Exposure to imipenem at sub-minimum inhibitory concentration leads to altered expression of major outer membrane proteins in Acinetobacter baumannii.

AIMS: Acinetobacter baumannii is a nosocomial pathogen known to be multidrug-resistant (MDR), especially to drugs of the carbapenem class. Several factors contribute to resistance, including efflux pumps, ß-lactamases, alteration of target sites, and permeability defects. In addition, outer membrane proteins (OMPs), like porins are involved in the passage of antibiotics, and their alteration could lead to resistance development. This study aimed to explore the possible involvement of porins and OMPs in developing carbapenem resistance due to differential expression. METHODS AND RESULTS: The antibiotic-susceptible and MDR isolates of A. baumannii were first studied for differences in their transcriptional levels of OMP expression and OMP profiles. The antibiotic-susceptible isolates were further treated with imipenem, and it was found that the omp genes were differentially expressed. Six of the nine genes studied were upregulated at 1 h of exposure to imipenem. Their expression gradually decreased with time, further confirmed by their OMP profile and two-dimensional gel electrophoresis. CONCLUSIONS: This study could identify OMPs that were differentially expressed on exposure to imipenem. Hence, this study provides insights into the role of specific OMPs in antibiotic resistance in A. baumannii.

Green synthesis of chitosan gum acacia based biodegradable polymeric nanoparticles to enhance curcumin's antioxidant property: an in vivo zebrafish (Danio rerio) study.

Green-synthesis of biodegradable polymeric curcumin-nanoparticles using affordable biodegradable polymers to enhance curcumin's solubility and anti-oxidative potential. The curcumin-nanoparticle was prepared based on the ionic-interaction method without using any chemical surfactants, and the particle-size, zeta-potential, surface-morphology, entrapmentefficiency, and in-vitro drug release study were used to optimise the formulation. The antioxidant activity was investigated using H2DCFDA staining in the zebrafish (Danio rerio) model. The mean-diameter of blank nanoparticles was 178.2 nm (±4.69), and that of curcuminnanoparticles was about 227.7 nm (±10.4), with a PDI value of 0.312 (±0.023) and 0.360 (±0.02). The encapsulation-efficacy was found to be 34% (±1.8), with significantly reduced oxidative-stress and toxicity (∼5 times) in the zebrafish model compared to standard curcumin. The results suggested that the current way of encapsulating curcumin using affordable, biodegradable, natural polymers could be a better approach to enhancing curcumin's water solubility and bioactivity, which could further be translated into potential therapeutics.

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.

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.

Differential expression of outer membrane proteins and quinolone resistance determining region mutations can lead to ciprofloxacin resistance in Salmonella Typhi.

Multi-drug resistance in Salmonella Typhi remains a public health concern globally. This study aimed to investigate the function of quinolone resistance determining region (QRDR) of gyrA and parC in ciprofloxacin (CIP) resistant isolates and examine the differential expression of outer membrane proteins (OMPs) on exposure to sub-lethal concentrations of CIP in S. Typhi. The CIP-resistant isolates were screened for mutations in the QRDR and analyzed for bacterial growth. Furthermore, major OMPs encoding genes such as ompF, lamB, yaeT, tolC, ompS1, and phoE were examined for differential expression under the sub-lethal concentrations of CIP by real-time PCR and SDS-PAGE. Notably, our study has shown a single-point mutation in gyrA at codon 83 (Ser83-tyrosine and Ser83-phenylalanine), also the rare amino acid substitution in parC gene at codon 80 (Glu80-glycine) in CIP-resistant isolates. Additionally, CIP-resistant isolates showed moderate growth compared to susceptible isolates. Although most of the OMP-encoding genes (tolC, ompS1, and phoE) showed some degree of upregulation, a significant level of upregulation (p < 0.05) was observed only for yaeT. However, ompF and lamB genes were down-regulated compared to CIP-susceptible isolates. Whereas OMPs profiling using SDS-PAGE did not show any changes in the banding pattern. These results provide valuable information on the QRDR mutation, and the difference in the growth, and expression of OMP-encoding genes in resistant and susceptible isolates of S. Typhi. This further provides insight into the involvement of QRDR mutation and OMPs associated with CIP resistance in S. Typhi.

Synthesis, Characterization, and Reactivity of High-Valent Carbene Dicarboxamide-Based Nickel Pincer Complexes.

