Structure elucidation of the syringafactin lipopeptides provides insight in the evolution of nonribosomal peptide synthetases.
Modular biosynthetic machineries such as polyketide synthases (PKSs) or nonribosomal peptide synthetases (NRPSs) give rise to a vast structural diversity of bioactive metabolites indispensable in the treatment of cancer or infectious diseases. Here, we provide evidence for different evolutionary processes leading to the diversification of modular NRPSs and thus, their respective products. Discovery of a novel lipo-octapeptide family from Pseudomonas, the virginiafactins, and detailed structure elucidation of closely related peptides, the cichofactins and syringafactins, allowed retracing recombinational diversification of the respective NRPS genes. Bioinformatics analyses allowed us to spot an evolutionary snapshot of these processes, where recombination occurred both within the same and between different biosynthetic gene clusters. Our systems feature a recent diversification process, which may represent a typical paradigm to variations in modular biosynthetic machineries.


Predictors of Reading Comprehension in Children With Cochlear Implants.
Children with a profound hearing loss who have been implanted with cochlear implants (CI), vary in terms of their language and reading skills. Some of these children have strong language skills and are proficient readers whereas others struggle with language and both the decoding and comprehension aspects of reading. Reading comprehension is dependent on a number of skills where decoding, spoken language comprehension and receptive vocabulary have been found to be the strongest predictors of performance. Children with CI have generally been found to perform more poorly than typically hearing peers on most predictors of reading comprehension including word decoding, vocabulary and spoken language comprehension, as well as working memory. The purpose of the current study was to investigate the relationships between reading comprehension and a number of predictor variables in a sample of twenty-nine 11-12-year-old children with profound hearing loss, fitted with CI. We were particularly interested in the extent to which reading comprehension in children with CI at this age is dependent on decoding and receptive vocabulary. The predictor variables that we set out to study were word decoding, receptive vocabulary, phonological skills, and working memory. A second purpose was to explore the relationships between reading comprehension and demographic factors, i.e., parental education, speech perception and age of implantation. The results from these 29 children indicate that receptive vocabulary is the most influential predictor of reading comprehension in this group of children although phonological decoding is, of course, fundamental.


Visual/cognitive correlates of vehicle accidents in older drivers.
Older drivers have more accidents per miles driven than any other age group and tend to have significant impairments in their visual function, which could interfere with driving. Previous research has largely failed to document a link between vision and driving in the elderly. We have taken a comprehensive approach by examining how accident frequency in older drivers relates to the visual/cognitive system at a number of levels: ophthalmological disease, visual function, visual attention, and cognitive function. The best predictor of accident frequency as recorded by the state was a model incorporating measures of early visual attention and mental status, which together accounted for 20% of the variance, a much stronger model than in earlier studies. Those older drivers with a visual attentional disorder or with poor scores on a mental status test had 3-4 times more accidents (of any type) and 15 times more intersection accidents than those without these problems.


Diabetes mellitus and bone health: epidemiology, etiology and implications for fracture risk stratification.
Skeletal fractures can result when there are co-morbid conditions that negatively impact bone strength. Fractures represent an important source of morbidity and mortality, especially in older populations. Diabetes mellitus is a metabolic disorder that has reached worldwide epidemic proportions and is increasingly being recognized as a risk factor for fracture. Type 1 and Type 2 diabetes have different effects on bone mineral density but share common pathways, which lead to bone fragility. In this review, we discuss the available data on diabetes and fractures, bone density and the clinical implications for fracture risk stratification in current practice.


An isolated insect leg's passive recovery from dorso-ventral perturbations.
Cockroaches recover rapidly from perturbations during high-speed running that allows them to cross unstructured terrains with no change in gait. Characterization of the exoskeletal material properties of the legs suggests that passive mechanical feedback could contribute to the self-stabilizing behavior. We imposed large, dorsal-ventrally directed impulsive perturbations to isolated hind legs having both a fixed and free body-coxa joint and measured their recovery. We tested a frequency-independent hysteretic damping model that effectively predicted the behavior of sinusoidal oscillations of isolated legs. Leg position reached its peak amplitude within 4-6 ms following an impulse. Position was 99% recovered within 16+/-3.3 ms for the stiffest possible leg configuration and within 46+/-6.6 ms for the most compliant leg configuration. The rapid recovery supports the hypothesis that passive musculo-skeletal properties play an important role in simplifying the control of high-speed locomotion.


Stress gates an astrocytic energy reservoir to impair synaptic plasticity.
Astrocytes support the energy demands of synaptic transmission and plasticity. Enduring changes in synaptic efficacy are highly sensitive to stress, yet whether changes to astrocyte bioenergetic control of synapses contributes to stress-impaired plasticity is unclear. Here we show in mice that stress constrains the shuttling of glucose and lactate through astrocyte networks, creating a barrier for neuronal access to an astrocytic energy reservoir in the hippocampus and neocortex, compromising long-term potentiation. Impairing astrocytic delivery of energy substrates by reducing astrocyte gap junction coupling with dominant negative connexin 43 or by disrupting lactate efflux was sufficient to mimic the effects of stress on long-term potentiation. Furthermore, direct restoration of the astrocyte lactate supply alone rescued stress-impaired synaptic plasticity, which was blocked by inhibiting neural lactate uptake. This gating of synaptic plasticity in stress by astrocytic metabolic networks indicates a broader role of astrocyte bioenergetics in determining how experience-dependent information is controlled.


