A high-throughput plate method for nucleic acid extraction from beet leafhopper (Hemiptera: Cicadellidae) and potato psyllid (Hemiptera: Triozidae) for pathogen detection.

Plant pathogens that are transmitted by insect vectors cause considerable damage to crops when pests or pathogens are not detected early in the season and populations are not controlled. Knowledge of pathogen prevalence in insect pest populations can aid growers in their insect pest management decisions but requires the timely dissemination of results. This process requires that specimen capture, identification, nucleic acid extraction, and molecular detection of a pathogen(s) occur alongside a platform for sharing results. The potato psyllid (Bactericera cockerelli, Sulc; Hemiptera: Triozidae) and beet leafhopper (Circulifer tenellus, Baker; Hemiptera: Cicadellidae) transmit pathogens to potato and other vegetable or seed crops each season in the northwestern United States. While the potato psyllid has been tested for pathogen occurrence for the past decade, testing of the beet leafhopper is a new endeavor and substantially increases the specimen number that must be tested by our laboratories each season. To aid in the rapid processing of individual insect specimens, we optimized and validated a new high-throughput 96-well plate nucleic acid extraction method for use in place of a standard 1.5-ml single-tube extraction method. Processing efficiency, in terms of total specimens processed over a 2-day period, improved 2.5-fold, and the cost associated with processing a single sample was nearly cut in half with this newly developed plate nucleic acid extraction method. Overall, this method has proven to be an excellent tool for the rapid testing of large numbers of small, individual insect vectors to enable timely dissemination of data on pathogen prevalence to growers.

New Assays for Rapid Detection of Beet Leafhopper-Associated Plant Pathogens, 'Candidatus Phytoplasma trifolii', Beet Curly Top Virus, and Spiroplasma citri.

The beet leafhopper Circulifer tenellus is an important pest of agricultural crops in the United States, where it transmits beet curly top virus, beet leafhopper-transmitted virescence agent phytoplasma, and Spiroplasma citri to numerous crops, affecting yield and quality. Each of these pathogens have been linked to serious disease outbreaks within Washington State in the past century. To mitigate the risk of disease, growers target the beet leafhopper in their insect pest management programs. Knowledge of pathogen prevalence in beet leafhopper populations could help growers make better management decisions, but timely diagnostics is required. Four new assays were developed for the rapid detection of the beet leafhopper-associated pathogens. These include two assays that detect Beet leafhopper transmitted virescence agent (a PCR and a real-time PCR SYBR green assay), a duplex PCR assay that simultaneously detects beet curly top virus and Spiroplasma citri, and a multiplex real-time PCR assay for the simultaneous detection of all three pathogens. The screening of dilution series generated from plant total nucleic acid extracts with these new assays typically led to detection at levels 10- to 100-fold more sensitive than the conventional PCR assays currently used. These new tools will allow the rapid detection of beet leafhopper-associated pathogens in both plant and insect specimens and will have the potential to be used in diagnostic laboratories seeking to disseminate fast and accurate results to growers for implementation in their insect pest monitoring programs.

Immunoglobulin M seroneutralization for improved confirmation of Japanese encephalitis virus infection in a flavivirus-endemic area.

BACKGROUND: The mainstay of diagnostic confirmation of acute Japanese encephalitis (JE) involves detection of anti-JE virus (JEV) immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA). Limitations in the specificity of this test are increasingly apparent with the introduction of JEV vaccinations and the endemicity of other cross-reactive flaviviruses. Virus neutralization testing (VNT) is considered the gold standard, but it is challenging to implement and interpret. We performed a pilot study to assess IgG depletion prior to VNT for detection of anti-JEV IgM neutralizing antibodies (IgM-VNT) as compared with standard VNT. METHODS: We evaluated IgM-VNT in paired sera from anti-JEV IgM ELISA-positive patients (JE n=35) and negative controls of healthy flavivirus-naïve (n=10) as well as confirmed dengue (n=12) and Zika virus (n=4) patient sera. IgM-VNT was subsequently performed on single sera from additional JE patients (n=76). RESULTS: Anti-JEV IgG was detectable in admission serum of 58% of JE patients. The positive, negative and overall percentage agreement of IgM-VNT as compared with standard VNT was 100%. A total of 12/14 (86%) patient samples were unclassified by VNT and, with sufficient sample available for IgG depletion and IgG ELISA confirming depletion, were classified by IgM-VNT. IgM-VNT enabled JE case classification in 72/76 (95%) patients for whom only a single sample was available. CONCLUSIONS: The novel approach has been readily adapted for high-throughput testing of single patient samples and it holds promise for incorporation into algorithms for use in reference centres.