High-valent metal-fluoride complexes are currently being explored for concerted proton-electron transfer (CPET) reactions, the driving force being the high bond dissociation energy of H-F (BDEH-F = 135 kcal/mol) that is formed after the reaction. Ni(III)-fluoride-based complexes on the pyridine dicarboxamide pincer ligand framework have been utilized for CPET reactions toward phenols and hydrocarbons. We have replaced the central pyridine ligand with an N-heterocyclic carbene carbene to probe its effect in both stabilizing the high-valent Ni(III) state and its ability to initiate CPET reactions. We report a monomeric carbene-diamide-based Ni(II)-fluoride pincer complex that was characterized through 1H/19F NMR, mass spectrometry, UV-vis, and X-ray crystallography analysis. Although carbenes and deprotonated carboxamides in the Ni(II)-fluoride complex are expected to stabilize the Ni(III) state upon oxidation, the Ni(III)/Ni(II) redox process occurred at very high potential (0.87 V vs Fc+/Fc, dichloromethane) and was irreversible. Structural studies indicate significant distortion in the imidazolium "NCN" carbene plane of Ni(II)-fluoride caused by the formation of six-membered metallacycles. The high-valent NiIII-fluoride analogue was synthesized by the addition of 1.0 equiv CTAN (ceric tetrabutylammonium nitrate) in dichloromethane at -20 °C which was characterized by UV-vis, mass spectrometry, and EPR spectroscopy. Density functional theory studies indicate that the Ni-carbene bond elongated, while the Ni-F bond shortened upon oxidation to the Ni(III) species. The high-valent Ni(III)-fluoride was found to react with the substituted phenols. Analysis of the KIE and linear free energy relationship correlates well with the CPET nature of the reaction. Preliminary analysis indicates that the CPET is asynchronous and is primarily driven by the E0' of the Ni(III)-fluoride complex.

Isothermal amplification-based assays for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2: Opportunities and recent developments.

The coronavirus disease 2019 (COVID-19) is a global pandemic caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, the virus has been detected in 219 countries of the world. Therefore, managing the disease becomes the priority, in which detecting the presence of the virus is a crucial step. Presently, real-time RT polymerase chain reaction (RT-qPCR) is considered a gold standard nucleic acid amplification test (NAAT). The test protocol of RT-qPCR is complicated, places high demands on equipment, testing reagents, research personnel skills and is expensive. Therefore, simpler point-of-care (POC) tests are needed to accelerate clinical decision-making and take some of the workload from centralized test laboratories. Various isothermal amplification-based assays have been developed for the sensitive detection of different microorganisms, and recently some of them have been applied for detection of SARS-CoV-2. These do not require any programable thermocycler, can produce the results in a single temperature, and therefore, are considered simple. Unlike RT-qPCR, these methods are highly sensitive, specific, less time-consuming, simple and affordable, and can be used as POC diagnostic kit for COVID-19. In this review, we have discussed the potential of isothermal amplification-based assays as an alternative to RT-qPCR for the detection of SARS-CoV-2.

Mechanistic Insight and Intersystem Crossing Dynamics of the C(3P) + H2CO/D2CO Reaction.

The reaction of atomic carbon, C(3P), with H2CO has been investigated using the direct dynamics trajectory surface hopping (DDTSH) method with Tully's fewest switches algorithm. The lowest lying ground triplet and single states are considered for the dynamics study at a reagent collision energy of 8.0 kcal/mol. From the trajectory calculations, we observed that CH2 + CO and H + HCCO are the two major product channels for the title reaction. The insertion mechanism of the C(3P) + H2CO reaction is rather complex and is followed by three distinct intermediates with no entrance channel barrier to the reaction on the B3LYP/6-31G(d,p) potential energy surfaces. The triplet insertion complexes are formed by three different approaches; "Sideways", "End-on" and "Head-on" attack of the triplet carbon atom toward H2CO molecule. Our dynamics calculations predict a new product channel (H + HCCO(X 2A'')) with a contribution of ∼46% of the overall products formation via ketocarbene intermediate through "Head-on" approach. Despite the weak spin-orbit coupling (SOC) interactions, intersystem crossing (ISC) via a ketocarbene intermediate has a small but significant contribution, about 2.3%, for the CH2 + CO channel. To understand the kinetic isotope effects on the reaction dynamics, we have extended our study for the C(3P) + D2CO reaction. It is seen that isotopic substitution of both the H atoms has a small reduction in the extent of ISC dynamics for the carbene formation. Our results, certainly, reveal the importance of the ketocarbene intermediate and the H + HCCO products channel as one of the major product formation channels in the title reaction, which was not reported earlier.