A randomized controlled trial evaluating the effects of a family-centered HIV care model on viral suppression and retention in care of HIV-positive children in Eswatini.
A family-centered care model (FCCM) providing family-based HIV services, rather than separate adult/pediatric services, has been proposed to increase pediatric retention and treatment adherence. Eight health-care facilities in the Hhohho region of Eswatini were randomized to implement FCCM (n = 4) or continue standard-of-care (SOC) separate adult/pediatric clinics (n = 4). HIV-positive children and caregivers were enrolled; caregiver interview and child/caregiver chart abstraction were done at enrollment and every three months; pediatric viral load was evaluated at enrollment and every six months through 12 months. Because of study group differences in 12-month viral load data availability (89.4% FCCM and 72.0% SOC children had 12-month viral load), we used three separate analyses to evaluate the effects of FCCM on children's viral suppression (<1,000 copies/mL) and undetectable virus (<400 copies/mL) at 12 months. In the first analysis, all children with missing viral outcome data were excluded from the analysis (modified intent to treat, mITT). The second analysis used inverse probability of missingness weighted logistic regression to estimate the effect of FCCM on 12-month viral outcomes compared to SOC (weighted mITT). For the third approach, missing virologic outcome data were imputed as virologic failure (imputed ITT). We also examined factors associated with viral suppression at 12 months using multivariable logistic regression. We enrolled 379 HIV-positive children and 363 caregivers. Among all children at enrollment, viral suppression and undetectability was 78.4% and 73.9%, respectively, improving to 90.2% and 87.3% at 12 months. In mITT and weighted mITT analyses, there was no significant difference in children's 12-month viral suppression between FCCM and SOC groups (89.2% and 91.6%, respectively). Using imputed ITT, there was a modest increase in 12-month viral suppression in FCCM versus SOC children (79.7% and 69.8%, respectively, p = 0.051) and 12-month undetectability (78.7% and 65.7%, respectively, p = 0.015). Among the 255 children suppressed at enrollment, more FCCM versus SOC children (98.0% versus 95.3%) were suppressed at 12-months, but this was not statistically significant in mITT or weighted mITT analyses, with a marginally significant difference using imputed mITT analysis (p = 0.042). A higher proportion of children suppressed at enrollment had undetectable viral load at 12 months in FCCM versus SOC children (98.0% versus 92.5%), a statistically significant difference across analytical methods. Among the 61 children unsuppressed at enrollment, achieving suppression was higher among SOC versus FCCM children, but this difference was not statistically significant and included only 38 children; and there were no significant differences in detectable viral load at 12 months. There were no significant differences between study groups in retention or ART adherence at 12 months for children or caregivers. Factors associated with lack of viral suppression/detectability at 12 months included lack of viral suppression at enrollment and having a younger caregiver (age <25 years). FCCM in Eswatini was associated with a modest increase in viral suppression/undetectability at 12-months; 12-month retention and adherence did not differ by study group for children or caregivers. High levels of suppression and retention in both groups may have limited our ability to detect a difference. NCT03397420; ClinicalTrials.gov.


Knowledge of the signs and symptoms and risk factors of lung cancer in Australia: mixed methods study.
Lung cancer is the leading cause of cancer death in Australia. There is potential that health promotion about the risks and warning signs of lung cancer could be used to reduce delays in symptom presentation when symptoms are first detected. This study investigated knowledge, attitudes and beliefs which might impact help-seeking behaviour and could provide insight into possible public health interventions in New South Wales (NSW). A convergent mixed method study design was used wherein data from 16 qualitative focus groups of residents (40+ years), purposefully recruited and stratified by smoking status, age and geography (metropolitan/regional), were compared with a CATI administered population-wide telephone survey (n = 1,000) using the Cancer Research UK cancer awareness measure (LungCAM). Qualitative findings were analysed thematically using NVIVO. Logistic regression analysis was used to investigate predictors of symptom knowledge in STATA. Findings were integrated using triangulation techniques. Across focus groups, haemoptysis was the only symptom creating a sense of medical urgency. Life experiences evoked a 'wait and see' attitude to any health deterioration. Perceived risk was low amongst those at risk with current smokers preferring to deny their risk while former smokers were generally unaware of any ongoing risk. The quantitative sample consisted of females (62 %), 40-65 years (53 %), low SES (53 %), former (46 %) and current smokers (14 %). In quantitative findings, haemoptysis and dyspnoea were the most recognised symptoms across the sample population. Age (<65 years), sex (female) and high socio-economic status contributed to a higher recognition of symptoms. Smoking was recognised as a cause of lung cancer, yet ever-smokers were less likely to recognise the risk of lung cancer due to second-hand smoke (OR 0.7 95 % CI 0.5-0.9). While there was some recognition of risk factors and symptoms indicative of lung cancer, there was disparity across the sample population. The qualitative findings also suggest that knowledge may not lead to earlier presentation; a lack of urgency about symptoms considered trivial, and smoking-related barriers such as stigma may also contribute to time delays in presentation. Public health interventions may be required to increase awareness of risk and emphasise the importance of seeking medical attention for ongoing symptoms.


Structure of Erm-modified 70S ribosome reveals the mechanism of macrolide resistance.
Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058, located in the drug-binding site, a reaction catalyzed by Erm-type rRNA methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4 Å resolution, together with the structures of unmethylated 70S ribosome functional complexes alone or in combination with macrolides. Altogether, our structural data do not support previous models and, instead, suggest a principally new explanation of how A2058 dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome reveal a previously unknown role of the desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.


Designing Real-time Decision Support for Trauma Resuscitations.
Use of electronic clinical decision support (eCDS) has been recommended to improve implementation of clinical decision rules. Many eCDS tools, however, are designed and implemented without taking into account the context in which clinical work is performed. Implementation of the pediatric traumatic brain injury (TBI) clinical decision rule at one Level I pediatric emergency department includes an electronic questionnaire triggered when ordering a head computed tomography using computerized physician order entry (CPOE). Providers use this CPOE tool in less than 20% of trauma resuscitation cases. A human factors engineering approach could identify the implementation barriers that are limiting the use of this tool. The objective was to design a pediatric TBI eCDS tool for trauma resuscitation using a human factors approach. The hypothesis was that clinical experts will rate a usability-enhanced eCDS tool better than the existing CPOE tool for user interface design and suitability for clinical use. This mixed-methods study followed usability evaluation principles. Pediatric emergency physicians were surveyed to identify barriers to using the existing eCDS tool. Using standard trauma resuscitation protocols, a hierarchical task analysis of pediatric TBI evaluation was developed. Five clinical experts, all board-certified pediatric emergency medicine faculty members, then iteratively modified the hierarchical task analysis until reaching consensus. The software team developed a prototype eCDS display using the hierarchical task analysis. Three human factors engineers provided feedback on the prototype through a heuristic evaluation, and the software team refined the eCDS tool using a rapid prototyping process. The eCDS tool then underwent iterative usability evaluations by the five clinical experts using video review of 50 trauma resuscitation cases. A final eCDS tool was created based on their feedback, with content analysis of the evaluations performed to ensure all concerns were identified and addressed. Among 26 EPs (76% response rate), the main barriers to using the existing tool were that the information displayed is redundant and does not fit clinical workflow. After the prototype eCDS tool was developed based on the trauma resuscitation hierarchical task analysis, the human factors engineers rated it to be better than the CPOE tool for nine of 10 standard user interface design heuristics on a three-point scale. The eCDS tool was also rated better for clinical use on the same scale, in 84% of 50 expert-video pairs, and was rated equivalent in the remainder. Clinical experts also rated barriers to use of the eCDS tool as being low. An eCDS tool for diagnostic imaging designed using human factors engineering methods has improved perceived usability among pediatric emergency physicians.


