A CRISPR/Cas9 library to map the HIV-1 provirus genetic fitness.

The integrated proviral genome is the major barrier to a cure for HIV-1 infection. Genome editing technologies, such as CRISPR/Cas9, may disable or remove the HIV-1 provirus by introducing DNA double strand breaks at sequence specific sites in the viral genome. Host DNA repair by the error-prone non-homologous end joining pathway generates mutagenic insertions or deletions at the break. CRISPR/Cas9 editing has been shown to reduce replication competent viral genomes in cell culture, but only a minority of possible genome editing targets have been assayed. Currently there is no map of double strand break genetic fitness for HIV-1 to inform the choice of editing targets. However, CRISPR/Cas9 genome editing makes it possible to target double strand breaks along the length of the provirus to generate a double strand break genetic fitness map. We identified all possible HIV-1 targets with different bacterial species of CRISPR/Cas9. This library of guide RNAs was evaluated for GC content and potential off-target sites in the human genome. Complexity of the library was reduced by eliminating duplicate guide RNA targets in the HIV-1 long terminal repeats and targets in the env gene. Although the HIV-1 genome is AT-rich, the S. pyogenes CRISPR/Cas9 with the proto-spacer adjacent motif NGG offers the most HIV-1 guide RNAs. This library of HIV-1 guide RNAs may be used to generate a double strand break genetic fragility map to be further applied to any genome editing technology designed for the HIV-1 provirus. Keywords: HIV-1; genome editing; CRISPR; genetic fitness; guide RNAs.

Prototype foamy virus integrase is promiscuous for target choice.

Retroviruses have two essential activities: reverse transcription and integration. The viral protein integrase (IN) covalently joins the viral cDNA genome to the host DNA. Prototype foamy virus (PFV) IN has become a model of retroviral intasome structure. However, this retroviral IN has not been well-characterized biochemically. Here we compare PFV IN to previously reported HIV-1 IN activities and discover significant differences. PFV IN is able to utilize the divalent cation calcium during strand transfer while HIV-1 IN is not. HIV-1 IN was shown to completely commit to a target DNA within 1 min, while PFV IN is not fully committed after 60 min. These results suggest that PFV IN is more promiscuous compared to HIV-1 IN in terms of divalent cation and target commitment.

First Report of Reduced Sensitivity to a QoI Fungicide in Apple Scab (Venturia inaequalis) in Virginia and Maryland.

Apple scab caused by Venturia inaequalis (Cooke) Winter continues to be a significant concern for apple growers in Virginia and Maryland. Management of scab has relied on foliar fungicides including strobilurins (QoIs) such as trifloxystrobin (TFX). In recent years, populations of V. inaequalis with reduced sensitivity to the QoIs have been reported in other apple-growing regions of the United States (1,2). Although QoIs generally remain effective in the mid-Atlantic, concerns about the development of resistance in some Virginia and Maryland orchards prompted this study. Twenty-five isolates of V. inaequalis were obtained from scabby leaves from commercial and experimental orchards in Virginia in 2010 (n = 6) and 2011 (n = 14) and from a commercial orchard in Maryland (n = 5) in 2011. Orchards had previously been treated with QoI or sterol-inhibiting (SI) fungicides. Isolates of V. inaequalis were grown on potato dextrose agar (PDA) amended with 0, 0.1, or 1.0 µg ml-1 TFX with 100 µg ml-1 salicylhydroxamic acid (SHAM) and incubated at 19°C. Colony growth was measured weekly for 4 weeks. To account for the SI use at some orchards, isolates of V. inaequalis were also evaluated on PDA amended with 0, 0.5, or 1.0 µg ml-1 myclobutanil. Fungicide sensitivities were expressed as a percentage of the difference in colony growth using a discriminatory dose of 1.0 µg ml-1 TFX with SHAM or 1.0 µg ml-1 myclobutanil at 28 days. Isolates with <25% growth suppression (GS) were classified as fully resistant, whereas those with >70% GS were classified as sensitive. Isolates with 25 to 70% GS were classified as partially resistant. Effective concentration (EC50) values (TFX concentration inhibiting colony growth by 50%) were also calculated for a subset of fully resistant and sensitive isolates. Of the 25 isolates tested, six were fully resistant to TFX (mean EC50 value greater than 10.0 µg ml-1) and 10 were sensitive (mean EC50 value of 0.04 µg ml-1 ± 0.05 µg ml-1). Nine isolates were classified as partially resistant. Some isolates showed more than a 200-fold increase in resistance to TFX, and one isolate grew almost as well on 10.0 µg ml-1 TFX as on the unamended control (GS of 3%). Current-season use of QoIs on isolate source trees was significantly associated with a lack of sensitivity Ç2 (1) = 3.72 (P < 0.06). All six fully resistant isolates originated from QoI-treated commercial orchards, which had shown control failures. Seven of 10 isolates sensitive to QoIs originated from trees that had been treated with SIs during the isolation year. Resistance to myclobutanil was not significantly associated with resistance to TFX Ç2 (1) = 1.220 (P < 0.5), and only one isolate was resistant (i.e. >25% GS) to both. Despite the long history of QoI use at the experimental orchards, no isolates fully resistant to TFX were identified there. To our knowledge, this is the first report of V. inaequalis isolates with resistance to TFX in Virginia and Maryland. Since SI resistance has been documented in Virginia (3) and resistance to both the SI and QoI chemical classes is a concern in the mid-Atlantic region (4), tank-mixing or alternating QoIs with broad-spectrum fungicides with different modes of action is recommended. References: (1). K. M. Cox et al. Phythopathology 99:S25, 2009. (2). K. E. Lesniak et al. Plant Dis. 95:927, 2011. (3) S. C. Marine et al. Plant Health Progress. doi:10.1094/PHP-2007-1113-01-RS, 2007. (4) E. E. Pfeufer and H. K. Ngugi. Phytopathology 102:272, 2012.