Polymerase chain reaction-based typing methods and protein profiling analysis of Acinetobacter baumannii isolated from environmental and clinical sources from South India.

Acinetobacter baumannii is an opportunistic pathogen known for causing hospital-acquired infections. The natural habitat includes soil, water, sewage, and drains, but it is also detected in infected individuals' blood, pus, and respiratory pathways. Due to its resilient nature, it is known to be a causative agent for outbreaks. Therefore, it is crucial to understand the genetic similarity between clinical and environmental isolates. The study aimed to find the genetic relationships between clinical and environmental isolates using PCR-based typing methods such as enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), random amplified polymorphic DNA (RAPD), and repetitive extragenic palindromic sequence-based PCR (Rep-PCR). Additionally, outer membrane protein (OMP) and whole cell protein (WCP) profiles were also used. The PCR-based methods, ERIC-PCR and Rep-PCR, showed decreased genetic similarity between clinical and environmental isolates (66% and 58%, respectively). However, RAPD showed relatively higher genetic similarity (91%). The OMP and WCP profiles showed varied banding patterns between the clinical and environmental isolates in the 29-43 kDa region. The PCR-based methods proved to be a reliable and reproducible technique. The OMP and WCP profiles, though not as discriminatory as the molecular typing methods, could help identify the most and least commonly occurring protein bands and thus help in typing clinical and environmental A. baumannii isolates.

Evaluation of loop-mediated isothermal amplification assay for visual detection of Acinetobacter baumannii directly from soil and water sample from Mangalore.

Acinetobacter baumannii is a well-known nosocomial pathogen that commonly inhabits soil and water and has been implicated in numerous hospital-acquired infections. The existing methods for detecting A. baumannii have several drawbacks, such as being time-consuming, expensive, labor-intensive, and unable to distinguish between closely related Acinetobacter species. Thus, it is important to have a simple, rapid, sensitive, and specific method for its detection. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay using hydroxynaphthol blue dye to visualize A. baumannii by targeting its pgaD gene. The LAMP assay was performed using a simple dry bath and was shown to be specific and highly sensitive, as it could detect up to 10 pg/µl of A. baumannii DNA. Further, the optimized assay was used to detect A. baumannii in soil and water samples by culture-medium enrichment. Out of 27 samples tested, 14 (51.85%) samples were positive for A. baumannii through LAMP assay, while only 5 (18.51%) samples were found to be positive through conventional methods. Thus, the LAMP assay has been found to be a simple, rapid, sensitive, and specific method that can be used as a point-of-care diagnostic tool for detecting A. baumannii.

Trajectory surface-hopping study of 1-pyrazoline photodissociation dynamics.

The photodissociation dynamics of 1-pyrazoline has been studied from its first excited electronic state (S1) using the Direct Dynamics Trajectory Surface-Hopping method in conjunction with Tully's fewest switches algorithm at the CASSCF(8,8)/6-31G* level of theory. After excitation of the molecule into the Franck-Condon region of the first excited state, S1, the molecule hops to the ground (S0) state quickly. The dissociation of one of the C-N bonds initially starts in the first excited state. Then, the molecule comes to the ground state (S0) via S1/S0 conical intersections, followed by complete dissociation in the ground state. Two different conical intersections are identified between the first excited singlet (S1) and the ground (S0) electronic states. One primary and three secondary dissociation channels are observed from our dynamics calculations of photodissociation of 1-pyrazoline that are in accord with the experimentally observed channels. After internal conversion to the ground electronic state (S0), the molecule dissociates to N2 and trimethylene biradical as the primary dissociation products. The trimethylene biradical then rearranges, leading to three secondary dissociation channels, N2 + cyclopropane, N2 + CH2 + C2H4, and N2 + CH3CHCH2. The major products formed from the trimethylene biradical in the secondary process is cyclopropane contributing about 78% of the overall products formation along with ∼12% propene and the rest ∼10% methylene (CH2) with ethene (C2H4).

Effect of sub-minimum inhibitory concentration of ceftriaxone on the expression of outer membrane proteins in Salmonella enterica serovar Typhi.