Degradable Anti-Biofouling Polyester Coatings with Controllable Lifetimes.

To achieve degradable, anti-biofouling coatings with longer lifetimes and better mechanical properties, we synthesized a series of degradable co-polyesters composed of cyclic ketene acetals, di-(ethylene glycol) methyl ether methacrylate, and a photoactive curing agent, 4-benzoylphenyl methacrylate, using a radical ring-opening polymerization. The precursor co-polyesters were spin-coated on a benzophenone-functionalized silicon wafer to form ca. 60 nm films and drop-casted on glass to form ∼32 µm films. The copolymers were cross-linked via UV irradiation at 365 nm. The degradation of films was studied by immersing the specimens in aqueous buffers of different pH values. The results show that both the pH of buffer solutions and gel fractions of networks affect the degradation rate. The coatings show good bovine serum albumin resistance capability. By adjusting the fractions of monomers, the degradation rate and degree of hydration (e.g., swelling ratio) are controllable.

Dynamic Surfaces-Degradable Polyester Networks that Resist Protein Adsorption.

We synthesized a series of novel degradable alternating copolyesters composed of diglycolic anhydride (DGA) and two epoxides, epoxymethoxytriethylene glycol (ETEG) and a photoactive crosslinking agent epoxy benzophenone (EBP). After UV crosslinking, soaking the films in a good solvent (tetrahydrofuran) removed uncrosslinked material, and the resulting film gel fractions were calculated. These network films were then degraded in buffer solutions of varying pH values. The degradation of networks with lower gel fraction (fewer crosslinks) was faster and followed first-order kinetics. In contrast, the denser network degraded slower and followed zeroth-order kinetics. The lower gel fraction networks possess a higher swelling ratio and resist bovine serum albumin (BSA) adsorption better by entropic shielding and faster degradation. In comparison, higher gel fraction networks with higher EBP mole fractions adsorb more BSA due to hydrophobic interactions and slower degradation.

Amphiphilic phospholipid-iodinated polymer conjugates for bioimaging.

Amphiphilic phospholipid-iodinated polymer conjugates were designed and synthesized as new macromolecular probes for a highly radiopaque and biocompatible imaging technology. Bioconjugation of PEG 2000-phospholipids and iodinated polyesters by click chemistry created amphiphilic moieties with hydrophobic polyesters and hydrophilic PEG units, which allowed their self-assemblies into vesicles or spiked vesicles. More importantly, the conjugates exhibited high radiopacity and biocompatibility in in vitro X-ray and cell viability measurements. This new type of bioimaging contrast agent with a Mn value of 11 289 g mol-1 was found to have a significant X-ray signal at 3.13 mg mL-1 of iodine equivalent than baseline and no cytotoxicity after 48 hours incubation of with HEK and 3T3 cells at 20 µM (20 picomoles) concentration of conjugates per well. The potential of adopting the described macromolecular probes for bioimaging was demonstrated, which could further promote the development of a field-friendly and highly sensitive bioimaging contrast agent for point-of-care diagnostic applications.

Whole genome sequencing and phylogenetic characterization of a novel bat-associated picornavirus-like virus with an unusual genome organization.

The order Picornavirales is one of the most important viral orders in terms of virus diversity and genome organizations, ranging from a mono- or bi-cistronic expression strategies to the recently described poly-cistronic Polycipiviridae viruses. We report here the description and characterization of a novel picorna-like virus identified in rectal swabs of frugivorous bats in Cambodia that presents an unusual genome organization. Kandabadicivirus presents a unique genome architecture and distant phylogenetic relationship to the proposed Badiciviridae family. These findings highlight a high mosaicism of genome organizations among the Picornavirales.

Synthesis of 5,12-Diazapentacenes and Their Properties.

An efficient synthesis via a precursor route to a new class of linear dialkyldiaminoazapentacenes is reported. The synthetic route involves the coupling of 4-substituted aniline derivatives to 2,5-dibromoterephthalonitrile via Buchwald-Hartwig amination followed by an acid-mediated cyclization to furnish the diazapentacenes. These reactions occur under short reaction times (<2 h), provide high yields (77-99%), and do not require column chromatography for purification. The electrochemical and optical properties of the diazapentacenes were evaluated, and the values indicate that these molecules are promising n-type materials for organic electronic devices. The photostability of these molecules was significantly greater than unsubstituted acenes. Their method of degradation via endoperoxide generation was confirmed by X-ray crystallography and mass spectrometry.