Hip Pain in a High School Football Player: A Case Report.
OBJECTIVE: To describe the evaluation, diagnosis, and conservative treatment of a 15-year-old male high school football player with an avulsion fracture of the ischial tuberosity. BACKGROUND: Avulsion fracture of the ischial tuberosity is a rare and often missed diagnosis. A literature review offered limited information concerning the evaluation and conservative treatment of such an injury. DIFFERENTIAL DIAGNOSIS: Avulsion fracture of the ischial tuberosity. TREATMENT: The athlete's treatment goal was to return to football and weight lifting without surgical intervention. Treatment initially focused on controlling pain and normalizing gait. The athlete then advanced to a progressive resistance exercise program and functional sporting drills as he improved in hip range of motion, strength, and neuromuscular control. He returned to unrestricted sporting activities 14 weeks after the injury. UNIQUENESS: Avulsion of the ischial tuberosity is a rare injury. Most published case reports have recommended surgical intervention for this injury, with little information describing conservative treatment. CONCLUSIONS: Sports medicine practitioners must obtain an accurate history, perform a thorough physical examination, and obtain appropriate radiographs in order to correctly diagnose an ischial tuberosity avulsion fracture. Furthermore, they should consider conservative treatment for minimally displaced ischial tuberosity avulsion fractures. Should the athlete not show significant functional gains within a month of conservative treatment, the health care provider should consider surgical treatment.


Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress.
Members of the family of calcium dependent protein kinases (CDPK's) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover, extracellular Tgcdpk3 mutant parasites have motility defects that are complemented by expression of S21+S743 phosphomimetic of TgMyoA. Thus, our studies establish that phosphorylation of TgMyoA by TgCDPK3 is responsible for initiation of motility and parasite egress from the host-cell and provides mechanistic insight into how this unique kinase regulates the lytic cycle of Toxoplasma gondii.


The relationships between mood disturbances and pain, hope, and quality of life in hospitalized cancer patients with pain on regularly scheduled opioid analgesic.
The study purposes were to describe the percentage of patients in one of four mood groups (i.e., neither anxiety nor depression [NEITHER], only anxiety [ANX], only depression [DEP], both anxiety and depression [BOTH]) and to evaluate how differences in mood states are related to pain, hope, and quality of life (QOL). Oncology inpatients (n = 225) completed Brief Pain Inventory, Herth Hope Index (HHI), and the European Organization for Research and Treatment of Cancer Core QOL Questionnaire-C30. Research nurses completed Symptom Severity Checklist, Karnofsky Performance Status score, and medical record reviews. Data were analyzed using chi(2), Kruskal-Wallis, one-way analyses of variance (ANOVAs), and analyses of covariance (ANCOVA). Thirty-two percent of patients were categorized in the NEITHER group, 12% in the ANX group, 12% in the DEP group, and 44% in the BOTH group. Younger patients and women were more likely to be in the BOTH group. While only minimal differences were found among the mood groups on pain intensity scores, patients in the NEITHER group in general, reported lower pain interference scores than those in the other three groups. Significant differences were found in HHI scores between the patients in the NEITHER group and the BOTH group. In addition, patients with both mood disorders reported significantly poorer QOL scores. Because 44% of the patients had both anxiety and depression, clinicians need to evaluate patients for the co-occurrence of these two symptoms, evaluate its impact on pain management, hope, and QOL, and develop appropriate interventions to manage these symptoms.


Burkholderia phytofirmans PsJN primes Vitis vinifera L. and confers a better tolerance to low nonfreezing temperatures.
Several endophytic bacteria reportedly induce resistance to biotic stress and abiotic stress tolerance in several plant species. Burkholderia phytofirmans PsJN is a plant-growth-promoting rhizobacterium (PGPR) that is able to colonize grapevine tissues and induce resistance to gray mold. Further, PsJN induces physiological changes that increase grapevine tolerance to low nonfreezing temperatures. To better understand how bacteria induced the observed phenomena, stress-related gene expression and metabolite accumulation were monitored in 6-week-old Chardonnay grapevine plantlets after exposure to low nonfreezing temperatures. Under normal conditions (26°C), plantlet bacterization had no significant effect on the monitored parameters. By contrast, at 4°C, both stress-related gene transcripts and metabolite levels increased earlier and faster, and reached higher levels in PsJN-bacterized plantlets than in nonbacterized counterparts, in accordance with priming phenomena. The recorded changes may be correlated with the tolerance to cold stress conferred by the presence of PsJN. This is the first time that PGPR-induced priming has been shown to protect plants against low-temperature stress. Moreover, 1 week after cold exposure, levels of stress-related metabolites had declined more in PsJN-bacterized plants, suggesting that the endophyte is involved in the cold acclimation process via the scavenging system.