Multi-drug resistance (MDR) in Salmonella is one of the major reasons for foodborne outbreaks worldwide. Decreased susceptibility of Salmonella Typhi to first-line drugs such as ceftriaxone, ciprofloxacin, and azithromycin has raised concern. Reduced outer membrane proteins (OMPs) permeability and increased efflux pump transportation are considered to be the main reasons for the emergence of antibiotic resistance in Salmonella. The present study aimed to assess the expression of OMPs at sub-lethal concentrations of ceftriaxone in S. Typhi (Sl5037/BC, and Sl05). The S. Typhi strains were exposed to sub-MIC and half of the sub-MIC concentrations of ceftriaxone at three different time intervals (0 min, 40 min, and 180 min) and analyzed for differential expression of OMPs. Further, the expression variation of OMP encoding genes (yaeT, ompX, lamb, ompA, and ybfM) in response to ceftriaxone was evaluated using real-time PCR. The genes like lamB, ompX, and yaeT showed significant downregulation (p < 0.05) compared to the control without antibiotic exposure, whereas ybfM and ompA showed a moderate downregulation. The expression of omp genes such as lamB, ompA, ompX, ybfM, and yaeT were found to be low in the presence of ceftriaxone, followed by time and dose-dependent. The study provides insights into the possible involvement of OMPs in drug resistance of S. Typhi, which could help develop a therapeutic strategy to combat MDR isolates of S. Typhi.

Oral Tecovirimat for the Treatment of Smallpox.

BACKGROUND: Smallpox was declared eradicated in 1980, but variola virus (VARV), which causes smallpox, still exists. There is no known effective treatment for smallpox; therefore, tecovirimat is being developed as an oral smallpox therapy. Because clinical trials in a context of natural disease are not possible, an alternative developmental path to evaluate efficacy and safety was needed. METHODS: We investigated the efficacy of tecovirimat in nonhuman primate (monkeypox) and rabbit (rabbitpox) models in accordance with the Food and Drug Administration (FDA) Animal Efficacy Rule, which was interpreted for smallpox therapeutics by an expert advisory committee. We also conducted a placebo-controlled pharmacokinetic and safety trial involving 449 adult volunteers. RESULTS: The minimum dose of tecovirimat required in order to achieve more than 90% survival in the monkeypox model was 10 mg per kilogram of body weight for 14 days, and a dose of 40 mg per kilogram for 14 days was similarly efficacious in the rabbitpox model. Although the effective dose per kilogram was higher in rabbits, exposure was lower, with a mean steady-state maximum, minimum, and average (mean) concentration (Cmax, Cmin, and Cavg, respectively) of 374, 25, and 138 ng per milliliter, respectively, in rabbits and 1444, 169, and 598 ng per milliliter in nonhuman primates, as well as an area under the concentration-time curve over 24 hours (AUC0-24hr) of 3318 ng×hours per milliliter in rabbits and 14,352 ng×hours per milliliter in nonhuman primates. These findings suggested that the nonhuman primate was the more conservative model for the estimation of the required drug exposure in humans. A dose of 600 mg twice daily for 14 days was selected for testing in humans and provided exposures in excess of those in nonhuman primates (mean steady-state Cmax, Cmin, and Cavg of 2209, 690, and 1270 ng per milliliter and AUC0-24hr of 30,632 ng×hours per milliliter). No pattern of troubling adverse events was observed. CONCLUSIONS: On the basis of its efficacy in two animal models and pharmacokinetic and safety data in humans, tecovirimat is being advanced as a therapy for smallpox in accordance with the FDA Animal Rule. (Funded by the National Institutes of Health and the Biomedical Advanced Research and Development Authority; ClinicalTrials.gov number, NCT02474589 .).

Evaluation of loop-mediated isothermal amplification assay along with conventional and real-time PCR assay for sensitive detection of pathogenic Vibrio parahaemolyticus from seafood sample without enrichment.

The primary reason for foodborne illness is improper seafood safety testing, and hence, an appropriate tool for testing is the key to control the outbreaks. The current study aimed to develop a loop-mediated isothermal amplification (LAMP) assay to detect pathogenic Vibrio parahaemolyticus, important foodborne pathogen, targeting tdh, and trh genes. The specificity of the LAMP assay was good without any false-positive and false-negative results. The assay was highly sensitive and could detect the pathogenic V. parahaemolyticus as low as 1 CFU/reaction in spiked seafood samples and 1 pg of extracted DNA. Out of 62 seafood samples from India's southwest coastal region tested with LAMP assay, eight (12.9%) were positive for trh, and seven (11.29%) samples were positive tdh gene. LAMP-based on tdh and trh was found to be significantly more sensitive (p < 0.05) than conventional PCR and nearly equal sensitive as real-time PCR (RT-PCR) for the detection of pathogenic V. parahaemolyticus. Our study shows that LAMP assay can be a better approach as a point-of-care (POC) diagnostic tool and could detect pathogenic V. parahaemolyticus on seafood samples directly without enrichment and isolation. The high sensitivity and simplicity make LAMP assay a better alternative method than the conventional method and RT-PCR for the detection of pathogens. LAMP assay can be considered as a good alternative to PCR for the routine detection of pathogenic V. parahaemolyticus in seafood.