Early trends in landcover change and forest fragmentation due to shale-gas development in Pennsylvania: a potential outcome for the Northcentral Appalachians.

Worldwide shale-gas development has the potential to cause substantial landscape disturbance. The northeastern U.S., specifically the Allegheny Plateau in Pennsylvania, West Virginia, Ohio, and Kentucky, is experiencing rapid exploration. Using Pennsylvania as a proxy for regional development across the Plateau, we examine land cover change due to shale-gas exploration, with emphasis on forest fragmentation. Pennsylvania's shale-gas development is greatest on private land, and is dominated by pads with 1-2 wells; less than 10 % of pads have five wells or more. Approximately 45-62 % of pads occur on agricultural land and 38-54 % in forest land (many in core forest on private land). Development of permits granted as of June 3, 2011, would convert at least 644-1072 ha of agricultural land and 536-894 ha of forest land. Agricultural land conversion suggests that drilling is somewhat competing with food production. Accounting for existing pads and development of all permits would result in at least 649 km of new road, which, along with pipelines, would fragment forest cover. The Susquehanna River basin (feeding the Chesapeake Bay), is most developed, with 885 pads (26 % in core forest); permit data suggests the basin will experience continued heavy development. The intensity of core forest disturbance, where many headwater streams occur, suggests that such streams should become a focus of aquatic monitoring. Given the intense development on private lands, we believe a regional strategy is needed to help guide infrastructure development, so that habitat loss, farmland conversion, and the risk to waterways are better managed.

Interactions of Glutamate and Gamma Amino Butyric Acid with the insulin-like growth factor system in traumatic brain injury (TBI) and/or cardiovascular accidents (CVA or stroke): A systematic review.

The brain maintains homeostasis of neural excitation in part through the receptor-mediated signaling of Glutamate (Glu) and Gamma Amino Butyric Acid (GABA), but localized injuries cause cellular release of excess Glu leading to neurotoxicity. The literature strongly supports the role of Insulin-like growth factor-1 (IGF-1) in adult brain neuroprotection and repair, and research supporting the existence of molecular interactions between Glu, GABA, and IGF-1 in vitro and in normal animals raises the question of whether and/or how the Glu/GABA system interacts with IGF-1 post-injury. This systematic review was undertaken to explore works addressing this question among adults with a history of traumatic brain injury (TBI) and/or cerebrovascular accident (CVA; stroke). The literature was searched for human and animal studies and only four animal papers met inclusion criteria. The SYRCLE criteria was used to evaluate risk of bias; results varied between categories and papers. All the included studies, one on TBI and three on stroke, supported the molecular relationship between the excitatory and IGF-1 systems; two studies provided direct, detailed molecular evidence. The results point to the importance of research on the role of this protective system in pathological brain injury; a hypothetical proposal for future studies is presented.