Does knowledge about antiretroviral therapy and mother-to-child transmission affect the relationships between HIV status and fertility preferences and contraceptive use? New evidence from Nigeria and Zambia.
The increasing availability of antiretroviral therapy (ART) and drug regimens to prevent mother-to-child transmission (PMTCT) has probably changed the context of childbearing for people living with HIV. Using data from 2009-2010 community-based surveys in Nigeria and Zambia, this study explores whether women's knowledge about ART and PMTCT influences the relationship between HIV status and fertility preferences and contraceptive behaviour. The findings show that women living with HIV are more likely to want more children in Nigeria and to want to limit childbearing in Zambia compared with HIV-negative women. While there is no significant difference in contraceptive use by women's HIV status in the two countries, women who did not know their HIV status are less likely to use contraceptives relative to women who are HIV-negative. Knowledge about ART reduces the childbearing desires of HIV-positive women in Nigeria and knowledge about PMTCT increases desire for more children among HIV-positive women in Zambia, as well as contraceptive use among women who do not know their HIV status. The findings indicate that knowledge about HIV prevention and treatment services changes how living with HIV affects childbearing desires and, at least in Zambia, pregnancy prevention, and highlight the importance of access to accurate knowledge about ART and PMTCT services to assist women and men to make informed childbearing decisions. Knowledge about ART and PMTCT should be promoted not only through HIV treatment and maternal and newborn care facilities but also through family planning centres and the mass media.


DNA-EIA to detect high and low risk HPV genotypes in cervical lesions with E6/E7 primer mediated multiplex PCR.
Oncogenicity of human papillomavirus (HPV) DNA in premalignant and malignant uterine cervical diseases is mainly induced by E6/E7 open reading frame (ORF). The presence of an oncogenic HPV DNA may be a diagnostic marker for the detection of cytologically negative smears. To evaluate an original polymerase chain reaction enzyme immunoassay (PCR-EIA) for the detection and typing of oncogenic and non-oncogenic HPV types. The test was an original multiplex labelled PCR-EIA for the detection and typing of oncogenic and non-oncogenic HPV using three consensus sequence primers within the oncogenic E6/E7 ORF. One primer was dinitrophenyl (DNP) labelled and the DNP labelled amplimers could be further hybridised with specific biotinylated oligoprobes mixed in only two cocktails: oncogenic (16, 18, 31, 33, 35, 52, and 58) and non-oncogenic (6 and 11) HPV types in only two wells; then biotinylated oligoprobes were deposited in streptavidin-coated microplates. The PCR-EIA was validated on HPV plasmids (types 6, 11, 16, 18, 31, 35, 52, and 58) and used to evaluate cervical scrapes from 181 patients (median age 32 years) at high risk for cervical cancer. HPV were detected in the cervical scrapes of 88 of 181 patients (48.6%); nine with non-oncogenic HPV (5.0%) and 79 with oncogenic HPV (43.6%) including 29 coinfections with oncogenic and non-oncogenic HPV. The number of oncogenic HPV infections increased with the presence of high grade lesions: 95.8% of the cervical scrapes from patients with high grade lesions contained oncogenic HPV compared with 32.1% of the specimens from patients without any lesions detectable by colposcopy and/or by cytological examination of the cervical smears. Moreover, 60% of cervical scrapes exhibiting low grade lesions contained oncogenic HPV. This test is simple, specific, sensitive, safe, fast, reproducible, and easy to use in routine practice. Thus, it is possible to detect simultaneously on a simple cervical scrape, two kinds of HPV--oncogenic and non-oncogenic--in just two microplate wells with non-isotopic oligoprobes.


Hypoxic modulation of ca(2+) signaling in human venous and arterial endothelial cells.
Our understanding of vascular endothelial cell physiology is based on studies of endothelial cells cultured from various vascular beds of different species for varying periods of time. Systematic analysis of the properties of endothelial cells from different parts of the vasculature is lacking. Here, we compare Ca(2+) homeostasis in primary cultures of endothelial cells from human internal mammary artery and saphenous vein and how this is modified by hypoxia, an inevitable consequence of bypass grafting (2.5% O(2), 24 h). Basal [Ca(2+)]( i ) and store depletion-mediated Ca(2+) entry were significantly different between the two cell types, yet agonist (ATP)-mediated mobilization from endoplasmic reticulum stores was similar. Hypoxia potentiated agonist-evoked responses in arterial, but not venous, cells but augmented store depletion-mediated Ca(2+) entry only in venous cells. Clearly, Ca(2+) signaling and its remodeling by hypoxia are strikingly different in arterial vs. venous endothelial cells. Our data have important implications for the interpretation of data obtained from endothelial cells of varying sources.


The zebrafish as a model for cancer.
For the last three decades significant parts of national science budgets, and international and private funding worldwide, have been dedicated to cancer research. This has resulted in a number of important scientific findings. Studies in tissue culture have multiplied our knowledge of cancer cell pathophysiology, mechanisms of transformation and strategies of survival of cancer cells, revealing therapeutically exploitable differences to normal cells. Rodent animal models have provided important insights on the developmental biology of cancer cells and on host responses to the transformed cells. However, the rate of death from some malignancies is still high, and the incidence of cancer is increasing in the western hemisphere. Alternative animal models are needed, where cancer cell biology, developmental biology and treatment can be studied in an integrated way. The zebrafish offers a number of features, such as its rapid development, tractable genetics, suitability for in vivo imaging and chemical screening, that make it an attractive model to cancer researchers. This Primer will provide a synopsis of the different cancer models generated by the zebrafish community to date. It will discuss the use of these models to further our understanding of the mechanisms of cancer development, and to promote drug discovery. The article was inspired by a workshop on the topic held in July 2009 in Spoleto, Italy, where a number of new zebrafish cancer models were presented. The overarching goal of the article is aimed at raising the awareness of basic researchers, as well as clinicians, to the versatility of this emerging alternative animal model of cancer.