DendriPeps: Expanding Dendrimer Functionality by Hybridizing Poly(amidoamine) (PAMAM) Scaffolds with Peptide Segments.

In this work, the first synthesis of poly(amidoamine) (PAMAM) dendrimers whose branches are hybridized with peptide segments (DendriPeps) is reported. The intercalation of amino acids within the branches of PAMAMs provides supplementary internal functionalities to the coronal groups. Four DendriPep prototypes are synthesized with lysine or glutamic acid as "guest" amino acids, displaying, respectively, a primary amine or a carboxyl group, on generation (G)2 and G3 PAMAMs as host scaffolds. The precise control over the number, type, and topological placement of functional groups expands the functional behavior of DendriPeps beyond current PAMAM dendrimers toward new frontiers or colloids, drug delivery vectors, and catalysis.

Corrigendum: Impact on clinical practice of a non-invasive gene expression melanoma rule-out test: 12-month followup of negative test results and utility data from a large US registry study.

The revised version of the article corrected Figure 2. This change appears in the revised online PDF copy of this article.

Impact on clinical practice of a non-invasive gene expression melanoma rule-out test: 12-month follow-up of negative test results and utility data from a large US registry study.

The Pigmented Lesion Assay (PLA, sensitivity 91-95%, specificity 69-91%, negative predictive value ?99%) is a commercially available, non-invasive gene expression test that helps dermatologists guide pigmented lesion management decisions and rule out melanoma. Earlier studies have demonstrated high clinical utility and no missed melanomas in a 3-6-month follow-up period. We undertook the current investigations to provide 12-month follow-up data on PLA(-) tests, and to further confirm utility. A 12-month chart review follow-up of 734 pigmented lesions that had negative PLA results from 5 US dermatology centers was performed. Thirteen of these lesions (1.8%) were biopsied in the follow-up period and submitted for histopathologic review. None of the lesions biopsied had a histopathologic diagnosis of melanoma. The test's utility was studied further in a registry (N=1575, 40 US dermatology offices, 62 participating providers), which demonstrated that 99.9% of PLA(-) lesions were clinically monitored, thereby avoiding a surgical procedure, and 96.5% of all PLA(+) lesions were appropriately biopsied, most commonly with a tangential shave. This long-term follow-up study confirms the PLA's high negative predictive value and high utility in helping guide the management of pigmented lesions to avoid unnecessary surgical procedures.

A Novel Polycipiviridae Virus Identified in Pteropus lylei Stools.

Polycipiviridae is a recently recognized viral family within the order Picornavirales with unusual genome organization and phylogenetic placement. Viruses belonging to this family were only reported from arthropod hosts. We describe here the first full genome of a distant polycipivirus-related virus identified in frugivorous bat stools in Cambodia.

Viral RNA Degradation Makes Urine a Challenging Specimen for Detection of Japanese Encephalitis Virus in Patients With Suspected CNS Infection.

BACKGROUND: Japanese encephalitis virus (JEV) is a leading cause of central nervous system (CNS) infections in Asia and results in significant morbidity and mortality. JEV RNA is rarely detected in serum or cerebrospinal fluid (CSF), and diagnosis of JEV infection is usually based on serological tests that are frequently difficult to interpret. Unlike serum or CSF, urine is relatively easy to obtain, but, to date, there has been minimal work on the feasibility of testing urine for JEV RNA. METHODS: We investigated the use of lysis buffer and a Microsep device to optimize urine storage for detection of JEV RNA by reverse transcription real-time polymerase chain reaction (RT-qPCR). The best of the studied methods was then evaluated in consecutive patients admitted to the hospital with suspected CNS infections in Laos. RESULTS: We demonstrated degradation of JEV RNA in urine after even short storage periods at 4°C or -80°C. Although there was no advantage in using a Microsep concentration device alone, immediate addition of lysis buffer to fresh urine improved the detection of JEV RNA at the limit of detection. CONCLUSIONS: In 2 studies of 41 patients with acute encephalitis syndrome, 11 (27%) were positive for JEV IgM in CSF and/or serum, and 2 (4.9%) were JEV RT-qPCR positive from throat swabs. JEV RNA was not detected in any of these patients' urine samples. However, lysis buffer was only used during a prospective study, that is, for only 17/41 (41%) patient urine samples. Our findings suggest a need for larger studies testing urine for JEV RNA, with urine collected at different times from symptom onset, and using lysis buffer, which stabilizes RNA, for storage.