Photodissociation Dynamics of Methyl Hydroperoxide at 193 nm: A Trajectory Surface-Hopping Study.

The photodissociation of methyl hydroperoxide (CH3OOH) at 193 nm has been studied using a direct dynamics trajectory surface-hopping (TSH) method. The potential energies, energy gradients, and nonadiabatic couplings are calculated on the fly at the MRCIS(6,7)/aug-cc-pVDZ level of theory. The hopping of a trajectory from one electronic state to another is decided on the basis of Tully's fewest switches algorithm. An analysis of the trajectories reveals that the cleavage of the weakest O-O bond leads to major products CH3O(2E) + OH(2Π), contributing about 72.7% of the overall product formation. This OH elimination was completed in the ground degenerate product state where both the ground singlet (S0) and first excited singlet (S1) states become degenerate. The O-H bond dissociation of CH3OOH is a minor channel contributing about 27.3% to product formation, resulting in products CH3OO + H. An inspection of the trajectories indicates that unlike the major channel OH elimination, the H-atom elimination channel makes a significant contribution (∼3% of the overall product formation) through the nonadiabatic pathway via conical intersection S1/S0 leading to ground-state products CH3OO(X 2A″) + H(2S) in addition to adiabatic dissociation in the first excited singlet state, S1, correlating to products CH3OO(1 2A') + H(2S). The computed translational energy of the majority of the OH products is found to be high, distributed in the range of 70 to 100 kcal/mol, indicating that the dissociation takes place on a strong repulsive potential energy surface. This finding is consistent with the nature of the experimentally derived translational energy distribution of OH with an average translational energy of 67 kcal/mol after the excitation of CH3OOH at 193 nm.

Importance of intersystem crossing in the C(3P) + SiH4 reaction.

The contribution of intersystem crossing (ISC) in the C(3P) + SiH4 reaction that leads to products formation in the singlet electronic state is investigated using a direct dynamics trajectory surface hopping (TSH) method with Tully's fewest switches algorithm. Interestingly, in contrast to the O(3P) + SiH4 reaction with no ISC effect, for the title reaction we observed ∼7% product formation through ISC despite weak spin-orbit coupling interactions (less than 25 cm-1) between the ground singlet and triplet states. This is presumably because of the topological differences in the potential energy surfaces of the two reactions at the entrance channel. The O(3P) + SiH4 reaction follows either an addition reaction (with shallow attractive potential and a late singlet-triplet crossing) or a direct abstraction pathway with singlet-triplet crossing at near or after the top of the barrier making ISC ineffective. On the other hand, an insertion mechanism is exclusively followed by the C(3P) + SiH4 reaction with no entrance barrier to the reaction in the triplet state. The triplet insertion complex initially formed (3H3SiCH) can go to the singlet state through ISC due to the fact that the triplet-singlet crossing is accessed several times during the course of the reaction. Our computed overall product angular distributions for H and H2 elimination channels are found to be broad and flat or nearly isotropic in nature indicating the formation of stable intermediate complexes, which corroborates the most recent crossed molecular-beam study.

Rapid visual detection of Vibrio parahaemolyticus in seafood samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye.

The detection and monitoring of Vibrio parahaemolyticus pathogen in aquatic foods have become essential for preventing outbreaks. In this study, loop-mediated isothermal amplification (LAMP) assay with the azo dye, hydroxynaphthol blue (HNB) was developed targeting species-specific tlh gene. The assay was carried out on 62 seafood samples that included clam and shrimp and compared with conventional LAMP assay performed with the commonly used fluorescent dye, conventional PCR, and real-time PCR (RT-PCR). Of 62 samples studied for tlh gene, 32 (51.61%) gave positive by HNB-LAMP, which comprised 22 (70.96%) clam samples and 10 (32.25%) shrimp samples. The HNB-LAMP assay was found to be highly sensitive, specific, and superior to conventional PCR (p > 0.05). RT-PCR presented higher sensitivity than HNB-LAMP; however, it has the limitation of being cost-intensive and requiring technical expertise to perform. HNB-LAMP is affordable, rapid, simple, and easy to perform, allowing naked eye visualization.