Disordered reward processing and functional connectivity in trichotillomania: a pilot study.
The neurobiology of Trichotillomania is poorly understood, although there is increasing evidence to suggest that TTM may involve alterations of reward processing. The current study represents the first exploration of reward processing in TTM and the first resting state fMRI study in TTM. We incorporate both event-related fMRI using a monetary incentive delay (MID) task, and resting state fMRI, using two complementary resting state analysis methodologies (functional connectivity to the nucleus accumbens and dual regression within a reward network) in a pilot study to investigate differences in reward processing between TTM and healthy controls (HC). 21 unmedicated subjects with TTM and 14 HC subjects underwent resting state fMRI scans. A subset (13 TTM and 12 HC) also performed the MID task. For the MID task, TTM subjects showed relatively decreased nucleus accumbens (NAcc) activation to reward anticipation, but relative over-activity of the NAcc to both gain and loss outcomes. Resting state functional connectivity analysis showed decreased connectivity of the dorsal anterior cingulate (dACC) to the NAcc in TTM. Dual regression analysis of a reward network identified through independent component analysis (ICA) also showed decreased dACC connectivity and more prominently decreased basolateral amygdala connectivity within the reward network in TTM. Disordered reward processing at the level of NAcc, also involving decreased modulatory input from the dACC and the basolateral amygdala may play a role in the pathophysiology of TTM.


Accountable Care Organizations' Performance in Depression: Lessons for Value-Based Payment and Behavioral Health.
Value-based payment initiatives, such as the Medicare Shared Savings Program (MSSP), offer the possibility of using financial incentives to drive improvements in mental health and substance use outcomes. In the past 2 years, Accountable Care Organizations (ACOs) participating in the MSSP began to publicly report on one behavioral health outcome-Depression Remission at Twelve Months, which may indicate how value-based payment incentives have impacted mental health and substance use, and if reforms are needed. For ACOs that meaningfully reported performance on the depression remission measure in 2017, the median rate of depression remission at 12 months was 8.33%. A recent meta-analysis found that the average rate of spontaneous depression remission at 12 months absent treatment was approximately 53%. Although a number of factors likely explain these results, the current ACO design does not appear to incentivize improved behavioral health outcomes. Four changes in value-based payment incentive design may help to drive better outcomes: (1) making data collection easier, (2) increasing the salience of incentives, (3) building capacity to implement new interventions, and (4) creating safeguards for inappropriate treatment or reporting.


Wilms tumor chromatin profiles highlight stem cell properties and a renal developmental network.
Wilms tumor is the most common pediatric kidney cancer. To identify transcriptional and epigenetic mechanisms that drive this disease, we compared genome-wide chromatin profiles of Wilms tumors, embryonic stem cells (ESCs), and normal kidney. Wilms tumors prominently exhibit large active chromatin domains previously observed in ESCs. In the cancer, these domains frequently correspond to genes that are critical for kidney development and expressed in the renal stem cell compartment. Wilms cells also express "embryonic" chromatin regulators and maintain stem cell-like p16 silencing. Finally, Wilms and ESCs both exhibit "bivalent" chromatin modifications at silent promoters that may be poised for activation. In Wilms tumor, bivalent promoters correlate to genes expressed in specific kidney compartments and point to a kidney-specific differentiation program arrested at an early-progenitor stage. We suggest that Wilms cells share a transcriptional and epigenetic landscape with a normal renal stem cell, which is inherently susceptible to transformation and may represent a cell of origin for this disease.


Hypocretin/orexin overexpression induces an insomnia-like phenotype in zebrafish.
As many as 10% of humans suffer chronic sleep disturbances, yet the genetic mechanisms that regulate sleep remain essentially unknown. It is therefore crucial to develop simple and cost-effective vertebrate models to study the genetic regulation of sleep. The best characterized mammalian sleep/wake regulator is hypocretin/orexin (Hcrt), whose loss results in the sleep disorder narcolepsy and that has also been implicated in feeding behavior, energy homeostasis, thermoregulation, reward seeking, addiction, and maternal behavior. Here we report that the expression pattern and axonal projections of embryonic and larval zebrafish Hcrt neurons are strikingly similar to those in mammals. We show that zebrafish larvae exhibit robust locomotive sleep/wake behaviors as early as the fifth day of development and that Hcrt overexpression promotes and consolidates wakefulness and inhibits rest. Similar to humans with insomnia, Hcrt-overexpressing larvae are hyperaroused and have dramatically reduced abilities to initiate and maintain rest at night. Remarkably, Hcrt function is modulated by but does not require normal circadian oscillations in locomotor activity. Our zebrafish model of Hcrt overexpression indicates that the ancestral function of Hcrt is to promote locomotion and inhibit rest and will facilitate the discovery of neural circuits, genes, and drugs that regulate Hcrt function and sleep.


Identification of new TRIP12 variants and detailed clinical evaluation of individuals with non-syndromic intellectual disability with or without autism.
The ubiquitin pathway is an enzymatic cascade including activating E1, conjugating E2, and ligating E3 enzymes, which governs protein degradation and sorting. It is crucial for many physiological processes. Compromised function of members of the ubiquitin pathway leads to a wide range of human diseases, such as cancer, neurodegenerative diseases, and neurodevelopmental disorders. Mutations in the thyroid hormone receptor interactor 12 (TRIP12) gene (OMIM 604506), which encodes an E3 ligase in the ubiquitin pathway, have been associated with autism spectrum disorder (ASD). In addition to autistic features, TRIP12 mutation carriers showed intellectual disability (ID). More recently, TRIP12 was postulated as a novel candidate gene for intellectual disability in a meta-analysis of published ID cohorts. However, detailed clinical information characterizing the phenotype of these individuals was not provided. In this study, we present seven novel individuals with private TRIP12 mutations including two splice site mutations, one nonsense mutation, three missense mutations, and one translocation case with a breakpoint in intron 1 of the TRIP12 gene and clinically review four previously published cases. The TRIP12 mutation-positive individuals presented with mild to moderate ID (10/11) or learning disability [intelligence quotient (IQ) 76 in one individual], ASD (8/11) and some of them with unspecific craniofacial dysmorphism and other anomalies. In this study, we provide detailed clinical information of 11 TRIP12 mutation-positive individuals and thereby expand the clinical spectrum of the TRIP12 gene in non-syndromic intellectual disability with or without ASD.