A citizen science model for implementing statewide educational DNA barcoding.

Our aim was to develop a widely available educational program in which students conducted authentic research that met the expectations of both the scientific and educational communities. This paper describes the development and implementation of a citizen science project based on DNA barcoding of reptile specimens obtained from the Museums Victoria frozen tissue collection. The student program was run by the Gene Technology Access Centre (GTAC) and was delivered as a "one day plus one lesson" format incorporating a one-day wet laboratory workshop followed by a single lesson at school utilising online bioinformatics tools. The project leveraged the complementary resources and expertise of the research and educational partners to generate robust scientific data that could be analysed with confidence, meet the requirements of the Victorian state education curriculum, and provide participating students with an enhanced learning experience. During two 1-week stints in 2013 and 2014, 406 students mentored by 44 postgraduate university students participated in the project. Students worked mainly in pairs to process ~200 tissue samples cut from 53 curated reptile specimens representing 17 species. A total of 27 novel Cytochrome Oxidase subunit 1 (CO1) sequences were ultimately generated for 8 south-east Australian reptile species of the families Scincidae and Agamidae.

Liquid Metal Nanoparticles as Initiators for Radical Polymerization of Vinyl Monomers.

Sonication of gallium or gallium-based liquid metals in an aqueous solution of vinyl monomers leads to rapid free radical polymerization (FRP), without the need for conventional molecular initiators. Under ambient conditions, a passivating native oxide separates these metals from solution and renders the metal effectively inert. However, sonication generates liquid metal nanoparticles (LMNPs) of ∼100 nm diameter and thereby increases the surface area of the metal. The exposed metal initiates polymerization, which proceeds via a FRP mechanism and yields high molecular weight polymers that can form physical gels. Spin trapping EPR reveals the generation of free radicals. Time-of-flight secondary ion mass spectrometry measurements confirm direct polymer bonding to gallium, verifying the formation of surface-anchored polymer grafts. The grafted polymers can modify the interfacial properties, that is, the preference of the metal particles to disperse in aqueous versus organic phases. The polymer can also be degrafted and isolated from the particles using strong acid or base. The concept of physically disrupting passivated metal surfaces offers new routes for surface-initiated polymerization and has implications for surface modification, reduction reactions, and fabrication of mechanically responsive materials.

Real-world performance and utility of a noninvasive gene expression assay to evaluate melanoma risk in pigmented lesions.

About 3 million surgical pigmented skin lesion biopsies are performed each year in the USA alone to diagnose fewer than 200 000 new cases of invasive melanoma and melanoma in situ using the current standard of care that includes visual assessment and histopathology. A recently described noninvasive adhesive patch-based gene expression rule-out test [pigmented lesion assay (PLA)] may be helpful in identifying high-risk pigmented skin lesions to aid with surgical biopsy decisions. The main objective of this utility study was to determine the real-world clinical performance of PLA use and assess how the PLA changes physician behavior in an observational cohort analysis of 381 patients assessed with the PLA. All (100%) of 51 PLA(+) test results were clinically managed with surgical biopsy. Of these, 19 (37%) were melanomas, corresponding to a number needed to biopsy of 2.7 and a biopsy ratio of 1.7. All melanomas were histopathologically classified as melanoma in situ or stage 1. Nearly all (99%) of 330 PLA(-) test results were clinically managed with surveillance. None of the three follow-up biopsies performed in the following 3-6 months, were diagnosed as melanoma histopathologically. The estimated sensitivity and specificity of the PLA from these data sets are 95 and 91%, respectively. Overall, 93% of PLA results positive for both LINC00518 and PRAME were diagnosed histopathologically as melanoma. PRAME-only and LINC00518-only lesions were melanomas histopathologically in 50 and 7%, respectively. The PLA alters clinical management of pigmented lesions and shows high clinical performance. The likelihood of positive histopathologic diagnosis of melanoma is higher in PLA results that are positive for both LINC00518 and PRAME.

Detection of Japanese Encephalitis Virus RNA in Human Throat Samples in Laos - A Pilot study.

