Using a Genome-Based PCR Primer Prediction Pipeline to Develop Molecular Diagnostics for the Turfgrass Pathogen Acidovorax avenae.

Acidovorax avenae is the causal agent of bacterial etiolation and decline (BED) of creeping bentgrass, a poorly understood and often misdiagnosed disease that can result in considerable aesthetic and functional damage to golf course putting greens. Current diagnostics of BED are based on laborious culture-based methods. In this work, we employed a novel alignment-free primer prediction pipeline to design diagnostic primers for turfgrass-pathogenic A. avenae using 15 draft genomes of closely related target and nontarget Acidovorax spp. as input. Twenty candidate primer sets specific to turfgrass-pathogenic A. avenae were designed. The specificity and sensitivity of these primer sets were validated via a traditional polymerase chain reaction (PCR) and a real-time PCR assay. Primer sets 0017 and 0019 coupled with an internal oligo probe showed optimal sensitivity and specificity when evaluated with the target pathogen, closely related bacterial species, and microorganisms that inhabit the same host and soil environment. Finally, the accuracy of the newly developed real-time PCR assay was evaluated to detect BED pathogens from BED-symptomatic and asymptomatic turfgrass samples. The diagnostic results produced by the real-time PCR assay were consistent with results of a cultural-based method. This assay will allow quicker and more effective detection of the BED pathogen, thus potentially reducing misdiagnoses and unnecessary usage of fungicides.

Recombination of Virulence Genes in Divergent Acidovorax avenae Strains That Infect a Common Host.

Bacterial etiolation and decline (BED), caused by Acidovorax avenae, is an emerging disease of creeping bentgrass on golf courses in the United States. We performed the first comprehensive analysis of A. avenae on a nationwide collection of turfgrass- and maize-pathogenic A. avenae. Surprisingly, our results reveal that the turfgrass-pathogenic A. avenae in North America are not only highly divergent but also belong to two distinct phylogroups. Both phylogroups specifically infect turfgrass but are more closely related to maize pathogens than to each other. This suggests that, although the disease is only recently reported, it has likely been infecting turfgrass for a long time. To identify a genetic basis for the host specificity, we searched for genes closely related among turfgrass strains but distantly related to their homologs from maize strains. We found a cluster of 11 such genes generated by three ancient recombination events within the type III secretion system (T3SS) pathogenicity island. Ever since the recombination, the cluster has been conserved by strong purifying selection, hinting at its selective importance. Together our analyses suggest that BED is an ancient disease that may owe its host specificity to a highly conserved cluster of 11 T3SS genes.

Identification, Characterization, and Distribution of Acidovorax avenae subsp. avenae Associated with Creeping Bentgrass Etiolation and Decline.

Bacterial etiolation and decline caused by Acidovorax avenae subsp. avenae is an emerging disease of creeping bentgrass (Agrostis stolonifera) in and around the transition zone, a unique area of turfgrass culture between cool and warm regions of the United States. It is suspected that the disease has been present for many years, although diagnosis of the first occurrence was not reported until 2010. Solicitation of samples from golf courses in 2010 and 2011 was undertaken to investigate the prevalence and dissemination of Acidovorax avenae subsp. avenae on creeping bentgrass. At least 21 isolates from 13 states associated with these outbreaks on golf courses were confirmed as A. avenae subsp. avenae by pathogenicity assays and 16S rDNA sequence analysis at two independent locations. Pathogenicity testing of bacterial isolates from creeping bentgrass samples exhibiting heavy bacterial streaming confirmed A. avenae subsp. avenae as the only bacterium to cause significant disease symptoms and turfgrass decline. Host range inoculations revealed isolates of A. avenae subsp. avenae to be pathogenic on all Agrostis stolonifera cultivars tested, with slight but significant differences in disease severity on particular cultivars. Other turfgrass hosts tested were only mildly susceptible to Acidovorax avenae subsp. avenae infection. This study initiated research on A. avenae subsp. avenae pathogenicity causing a previously uncharacterized disease of creeping bentgrass putting greens in the United States.

The availability of global guidance for the promotion of women's, newborns', children's and adolescents' health and nutrition in conflicts.