External levator advancement vs Müller's muscle-conjunctival resection for correction of upper eyelid involutional ptosis.
To compare external levator advancement and Müller's muscle-conjunctival resection (conjunctivomullerectomy, or CJM) for correction of upper eyelid involutional ptosis. Retrospective, nonrandomized, comparative interventional case series. Review of medical records of 159 patients (272 surgical procedures) who underwent external levator advancement or CJM was performed. Functional and cosmetic outcome, marginal reflex distance one (MRD1), and surgical complications. A total of 159 patients (51 men, 108 women, mean age 70 years) underwent 272 surgical procedures for upper eyelid ptosis; concurrent blepharoplasty was performed in 141 cases. MRD1 increased an average of 1.6 (+/-1.5) mm, from 0.8 mm (+/-1.2) preoperatively to 2.3 mm (+/-1.2) postoperatively (P < .001). Fifteen patients (5.5%) underwent reoperation for residual ptosis, nine (18%) in the external levator advancement group, two (3%) in the CJM group, three (8%) in the external plus blepharoplasty group, and one (1%) in the CJM plus blepharoplasty group (P < .001). Patients who underwent external levator advancement had significantly more severe ptosis preoperatively but attained similar eyelid position postoperatively as compared with CJM patients. Complications included overcorrection in four cases (1.4%), lagophthalmos of 1 mm in 10 (3.6%), and pyogenic granuloma in two (<1%). External levator advancement and CJM performed alone or with concurrent blepharoplasty are effective treatments for upper eyelid ptosis. Residual ptosis or postoperative eyelid retraction occurs in up to 20% of cases and can be addressed successfully with a second operation.


Effect of diet and age on jejunal and circulating lymphocyte subsets in children with coeliac disease: persistence of CD4-8-intraepithelial T cells through treatment.
Monoclonal antibodies were used to determine the relative numbers of T lymphocyte subsets in 61 jejunal biopsies and in peripheral blood of 35 children with coeliac disease, and of 13 healthy controls. The T cell numbers in the lamina propria were unaffected by a change from gluten-free to gluten containing diet in the patients. The number of intraepithelial lymphocytes (where the CD8 cells predominated) were significantly raised in patients taking gluten. Ten to 20% of the patients' intraepithelial CD3 (mature T) cells expressed neither CD8 nor CD4 surface antigens. This CD4 8 T cell population persisted through gluten elimination and challenge. The circulating lymphocyte subsets showed little variation with the diet although there was a marked increase in the proportion (14.9%) of CD4 8 T cells in patients during gluten elimination. In the histologically normal jejunal mucosa from control subjects, the age of the subject showed a positive correlation with villus intraepithelial CD3+ and CD8+ cells, and crypt intraepithelial CD4+ cells. No clear cut effect of age was observed on lamina propria lymphocyte counts of the controls, or on the lymphocyte counts in jejunal mucosa of the coeliac patients. The observed CD3+4-8- lymphocytes may represent activated cells unable to present their surface antigens, or they may be gamma delta-receptor bearing T cells, which could have a significant role in the pathogenesis of coeliac disease.


Therapies for psoriatic enthesopathy. A systematic review.
Enthesitis is defined as inflammation at sites of tendon, ligament, joint capsule, or fascia insertion sites to bone, and is a hallmark feature of psoriatic arthritis. Several outcome measures have been developed to assess enthesitis, but none have been validated in psoriatic arthritis. In this evidence-based review, we assess the limited data on treatments for enthesitis and make recommendations for further studies in psoriatic enthesitis.


Protective effect of α-lipoic acid against arsenic trioxide-induced acute cardiac toxicity in rats.
The clinical use of arsenic trioxide (ATO) is often limited because of its adverse effects. We examined whether α-lipoic acid (LA) protects against the ATO-induced cardiac toxicity. In the chronic study, two of four rats suddenly died by the repeated dosing of ATO, whereas no deaths were observed in combination with LA. In the acute study, continuous ECG recording revealed that intravenous injection of ATO caused transient ST-T change, whereas pretreatment with LA abolished the ATO-induced ECG abnormality in all animals. These results suggest that LA protects against the ATO-induced acute cardiac toxicity and subsequent sudden death in rats.


BET Bromodomain Inhibition Suppresses the Function of Hematopoietic Transcription Factors in Acute Myeloid Leukemia.
The bromodomain and extraterminal (BET) protein BRD4 is a validated drug target in leukemia, yet its regulatory function in this disease is not well understood. Here, we show that BRD4 chromatin occupancy in acute myeloid leukemia closely correlates with the hematopoietic transcription factors (TFs) PU.1, FLI1, ERG, C/EBPα, C/EBPβ, and MYB at nucleosome-depleted enhancer and promoter regions. We provide evidence that these TFs, in conjunction with the lysine acetyltransferase activity of p300/CBP, facilitate BRD4 recruitment to their occupied sites to promote transcriptional activation. Chemical inhibition of BET bromodomains was found to suppress the functional output of each hematopoietic TF, thereby interfering with essential lineage-specific transcriptional circuits in this disease. These findings reveal a chromatin-based signaling cascade comprised of hematopoietic TFs, p300/CBP, and BRD4 that supports leukemia maintenance and is suppressed by BET bromodomain inhibition.


Setting the standard for recovery: Physicians' Health Programs.
A sample of 904 physicians consecutively admitted to 16 state Physicians' Health Programs (PHPs) was studied for 5 years or longer to characterize the outcomes of this episode of care and to explore the elements of these programs that could improve the care of other addicted populations. The study consisted of two phases: the first characterized the PHPs and their system of care management, while the second described the outcomes of the study sample as revealed in the PHP records. The programs were abstinence-based, requiring physicians to abstain from any use of alcohol or other drugs of abuse as assessed by frequent random tests typically lasting for 5 years. Tests rapidly identified any return to substance use, leading to swift and significant consequences. Remarkably, 78% of participants had no positive test for either alcohol or drugs over the 5-year period of intensive monitoring. At post-treatment follow-up 72% of the physicians were continuing to practice medicine. The unique PHP care management included close linkages to the 12-step programs of Alcoholics Anonymous and Narcotics Anonymous and the use of residential and outpatient treatment programs that were selected for their excellence.