Japanese encephalitis virus (JEV) is the most commonly identified cause of acute encephalitis syndrome (AES) in Asia. The WHO recommended test is anti-JEV IgM-antibody-capture-enzyme-linked-immunosorbent-assay (JEV MAC-ELISA). However, data suggest this has low positive predictive value, with false positives related to other Flavivirus infections and vaccination. JEV RT-PCR in cerebrospinal fluid (CSF) and/or serum is highly specific, but is rarely positive; 0-25% of patients that fulfil the WHO definition of JE (clinical Acute Encephalitis Syndrome (AES) and JEV MAC-ELISA positive). Testing other body fluids by JEV RT-qPCR may improve the diagnosis. As a pilot study thirty patients admitted to Mahosot Hospital 2014-2017, recruited to the South-East-Asia-Encephalitis study, were tested by JEV MAC-ELISA and two JEV real-time RT-PCR (RT-qPCR) assays (NS2A and NS3). Eleven (36.7%) were JEV MAC-ELISA positive. Available CSF and serum samples of these patients were JEV RT-qPCR negative but 2 (7%) had JEV RNA detected in their throat swabs. JEV RNA was confirmed by re-testing, and sequencing of RT-qPCR products. As the first apparent report of JEV RNA detection in human throat samples, the provides new perspectives on human JEV infection, potentially informing improving JEV detection. We suggest that testing patients' throat swabs for JEV RNA is performed, in combination with molecular and serological CSF and serum investigations, on a larger scale to investigate the epidemiology of the presence of JEV in human throats. Throat swabs are an easy and non-invasive tool that could be rolled out to a wider population to improve knowledge of JEV molecular epidemiology.

Isolation and full-genome sequences of Japanese encephalitis virus genotype I strains from Cambodian human patients, mosquitoes and pigs.

Japanese encephalitis remains the most important cause of viral encephalitis in humans in several southeast Asian countries, including Cambodia, causing at least 65 000 cases of encephalitis per year. This vector-borne viral zoonosis - caused by Japanese encephalitis virus (JEV) - is considered to be a rural disease and is transmitted by mosquitoes, with birds and pigs being the natural reservoirs, while humans are accidental hosts. In this study we report the first two JEV isolations in Cambodia from human encephalitis cases from two studies on the aetiology of central nervous system disease, conducted at the two major paediatric hospitals in the country. We also report JEV isolation from Culextritaeniorhynchus mosquitoes and from pig samples collected in two farms, located in peri-urban and rural areas. Out of 11 reverse-transcription polymerase chain reaction-positive original samples, we generated full-genome sequences from 5 JEV isolates. Five additional partial sequences of the JEV NS3 gene from viruses detected in five pigs and one complete coding sequence of the envelope gene of a strain identified in a pig were generated. Phylogenetic analyses revealed that JEV detected in Cambodia belonged to genotype I and clustered in two clades: genotype I-a, mainly comprising strains from Thailand, and genotype I-b, comprising strains from Vietnam that dispersed northwards to China. Finally, in this study, we provide proof that the sequenced JEV strains circulate between pigs, Culex tritaeniorhynchus and humans in the Phnom Penh vicinity.

Aetiology of acute meningoencephalitis in Cambodian children, 2010-2013.

Acute meningoencephalitis (AME) is associated with considerable morbidity and mortality in children in developing countries. Clinical specimens were collected from children presenting with AME at two Cambodian paediatric hospitals to determine the major aetiologies associated with AME in the country. Cerebrospinal fluid (CSF) and blood samples were screened by molecular and cell culture methods for a range of pathogens previously associated with AME in the region. CSF and serum (acute and convalescent) were screened for antibodies to arboviruses such as Japanese encephalitis virus (JEV), dengue virus (DENV), and chikungunya virus (CHIKV). From July 2010 through December 2013, 1160 children (one month to 15 years of age) presenting with AME to two major paediatric hospitals were enroled into the study. Pathogens associated with AME were identified using molecular diagnostics, cell culture and serology. According to a diagnostic algorithm, a confirmed or highly probable aetiologic agent was detected in 35.0% (n=406) of AME cases, with a further 9.2% (total: 44.2%, n=513) aetiologies defined as suspected. JEV (24.4%, n=283) was the most commonly identified pathogen followed by Orientia tsutsugamushi (4.7%, n=55), DENV (4.6%, n=53), enteroviruses (3.5%, n=41), CHIKV (2.0%, n=23) and Streptococcus pneumoniae (1.6%, n=19). The majority of aetiologies identified for paediatric AME in Cambodia were vaccine preventable and/or treatable with appropriate antimicrobials.