Absence of LEDGF/p75 Expression in Astrocytes May Affect HIV-1 Integration Efficiency.

In spite of effective anti-retroviral therapy, HIV-1 infection may still lead to neurological impairment in patients. The underlying mechanism of neurodegeneration remains mysterious. HIV-1 does not infect neurons, but does infect microglia cells in the brain. It is controversial whether HIV-1 productively infects astrocytes, an abundant glial cell type in the brain. Thirty years of investigation have led to conflicting reports concerning the entry, infection, and production of progeny virions from astrocytes. New models from studies in primary human fetal astrocytes suggest phagocytosis of HIV-1 with little productive infection. The retroviral life cycle requires integration of the viral genome to the host genome. The host protein LEDGF/p75 is required for efficient HIV-1 integration. In the absence of LEDGF/p75, HIV-1 integration and infection efficiency is reduced ten fold. Differentiated astrocytes do not appear to express LEDGF/p75, which suggests these cells are disabled for efficient integration. Phagocytosis of HIV-1 virions and the lack of LEDGF/p75 expression in astrocytes suggest that this cell type is not efficiently infected in vivo.

Estimation from PET data of transient changes in dopamine concentration induced by alcohol: support for a non-parametric signal estimation method.

We previously developed a model-independent technique (non-parametric ntPET) for extracting the transient changes in neurotransmitter concentration from paired (rest & activation) PET studies with a receptor ligand. To provide support for our method, we introduced three hypotheses of validation based on work by Endres and Carson (1998 J. Cereb. Blood Flow Metab. 18 1196-210) and Yoder et al (2004 J. Nucl. Med. 45 903-11), and tested them on experimental data. All three hypotheses describe relationships between the estimated free (synaptic) dopamine curves (FDA(t)) and the change in binding potential (DeltaBP). The veracity of the FDA(t) curves recovered by nonparametric ntPET is supported when the data adhere to the following hypothesized behaviors: (1) DeltaBP should decline with increasing DA peak time, (2) DeltaBP should increase as the strength of the temporal correlation between FDA(t) and the free raclopride (FRAC(t)) curve increases, (3) DeltaBP should decline linearly with the effective weighted availability of the receptor sites. We analyzed regional brain data from 8 healthy subjects who received two [11C]raclopride scans: one at rest, and one during which unanticipated IV alcohol was administered to stimulate dopamine release. For several striatal regions, nonparametric ntPET was applied to recover FDA(t), and binding potential values were determined. Kendall rank-correlation analysis confirmed that the FDA(t) data followed the expected trends for all three validation hypotheses. Our findings lend credence to our model-independent estimates of FDA(t). Application of nonparametric ntPET may yield important insights into how alterations in timing of dopaminergic neurotransmission are involved in the pathologies of addiction and other psychiatric disorders.

Knowledge and use of fluoride among Indiana dental professionals.

OBJECTIVES: This study assessed the knowledge of Indiana dentists and dental hygienists about fluoride's predominant mode of action and their protocols for the use of fluoride for dental caries prevention. METHODS: In 2000, questionnaires were mailed to 6,681 Indiana dentists and hygienists prior to the 2001 release of recommendations for the use of fluoride by the US Centers for Disease Control and Prevention. In 2005, the questionnaires were again sent to Indiana dental professionals to assess changes in knowledge and protocols. In addition, a 10 percent sample of Illinois dentists and hygienists were surveyed to determine the similarity of Indiana and Illinois responses. RESULTS: Questionnaires were anonymously completed and returned. In 2000, a minority of Indiana health professionals (17 percent) correctly identified that remineralization was fluoride's predominant mode of action. There was a significant increase in Indiana respondents correctly identifying this predominant mode of action between 2000 and 2005 (17 percent versus 25 percent, respectively, P < 0.0001). Fourteen percent of Illinois respondents answered correctly in 2005. Preeruptive incorporation of fluoride into enamel was the most frequently cited incorrect response (IN 2000, 79 percent; IN 2005, 71 percent; IL 2005, 82 percent). Some protocols for use of fluoride products reflected inadequate understanding of fluoride's predominant posteruptive mode of action. CONCLUSIONS: The majority of dental professionals surveyed were unaware of the current understanding of fluoride's predominant posteruptive mode of action through remineralization of incipient carious lesions. Additional research is indicated to assess fluoride knowledge and protocols of dental professionals nationwide. Educational efforts are needed to promote the appropriate use of fluoride.