BACKGROUND: Significant global gains in sexual, reproductive, maternal, newborn, child and adolescent health and nutrition (SRMNCAH&N) will be difficult unless conflict settings are adequately addressed. We aimed to determine the amount, scope and quality of publically available guidance documents, to characterise the process by which agencies develop their guidance and to identify gaps in guidance on SRMNCAH&N promotion in conflicts. METHODS: We identified guidance documents published between 2008 and 2018 through English-language Internet sites of humanitarian response organisations, reviewed them for their scope and assessed their quality with the AGREE II (Appraisal of Guidelines for REsearch and Evaluation II) tool. Additionally, we interviewed 22 key informants on guidance development, dissemination processes, perceived guidance gaps and applicability. FINDINGS: We identified 105 conflict-relevant guidance documents from 75 organisations. Of these, nine were specific to conflicts, others were applicable also to other humanitarian settings. Fifteen documents were technical normative guidelines, others were operational guides (67), descriptive documents (21) or advice on legal, human rights or ethics questions (2). Nutrition was the most addressed health topic, followed by communicable diseases and violence. The documents rated high quality in their 'scope and purpose' and 'clarity of presentation' and low for 'rigour of development' and 'editorial independence'. Key informants reported end user need as the primary driver for guideline development and WHO technical guidelines as their main evidence base. Insufficient local contextualisation, lack of inter-agency coordination and lack of systematic implementation were considered problems in guideline development. Several guidance gaps were noted, including abortion care, newborn care, early child development, mental health, adolescent health beyond sexual and reproductive health and non-communicable diseases. INTERPRETATION: Organisations are motivated and actively producing guidance for SRMNCAH&N promotion in humanitarian settings, but few documents address conflicts specifically and there are important guidance gaps. Improved inter-organisation collaboration for guidance on SRMNCAH&N promotion in conflicts and other humanitarian settings is needed.

A new species of anole from Parque Nacional Volcán Arenal, Costa Rica (Reptilia, Squamata, Dactyloidae: Norops).

We describe the new species Norops arenal sp. nov. from Parque Nacional Volcán Arenal, north-central Costa Rica. In external morphology and genetic similarity of the 16S DNA barcode, Norops arenal is most similar to N. altae, N. fortunensis, N. fuscoauratus, N. gruuo, N. kemptoni, N. monteverde, N. pseudokemptoni, and N. tenorioensis. In morphology it shares with these species the following characteristics: (1) short hind limbs; (2) a single elongate prenasal scale; (3) tiny, smooth, often juxtaposed body scales; and (4) a slender habitus, often delicate. Norops arenal differs from these species, among several scalation details, by having a blackish central area in the male dewlap in life and in preservative (vs. no suffusion of black pigment on male dewlap in the other species), and a small red female dewlap in life (vs. dirty white, cream colored, or orange); extremely short hind legs with the tip of fourth toe of the adpressed hind leg reaching only to level of shoulder (vs. usually at least to level of ear in the other species); a short tail with a tail length/SVL ratio of 1.53 in single specimen with complete tail (vs. this ratio >1.6 in the other species); and a tiny size with 41.5 mm in single known adult male and 38.5 mm in single known adult female (vs. SVL of adults usually >42.0 mm). It further differs from N. altae, N. fuscoauratus, N. gruuo, N. pseudokemptoni, and N. tenorioensis by having a unilobed hemipenis (vs. bilobed in these five species).

A comparison of the in vitro retentive strength of glass-ionomer cement, zinc-phosphate cement, and mineral trioxide aggregate for the retention of prefabricated posts in bovine incisors.

The purpose of this study was to compare the retentive strength of zinc-phosphate cement, glass-ionomer cement, and mineral trioxide aggregate (MTA) cement in the retention of prefabricated posts. The root canals of 60 bovine incisors were prepared and obturated with warm gutta-percha. Post space was prepared, the smear layer removed, and posts were luted with zinc-phosphate cement, glass-ionomer cement, or MTA. The specimens were stored at 37 degrees C and 100% humidity for 2 weeks, and then subjected to increasing axial tensile forces by an Instron machine until bond failure occurred. Data were analyzed by a one-way ANOVA and a Tukey-Kramer multiple comparison test. The retentive strengths of zinc phosphate and glass-ionomer cements were statistically equivalent, and significantly greater than MTA (p < 0.001), which suggests that zinc phosphate or glass-ionomer cement may be superior to MTA when used as luting agents for posts in endodontically treated teeth.