Intermittent oral iron supplementation during pregnancy.
Anaemia is a frequent condition during pregnancy, particularly among women from developing countries who have insufficient iron intake to meet increased iron needs of both the mother and the fetus.Traditionally, gestational anaemia has been prevented with the provision of daily iron supplements throughout pregnancy, but adherence to this regimen due to side effects, interrupted supply of the supplements, and concerns about safety among women with an adequate iron intake, have limited the use of this intervention. Intermittent (i.e. one, two or three times a week on non-consecutive days) supplementation with iron alone or in combination with folic acid or other vitamins and minerals has recently been proposed as an alternative to daily supplementation. To assess the benefits and harms of intermittent supplementation with iron alone or in combination with folic acid or other vitamins and minerals to pregnant women on neonatal and pregnancy outcomes. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (23 March 2012). We also searched the WHO International Clinical Trials Registry Platform (ICTRP) for ongoing studies and contacted relevant organisations for the identification of ongoing and unpublished studies (23 March 2012). Randomised or quasi-randomised trials. We assessed the methodological quality of trials using standard Cochrane criteria. Two review authors independently assessed trial eligibility, extracted data and conducted checks for accuracy. This review includes 21 trials from 13 different countries, but only 18 trials (with 4072 women) reported on our outcomes of interest and contributed data to the review. All of these studies compared daily versus intermittent iron supplementation.Three studies provided iron alone, 12 iron+folic acid and three more iron plus multiple vitamins and minerals. Their methodological quality was mixed and most had high levels of attrition. Overall, there was no clear evidence of differences between groups for infant primary outcomes: low birthweight (average risk ratio (RR) 0.96; 95% confidence interval (CI) 0.61 to 1.52, seven studies), infant birthweight (mean difference MD -8.62 g; 95% CI -52.76 g to 35.52 g, eight studies), premature birth (average RR 1.82; 95% CI 0.75 to 4.40, four studies). None of the studies reported neonatal deaths or congenital anomalies.For maternal outcomes, there was no clear evidence of differences between groups for anaemia at term (average RR 1.22; 95% CI 0.84 to 1.80, four studies) and women receiving intermittent supplementation had less side effects (average RR 0.56; 95% CI 0.37 to 0.84, 11 studies) than those receiving daily supplements. Women receiving intermittent supplements were also at lower risk of having high haemoglobin (Hb) concentrations (greater than 130 g/L) during the second or third trimester of pregnancy (average RR 0.48; 95% CI 0.35 to 0.67, 13 studies). There were no significant differences in iron-deficiency anaemia between women receiving intermittent or daily iron+folic acid supplementation (average RR 0.71; 95% CI 0.08 to 6.63, 1 study). There were no maternal deaths (six studies) or women with severe anaemia in pregnancy (six studies). None of the studies reported on iron deficiency at term or infections during pregnancy.Where sufficient data were available for primary outcomes, we set up subgroups to look for possible differences between studies in terms of earlier or later supplementation; women's anaemia status at the start of supplementation; higher and lower weekly doses of iron; and the malarial status of the region in which the trials were conducted. There was no clear effect of these variables on the results of the review. The present systematic review is the most comprehensive summary of the evidence assessing the benefits and harms of intermittent iron supplementation regimens in pregnant women on haematological and pregnancy outcomes. The findings suggest that intermittent iron+folic acid regimens produce similar maternal and infant outcomes at birth as daily supplementation but are associated with fewer side effects. Women receiving daily supplements had increased risk of developing high levels of Hb in mid and late pregnancy but were less likely to present mild anaemia near term. Although the evidence is limited and the quality of the trials was low or very low, intermittent may be a feasible alternative to daily iron supplementation among those pregnant women who are not anaemic and have adequate antenatal care.


Use of real-time sensors for compliance monitoring of nitrate in finished drinking water.
Across the Midwestern United States, Public Water Systems (PWSs) struggle with high levels of nitrate in source waters from intense agricultural activity. Leveraging a sensor network deployed across Iowa surface waters, we evaluated the potential of the Hach Nitratax SC Plus, which uses UV-light absorption to quantify dissolved nitrate-nitrite (NOx-N) down to 0.1 mg-N L-1, for real-time monitoring of NOx-N in drinking water. For six different PWSs over multiple years, we compare NOx-N levels in source waters (surface and groundwater under surface influence) to those measured via traditional methods (e.g., ion chromatography (IC)) for US EPA compliance monitoring. At one large PWS, we also evaluated sensor performance when applied to near-finished drinking water (filter effluent). We find good agreement between traditional analytical methods and in situ sensors. For example, for 771 filter effluent samples from 2006-2011, IC analysis averaged NOx-N of 5.8 mg L-1 while corresponding sensor measurements averaged 5.7 mg L-1 with a mean absolute error of 0.23 (5.6%). We identify several benefits of using real-time sensors in PWSs, including improved frequency to capture elevated NOx-N levels and as decision-support tools for NOx-N management.


Water-Compression Gating of Nanopore Transport.
Electric field-driven motion of biomolecules is a process essential to many analytics methods, in particular, to nanopore sensing, where a transient reduction of nanopore ionic current indicates the passage of a biomolecule through the nanopore. However, before any molecule can be examined by a nanopore, the molecule must first enter the nanopore from the solution. Previously, the rate of capture by a nanopore was found to increase with the strength of the applied electric field. Here, we theoretically show that, in the case of narrow pores in graphene membranes, increasing the strength of the electric field can not only decrease the rate of capture, but also repel biomolecules from the nanopore. As the strong electric field polarizes water near and within the nanopore, the high gradient of the field also produces a strong dielectrophoretic force that compresses the water. The pressure difference caused by the sharp water density gradient produces a hydrostatic force that repels DNA or proteins from the nanopore, preventing, in certain conditions, their capture. We show that such local compression of fluid can regulate the transport of biomolecules through nanoscale passages in the absence of physical gates and sort proteins according to their phosphorylated states.