Experimental Bin Drenching System for Testing Biocontrol Agents to Control Postharvest Decay of Apples.

A portable drencher capable of drenching a single bin of fruit was built to simulate the commercial application of chemicals to harvested apples in small orchard operations in the central and eastern United States. The drencher required as little as 125 liters of the treatment solution and permitted various bin travel speeds. Wounded apples were placed midway between the bottom and top of the bin, in the center, and near the four corners of the bin (20 fruit per location) and covered with enough unwounded apples to fill the bin. The bins were drenched with a suspension containing Penicillium expansum at 2 × 104 conidia per ml in 2000, 5 × 103 conidia per ml in 2001, and 3 × 103 conidia per ml in 2002 and 2003. In 2000 and 2003, the additional treatments included a combination of P. expansum with the yeast Metschnikowia pulcherrima at ~;1.2 × 107 CFU/ml, and in 2003 a combination with 2% sodium bicarbonate (SB) or a mixture of the yeast and SB. After 3 months of storage at ~;2°C, at all P. expansum conidial concentrations, more than 90% of wounded fruit developed decay on 'Golden Delicious', 'Delicious', and 'Rome' apples in the 2000-02 experiments. In 2003, 66 and 33.1% of the wounded fruit developed decay on 'Delicious' and 'Golden Delicious', respectively. The application of the antagonist reduced decay to 39 and 3.3% on 'Golden Delicious' in 2000 and 2003, respectively, and to 26% on 'Delicious' in 2003. The addition of SB reduced decay on both cultivars and, in combination with the yeast, was the most effective treatment on 'Golden Delicious'. This portable drencher can be very useful for evaluating different treatments applied to apples after harvest at the commercial level.

Neural networks underlying implicit and explicit moral evaluations in psychopathy.

Psychopathy, characterized by symptoms of emotional detachment, reduced guilt and empathy and a callous disregard for the rights and welfare of others, is a strong risk factor for immoral behavior. Psychopathy is also marked by abnormal attention with downstream consequences on emotional processing. To examine the influence of task demands on moral evaluation in psychopathy, functional magnetic resonance imaging was used to measure neural response and functional connectivity in 88 incarcerated male subjects (28 with Psychopathy Checklist Revised (PCL-R) scores ⩾ 30) while they viewed dynamic visual stimuli depicting interpersonal harm and interpersonal assistance in two contexts, implicit and explicit. During the implicit task, high psychopathy was associated with reduced activity in the dorsolateral prefrontal cortex and caudate when viewing harmful compared with helpful social interactions. Functional connectivity seeded in the right amygdala and right temporoparietal junction revealed decreased coupling with the anterior cingulate cortex (ACC), anterior insula, striatum and ventromedial prefrontal cortex. In the explicit task, higher trait psychopathy predicted reduced signal change in ACC and amygdala, accompanied by decreased functional connectivity to temporal pole, insula and striatum, but increased connectivity with dorsal ACC. Psychopathy did not influence behavioral performance in either task, despite differences in neural activity and functional connectivity. These findings provide the first direct evidence that hemodynamic activity and neural coupling within the salience network are disrupted in psychopathy, and that the effects of psychopathy on moral evaluation are influenced by attentional demands.

Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism.