Using suture anchors for coracoclavicular fixation in treatment of complete acromioclavicular separation.

For the repair of acromioclavicular separations, we describe a new method of securing the clavicle to the coracoid process using suture anchors. We have repaired 11 consecutive complete acromioclavicular separations in this manner with very good results. We find this to be an easy and reproducible method of anatomical fixation.

Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs.

Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies.

The glial modulatory drug AV411 attenuates mechanical allodynia in rat models of neuropathic pain.

Controlling neuropathic pain is an unmet medical need and we set out to identify new therapeutic candidates. AV411 (ibudilast) is a relatively nonselective phosphodiesterase inhibitor that also suppresses glial-cell activation and can partition into the CNS. Recent data strongly implicate activated glial cells in the spinal cord in the development and maintenance of neuropathic pain. We hypothesized that AV411 might be effective in the treatment of neuropathic pain and, hence, tested whether it attenuates the mechanical allodynia induced in rats by chronic constriction injury (CCI) of the sciatic nerve, spinal nerve ligation (SNL) and the chemotherapeutic paclitaxel (Taxol). Twice-daily systemic administration of AV411 for multiple days resulted in a sustained attenuation of CCI-induced allodynia. Reversal of allodynia was of similar magnitude to that observed with gabapentin and enhanced efficacy was observed in combination. We further show that multi-day AV411 reduces SNL-induced allodynia, and reverses and prevents paclitaxel-induced allodynia. Also, AV411 cotreatment attenuates tolerance to morphine in nerve-injured rats. Safety pharmacology, pharmacokinetic and initial mechanistic analyses were also performed. Overall, the results indicate that AV411 is effective in diverse models of neuropathic pain and support further exploration of its potential as a therapeutic agent for the treatment of neuropathic pain.

Evidence of multiyear factor IX expression by AAV-mediated gene transfer to skeletal muscle in an individual with severe hemophilia B.

In a phase I study, administration of an AAV2-FIX vector into the skeletal muscle of eight hemophilia B subjects proved safe and achieved local gene transfer and FIX expression for at least 10 months after vector injection, the last time point assessed by muscle biopsy. In hemophilia B dogs we have demonstrated FIX in both muscle biopsies and circulation >4 years following AAV2-FIX injection. Because circulating FIX levels remained less than 1% of normal in human subjects from the study, the duration of AAV2-mediated transgene expression in humans is unknown. We sought to determine if FIX gene transfer and expression persisted locally at injection sites. Muscle biopsies were obtained from one subject 3.7 years following treatment and revealed transgene FIX DNA and protein by quantitative PCR, DNA fluorescence in situ hybridization, and immunohistochemistry for FIX. These results demonstrate, for the first time, multiyear FIX expression by AAV2 vector in humans and suggest that improved muscle delivery provides effective treatment for protein deficiencies or muscle-specific diseases.

Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy.

In a clinical study of recombinant adeno-associated virus-2 expressing human factor IX (AAV2-FIX), we detected 2 impediments to long-term gene transfer. First, preexisting anti-AAV neutralizing antibodies (NABs) prevent vector from reaching the target tissue, and second, CD8(+) T-cell responses to hepatocyte-cell surface displayed AAV-capsid-terminated FIX expression after several weeks. Because the vector is incapable of synthesizing viral proteins, a short course of immunosuppression, until AAV capsid is cleared from the transduced cells, may mitigate the host T-cell response, allowing long-term expression of FIX. To evaluate coad-ministration of immunosuppression, we studied AAV8 vector infusion in rhesus macaques, natural hosts for AAV8. We administered AAV8-FIX in 16 macaques via the hepatic artery and assessed the effects of (1) preexisting anti-AAV8 NABs, (2) a standard T-cell immunosuppressive regimen, and (3) efficacy and safety of AAV8-FIX. We found that low titers (1:5) of preexisting NABs abrogate transduction, whereas animals with undetectable NABs are safely and effectively transduced by AAV8-FIX. Coadministration of mycophenolate mofetil and tacrolimus with vector does not induce toxicity and does not impair AAV transduction or FIX synthesis. These findings enable a clinical study to assess the effects of immunomodulation on long-term FIX expression in patients with hemophilia B.

