A. hydrophila (AY987520.1 and DQ519366.1) reference strains. None of our isolates clustered with A. caviae (AJ868400.1ATCC) reference strain. These findings suggest that A. sobria, A. veronii and A. hydrophila could be the etiological agents of diseases observed in farmed fish and soft-shelled turtles (Pelodiscus sinensis) examined in this study. Overall, our findings accentuate the importance of combining phenotyping with genotyping for correct taxonomic classification of Aeromonas spp. in Aquaculture.

Tilapia lake virus downplays innate immune responses during early stage of infection in Nile tilapia (Oreochromis niloticus).

Tilapia lake virus (TiLV) causes high mortality and high economic losses in tilapines. We describe an experimental challenge study focusing on early post challenge innate immune responses. Nile tilapia (Oreochromis niloticus) were infected with 105 TCID50/mL TiLV intraperitoneally, followed by virus quantification, histopathology and gene expression analysis in target (brain/liver) and lymphoid (spleen/headkidney) organs at 3, 7, 12, 17, and 34 days post challenge (dpc). Onset of mortality was from 21 dpc, and cumulative mortality was 38.5% by 34 dpc. Liver and kidney histopathology developed over the period 3-17 dpc, characterized by anisocytosis, anisokaryocytosis, and formation of multinucleated hepatocytes. Viral loads were highest at early time (3 dpc) in liver, spleen and kidney, declining towards 34 dpc. In brain, viral titer peaked 17 dpc. Innate sensors, TLRs 3/7 were inversely correlated with virus titer in brain and headkidney, and IFN-ß and Mx showed a similar pattern. All organs showed increased mRNA IgM expression over the course of infection. Overall, high virus titers downplay innate responses, and an increase is seen when viral titers decline. In silico modeling found that TiLV segments 4, 5 and 10 carry nucleolar localization signals. Anti-viral effects of TiLV facilitate production of virus at early stage of infection.

Postinflammatory Hyperpigmentation following Electrodesiccation with a Eutectic Mixture of Local Anesthetics.

We assessed the effects of prior application of a eutectic mixture of local anesthetics (EMLA) on the appearance of dyschromia at the site of superficial electrodesiccation in an observer-blind, case-control study in 60 patients. Thirty subjects each were assigned to Groups A and B; both groups underwent radiofrequency (RFC) ablation for facial dermatosis papulosa nigrans (DPN). Group A received RFC ablation with prior application of EMLA, whereas Group B did not. No significant difference was observed in the dyschromia between both groups. EMLA cream was well tolerated by the study participants. (SKINmed. 2020;18:222-225).

Application of Outer Membrane Protein-Based Vaccines Against Major Bacterial Fish Pathogens in India.

Aquaculture is one of the fastest-growing food-producing sectors in the world. However, its growth is hampered by various disease problems due to infectious microorganisms, including Gram-negative bacteria in finfish aquaculture. Disease control in aquaculture by use of antibiotics is not recommended as it leads to antibiotic residues in the final product, selection, and spread of antibiotic resistance in the environment. Therefore, focus is on disease prevention by vaccination. All Gram-negative bacteria possess surface-associated outer membrane proteins (OMPs), some of which have long been recognized as potential vaccine candidates. OMPs are essential for maintaining the integrity and selective permeability of the bacterial membrane and play a key role in adaptive responses of bacteria such as solute and ion uptake, iron acquisition, antimicrobial resistance, serum resistance, and bile salt resistance and some adhesins have virulence attributes. Antigenic diversity among bacterial strains even within the same bacterial species has constrained vaccine developments, but OMPs that are conserved across serotypes could be used as potential candidates in vaccine development, and several studies have demonstrated their efficacy and potential as vaccine candidates. In this review, we will look into the application of OMPs for the design of vaccines based on recombinant proteins, subunit vaccines, chimeric proteins, and DNA vaccines as new-generation vaccine candidates for major bacterial pathogens of fish for sustainable aquaculture.

Comparing the capabilities of transfer learning models to detect skin lesion in humans.

Effective diagnosis of skin tumours mainly relies on the analysis of the characteristics of the lesion. Automatic detection of malignant skin lesion has become a mandatory task to reduce the risk of human deaths and increase their survival. This article proposes a study of skin lesion classification using transfer learning approach. The transfer learning model uses four different state-of-the-art architectures, namely Inception v3, Residual Networks (ResNet 50), Dense Convolutional Networks (DenseNet 201) and Inception Residual Networks (Inception ResNet v2). These models are trained under the dataset comprising seven different classes of skin lesions. The skin lesion images are pre-processed using image quantization, grayscaling and the Wiener filter before final training step. These models are compared for performance evaluation on different metrics. The present study shows the efficacy of the methodology for automated classification of lesion images.