Cyclospora cayetanensis is a coccidian parasite which causes severe gastroenteritis in humans. Molecular information on this newly emerging pathogen is scarce. Our objectives were to assess genetic variation within and between human-associated C. cayetanensis and baboon-associated Cyclospora papionis by examining the internal transcribed spacer (ITS) region of the ribosomal RNA operon, and to develop an efficient polymerase chain reaction- (PCR)-based method to distinguish C. cayetanensis from other closely related organisms. For these purposes, we studied C. cayetanensis ITS-1 nucleotide variability in 24 human faecal samples from five geographic locations and C. papionis ITS-1 variability in four baboon faecal samples from Tanzania. In addition, a continuous sequence encompassing ITS-1, 5.8S rDNA and ITS-2 was determined from two C. cayetanensis samples. The results indicate that C. cayetanensis and C. papionis have distinct ITS-1 sequences, but identical 5.8S rDNA sequences. ITS-1 is highly variable within and between samples, but variability does not correlate with geographic origin of the samples. Despite this variability, conserved species-specific ITS-1 sequences were identified and a single-round, C. cayetanensis-specific PCR-based assay with a sensitivity of one to ten oocysts was developed. This consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argues for polyparasitism and simultaneous transmission of multiple strains.

Is akathisia associated with poor clinical response to antipsychotics during acute hospital treatment?

Previous studies have suggested that akathisia is associated with poor acute clinical response to antipsychotics and that low serum iron levels are associated with emergence of akathisia. To examine these relationships during routine clinical treatment, we studied patients with DSM-IV schizophrenia or schizoaffective disorder undergoing hospital treatment for acute psychotic exacerbations with doctor's choice medications. There were 34 subjects observed for at least 2 weeks. They were assessed at baseline and weekly by one rater with the Anchored Brief Psychiatric Rating Scale and by another rater with the Barnes Rating Scale for akathisia, with the two raters blind to each other's ratings. Serum ferritin and transferrin levels were obtained at baseline. Seventeen subjects developed akathisia. Subjects with and without akathisia did not differ in change in thinking disturbance or anxiety-depression scores over 2 weeks, or in serum ferritin or transferrin levels. We conclude that mild akathisia by itself is not strongly associated with initial response to low to moderate doses of antipsychotics in the acute clinical setting. Limitations of the study are discussed.

Severe dental fluorosis in a Tanzanian population consuming water with negligible fluoride concentration.

OBJECTIVES: To identify risk factors for dental fluorosis that cannot be explained by drinking water fluoride concentration alone. METHODS: Two hundred eighty-four Tanzanian children ages 9 to 19 (mean 14.0+/-SD 1.69), who were lifetime residents at differing altitudes (Chanika, 100 m; Rundugai, 840 m; and Kibosho, 1,463 m; Sites 1, 2, and 3 respectively) were examined for dental fluorosis and caries. They were interviewed about their food habits, environmental characteristics and use of a fluoride-containing food tenderizer known locally as magadi. Meal, urine, water and magadi samples supplied by the participants were analyzed for fluoride content. Urine samples were also analyzed for creatinine concentration. Four magadi samples from Sites 1 and 3 were analyzed for complete element composition. RESULTS: Of the 13 water samples from Site 2, 10 contained > or =4 mg/L F, ranging from 1.26 to 12.36 mg/L with a mean+/-SD of 5.72+/-4.71 mg/L. Sites 1 and 3 had negligible water fluoride of 0.05+/-0.05 and 0.18+/-0.32 mg/L respectively. Mean TFI fluorosis scores (range 0-9) for Site 2 were high: 4.44+/-1.68. In Sites 1 and 3, which both had negligible water fluoride, fluorosis scores varied dramatically: Site 1 mean maximum TFI was 0.01+/-0.07 and Site 3 TFI was 4.39+/-1.52. Mean DMFS was 1.39+/-2.45, 0.15+/-0.73 and 0.19+/-0.61 at Sites 1, 2, and 3, respectively. There were no restorations present. Urinary fluoride values were 0.52+/-0.70, 4.34+/-7.62, and 1.43+/-1.80 mg/L F at Sites 1, 2, and 3, respectively. Mean urinary fluoride values at Site 3 were within the normal urinary fluoride reference value range in spite of pervasive severe pitting fluorosis. Meal and magadi analyses revealed widely varied fluoride concentrations. Concentrations ranged from 0.01 to 22.04 mg/L F for meals and from 189 to 83211 mg/L F for magadi. Complete element analysis revealed the presence of aluminum, iron, magnesium, manganese, strontium and titanium in four magadi samples. There were much higher concentrations of these elements in samples from Site 3, which was at the highest altitude and had severe enamel disturbances in spite of negligible water fluoride concentration. An analysis of covariance model supported the research hypothesis that the three communities differed significantly in mean fluorosis scores (P<0.0001). Controlling for urinary fluoride concentration and urinary fluoride:urinary creatinine ratio, location appeared to significantly affect fluorosis severity. Urinary fluoride:urinary creatinine ratio had a stronger correlation than urinary fluoride concentration with mean TFI fluorosis scores (r=0.43 vs r= 0.25). CONCLUSIONS: The severity of enamel disturbances at Site 3 (1463 m) was not consistent with the low fluoride concentration in drinking water, and was more severe than would be expected from the subjects' normal urinary fluoride values. Location, fluoride in magadi, other elements found in magadi, and malnutrition are variables which may be contributing to the severity of dental enamel disturbances occurring in Site 3. Altitude was a variable which differentiated the locations.