Genotypic and phenotypic characterization of Edwardsiella isolates from different fish species and geographical areas in Asia: Implications for vaccine development.

The genus Edwardsiella is one of the major causes of fish diseases globally. Herein, we examined 37 isolates from ten different fish species from India, South Korea and Taiwan to gain insight into their phenotypic and genotypic properties, of which 30 were characterized as E. tarda with phenotypic homology estimated at 85.71% based on API-20E biochemical tests. Genotyping using 16S rRNA put all isolates together with E. anguillarum, E. hoshinae, E. tarda, E. piscicida and E. ictaluri reference strains in a monophyletic group. In contrast, the gyrB phylogenetic tree clearly separated E. ictaluri, E. tarda and E. hoshinae reference strains from our isolates and put our isolates into two groups with group I being homologous with the E. anguillarum reference strain while group II was homologous with the E. piscicida reference strain. Hence, our findings point to E. piscicida and E. anguillarum as species infecting different fish species in Asia. Homology of the ompW protein suggested that strains with broad protective coverage could be identified as vaccine candidates. This study underscores the importance of combining genotyping with phenotyping for valid species classification. In addition, it accentuates the importance of phylogenetic comparison of bacterial antigens for identification of potential vaccine candidates.

Dual Fluorophore Containing Efficient Photoinduced Electron Transfer Based Molecular Probe for Selective Detection of Cr3+ and PO43- Ions through Fluorescence " Turn-On-Off" Response in Partial Aqueous and Biological Medium: Live Cell Imaging and Logic Application.

The present work describes a new photoinduced electron transfer (PET) based molecular probe in which naphthalimide (NPI) and anthracene (AN) chromophores are linked through a molecular bridge of piperazine and triazole units by the Click reaction. A typical meaningful structural variation has made the present probe highly selective for Cr3+ ion (limit of detection (LOD), 5.567 × 10-8 M) that displayed enhanced, " turn-On" emission (due to the PET- Off photophysical mechanism) and naked-eye sensitive bright green color fluorescence in the environment of interfering and competitive ions, in Tris-HCl buffer. The minimum energy structure obtained through theoretical calculations (density functional theory (DFT) and time-dependent (TD)-DFT) revealed a "tub" shape structure for probe 10. Upon complexation, the conformation of piperazine fragment changes from chair to boat in which the triazole and piperazine units create a cavity to tether Cr3+. Moreover, the probe showed excellent biocompatibility and cell permeability to sense Cr3+ sensitively in live cells and, thus, holds great promise for application in biological and environmental sciences. Additionally, the sensitive " Off-On-Off" sensing behavior of probe 10 providing two chemical inputs (Cr3+ and PO43-) helps to construct an INHIBIT logic gate. Also the probe has been utilized as printing material to decode secret information through the Cr3+ ion containing "marker ink" under UV light.

Influence of Salinity on the Mechanism of Surface Icing: Implication to the Disappearing Freezing Singularity.

The effect of salinity on surface icing has been investigated experimentally. Water droplets with a variable salinity are deposited on a cold polished copper substrate. Distinctive two-stage freezing, which can be seen in case of pure water, is not observed in heterogeneous freezing of saltwater droplets. Interestingly, the final freezing stage commences before the initial freezing front completely traverses the saline droplet. A considerable increase in delay for heterogeneous ice nucleation is observed with the increasing salinity. The reduction in the associated degree of metastability due to the depression in the freezing point of the bulk solution and the increase in the nucleation barrier due to the appearance of the solvation shells that are formed around the ions are two possible causes of this nucleation delay. Moreover, the solidification time associated with surface icing increases considerably with the increasing salinity. Because of the insolubility of salt in ice, the salt ions are rejected to the entrapped water in the ice scaffold locally and to the bulk unfrozen water explicitly. This collective implicit and explicit modes of brine rejection contributes to the overall slowdown of freezing of the saline water droplets. From the phase diagram, it can be found that the complete solidification of water within the saline droplet is not possible when the substrate temperature is in between the eutectic temperature and the equilibrium freezing temperature. As a result, the relative magnitude of tip singularity during freezing reduces considerably with the increasing salinity due to the increase in unfrozen water content within the droplet.