AAV2-mediated gene delivery to monkey putamen: evaluation of an infusion device and delivery parameters.

In this study, a modified infusion procedure and a novel infusion device designed for use in humans (Clinical Device B) were evaluated for delivery of recombinant adeno-associated virus (AAV2) to brain. The device is composed of 1.2 m of fused silica inserted through a 24.6-cm surgical steel cannula designed to fit a standard Leksell clinical stereotaxic frame and micro-infusion syringe pump. AAV2 encoding the human aromatic l-amino acid decarboxylase gene (AAV-hAADC-2) was infused into the putamen of 4 normal rhesus monkeys as a supportive study for a clinical trial in Parkinson's disease (PD) patients. Two infusion protocols were tested: a ramped procedure (slow stepwise increases in rate from 0.2 muL/min to 1 muL/min), thought to be essential for convection-enhanced delivery (CED), and a non-ramped infusion at a constant rate of 1 muL/min. The primary endpoints were safety evaluation of the infusion procedures and assessment of transgene expression at 5.5 weeks post-infusion. Clinical observations after vector infusions revealed no behavioral abnormalities during the study period. No differences in gross pathology with either the ramped or non-ramped infusion procedure were observed. Histopathology of the putamen was comparable with both procedures, and revealed only minimal localized inflammatory tissue reaction along the needle track in response to cannula placement and vector infusion. AADC immunohistochemistry demonstrated that vector was distributed throughout the putamen, with no significant difference in volume of immunostaining with either infusion procedure. Serum antibody levels against AAV2 vector exhibited a minor increase after infusion. These results validate the clinical utility of this new infusion device and non-ramped infusion conditions for intraputamenal gene therapy, and have the potential to impact a number of human diseases in which delivery of therapeutics to brain is indicated.

Phenotypic correction of a mouse model of hemophilia A using AAV2 vectors encoding the heavy and light chains of FVIII.

Using separate adeno-associated viral 2 (AAV2) vectors to deliver the heavy and light chains of factor VIII (FVIII) we have overcome the packaging limitations of AAV, achieving phenotypic correction of hemophilia A in mice. AAV vectors were constructed that use a liver-specific promoter and the cDNA sequences of either the human or canine heavy and light chains of FVIII. After intraportal vein injection of these vectors in hemophilia-A mice, therapeutic to superphysiologic levels of active FVIII were achieved in plasma in a dose-dependent manner. Phenotypic correction of the bleeding diathesis was demonstrated by survival of all treated mice after tail clipping. Biochemical analysis demonstrated lower levels of heavy-chain (25- to 100-fold) compared with light-chain protein in the plasma of treated animals. Differences in gene transfer and transcription did not account for the differences in protein expression. We hypothesize that improvements in FVIII activity could be achieved by improvements in FVIII heavy-chain expression. This work demonstrates that cotransduction of liver with AAV vectors expressing the heavy and light chains of FVIII corrects hemophilia A in vivo, providing an alternative approach to the use of a single vector. This strategy may potentially be useful for other large therapeutic proteins that contain functionally distinct domains.

Sustained phenotypic correction of canine hemophilia A using an adeno-associated viral vector.

Gene therapy for hemophilia A requires efficient delivery of the factor VIII gene and sustained protein expression at circulating levels of at least 1% to 2% of normal. Adeno-associated viral type 2 (AAV2) vectors have a number of advantages over other viral vectors, including an excellent safety profile and persistent gene expression. However, a major disadvantage is their small packaging capacity, which has hampered their use in treating diseases such as hemophilia A, cystic fibrosis, and muscular dystrophy, which are caused by mutations in large genes. Here we demonstrate that this can be overcome by using small regulatory elements to drive expression of a B-domain-deleted form of FVIII. The use of this vector for hepatic gene transfer in a canine model of hemophilia A resulted in the sustained (> 14 months) expression of biologically active FVIII. FVIII activity levels of 2% to 4% were achieved. These levels correlated with a partial correction in the whole-blood clotting time and cuticle bleeding time. In addition, immunoprecipitation analysis demonstrated the expression of canine FVIII of the predicted size in the plasma of injected animals. These data support the use of AAV2 vectors in human clinical trials to treat hemophilia A patients.