Comparing suicide attempters, suicide ideators, and nonsuicidal homeless and runaway adolescents.

This study considers variables that best distinguish among attempters, ideators, and nonsuicidal youth in a sample of 527 homeless and runaway adolescents from four Midwestern states. Univariate results indicate that attempters are significantly more likely than ideators and nonsuicidal youth to have experienced physical or sexual abuse by an adult caretaker, to have experienced sexual victimization while on their own, and to have a friend who attempted suicide. Multivariate analyses reveal five variables that best distinguish among the three groups: self-esteem, depression, physical abuse, sexual abuse, and having a friend who attempted suicide. Further analysis suggests that the accumulation of these risk factors greatly increases the chance that these youth will engage in suicidal behavior.

Legal aspects of anesthesia charting.

To the anesthesia practitioner, the possibility of malpractice claims constitutes a very real and ever-present problem for it lies within the risks inherent in the administration of anesthesia. Since the areas of anesthesia which may be subject to suit are legion, the problem of malpractice becomes too complex to afford easy answers. Clearly, though, it is a problem all too serious to ignore. This article examines one aspect of the problem: the importance of good documentation in anesthesia.

The oral health of Indiana's independent, disabled adults.

This epidemiological study was conducted to measure the oral health and related variables of one of Indiana's special population groups: adults who are developmentally disabled, work in sheltered workshops and do not live in staffed residential facilities. Fifteen sheltered workshops in ten Indiana counties cooperated with this study in which 393 individuals were interviewed and received oral screening. Information was gathered regarding oral health status, history of utilization of dental services, availability of dental insurance or Medicaid and use of tobacco products. This study was conducted by the Indiana Foundation of Dentistry for the Handicapped and was funded by the Indiana State Department of Health.

Monitoring of sound and carious surfaces under sealants over 44 months.

Although there is strong evidence for the effectiveness of sealants, one major barrier in sealant utilization is the concern of sealing over active caries lesions. This study evaluated detection and monitoring of caries lesions through a clear sealant over 44 mo. Sixty-four 7- to 10-year-old children with at least 2 permanent molars with International Caries Detection and Assessment System (ICDAS) scores 0-4 (and caries less than halfway through the dentin, radiographically) were examined with ICDAS, DIAGNOdent, and quantitative light-induced fluorescence (QLF) before sealant placement and 1, 12, 24, and 44 mo (except QLF) after. Bitewing radiographs were taken yearly. DIAGNOdent and QLF were able to distinguish between baseline ICDAS before and after sealant placement. There was no significant evidence of ICDAS progression at 12 mo, but there was small evidence of minor increases at 24 and 44 mo (14% and 14%, respectively) with only 2% ICDAS ≥ 5. Additionally, there was little evidence of radiographic progression (at 12 mo = 1%, 24 mo = 3%, and 44 mo = 9%). Sealant retention rates were excellent at 12 mo = 89%, 24 mo = 78%, and 44 mo = 70%. The small risk of sealant repair increased significantly as baseline ICDAS, DIAGNOdent, and QLF values increased. However, regardless of lesion severity, sealants were 100% effective at 12 mo and 98% effective over 44 mo in managing occlusal surfaces at ICDAS 0-4 (i.e., only 4 of 228 teeth progressed to ICDAS ≥ 5 associated with sealants in need of repair and none to halfway or more through the dentin, radiographically). This study suggests that occlusal surfaces without frank cavitation (ICDAS 0-4) that are sealed with a clear sealant can be monitored with ICDAS, QLF, or DIAGNOdent, which may aid in predicting the need for sealant repair.