Triangulation of cubic panorama for view synthesis.
An unstructured triangulation approach, new to our knowledge, is proposed to apply triangular meshes for representing and rendering a scene on a cubic panorama (CP). It sophisticatedly converts a complicated three-dimensional triangulation into a simple three-step triangulation. First, a two-dimensional Delaunay triangulation is individually carried out on each face. Second, an improved polygonal triangulation is implemented in the intermediate regions of each of two faces. Third, a cobweblike triangulation is designed for the remaining intermediate regions after unfolding four faces to the top/bottom face. Since the last two steps well solve the boundary problem arising from cube edges, the triangulation with irregular-distribution feature points is implemented in a CP as a whole. The triangular meshes can be warped from multiple reference CPs onto an arbitrary viewpoint by face-to-face homography transformations. The experiments indicate that the proposed triangulation approach provides a good modeling for the scene with photorealistic rendered CPs.


Motivational typologies of drinkers: do enhancement and coping drinkers form two distinct groups?
This study used a person-centered approach to test whether drinking motive typologies could be identified. Longitudinal study of college students within the intensive multivariate prospective alcohol college-transitions (IMPACTS) data set. University campus in the United States. University students (baseline n reporting alcohol motives = 2158; baseline age = 18.60 years old). The drinking motives questionnaire-revised (DMQ-R). Using Steinley & Brusco's cluster analysis approach [based on the theoretical ratio expected between the within sum of squares and the total sum of squares when the data are divided into two clusters when no cluster structure is present; the cut-off for the ratio is 0.25 for uniform (multivariate uniform) distributions and 0.36 for normal (multivariate normal) distributions], we examined whether there was evidence for distinct clusters of individuals that differed on their overall level of motives to drink. We tested the fit of a one-group (cluster) solution compared to multi-cluster solutions. Both cross-sectionally and prospectively, the data could not be partitioned into two or more clusters [regardless of whether the cut-off assuming a multivariate uniform distribution (i.e. 0.25) or the more liberal multivariate normal distribution (i.e., 0.36) was used]. These findings showed that enhancement and coping drinkers do not form two distinct groups but, rather, these motives exist on a continuum such that individuals who are high in one internal motive tend to be high in the other motive. Coping and enhancement drinkers do not form two distinct groups. Variable-centered approaches to drinking motives may be a better alternative to classifying all drinkers as either enhancement or coping drinkers for both clinical and research endeavors.


Prognostic Value of Serial Changes in High-Sensitivity Cardiac Troponin I and T over 3 Months Using Reference Change Values in Hemodialysis Patients.
Serial changes in cardiac troponin in hemodialysis (HD) patients have uncertain clinical implications. We evaluated associations of adverse outcomes in HD patients with reference change value (RCV) data and tertile concentrations for cardiac troponin I (cTnI) and cTnT measured by high-sensitivity (hs) assays. RCV data and tertiles for hs-cTnI and hs-cTnT were determined from plasma samples collected 3 months apart in 677 stable outpatient HD patients and assessed for their associations with adverse outcomes using adjusted Cox models. Primary outcomes were all-cause mortality and sudden cardiac death (SCD). During a median follow-up of 23 months, 18.6% of patients died. RCVs were: hs-cTnI +37% and -30%; hs-cTnT +25% and -20%. Patients with serial hs-cTnI and hs-cTnT changes >RCV (increase or decrease) had all-cause mortality of 25.2% and 23.8% respectively, compared to 15.0% and 16.5% with changes ≤RCV [adjusted hazard ratios (aHRs): 1.9, P = 0.0003 and 1.7, P = 0.0066), respectively]. Only hs-cTnI changes >RCV were predictive of SCD (aHR 2.6, P = 0.005). hs-Cardiac troponin changes >RCV improved all-cause mortality prognostication compared to changes ≤RCV in tertile 2: hs-cTnI aHR, 2.70 (P = 0.003); hs-cTnT aHR, 1.98 (P = 0.043). The aHR of changes in hs-cTnI in tertile 2 >RCV for SCD was 5.62 (P = 0.039). Changes over 3 months in hs-cTnI and hs-cTnT of >RCV identified patients at greater risk of all-cause mortality, and for hs-cTnI were also predictive of SCD. Among patients with middle tertile cardiac troponin concentrations, hs-cTnI changes >RCV provided additive prognostic value for both SCD and all-cause mortality, whereas those for hs-cTnT provided additive prognostic value only for all-cause mortality.


Cellular redox modulator, ortho Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, MnTnHex-2-PyP(5+) in the treatment of brain tumors.
Despite intensive efforts to improve multimodal treatment of brain tumor, survival remains limited. Current therapy consists of a combination of surgery, irradiation and chemotherapy with predisposition to long-term complications. Identifying novel targeted therapies is therefore at the forefront of brain tumor research. This study explores the utility of a manganese porphyrin in a brain tumor model. The compound used is ortho isomer, mangnese(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, MnTnHex-2-PyP(5+). It is a powerful SOD mimic and peroxynitrite scavenger and a potent modulator of redox-based cellular transcriptional activity, able to suppress excessive immune and inflammatory responses and in turn proliferative pathways. It is further one of the most lipophilic compound among cationic Mn(III) N-alkylpyridylporphyrins, and thus accumulates predominantly in mitochondria relative to cytosol. In mitochondria, MnTnHex-2-PyP(5+) mimics our key antioxidant system, mitochondrial superoxide dismutase, MnSOD, whose overexpression has been widely shown to suppress tumor growth. Importantly, MnTnHex-2-PyP(5+) crosses blood brain barrier in sufficient amounts to demonstrate efficacy in treating CNS injuries. For those reasons we elected to test its effects in inhibiting brain tumor growth. This study is the first report of the antitumor properties of MnTnHex-2-PyP(5+) as a single agent in adult and pediatric glioblastoma multiforme (D-54 MG, D-245 MG, D-256 MG, D-456 MG) and pediatric medulloblastoma (D-341 MED), and is the first case where a redox-able metal complex has been used in glioma therapy. When given subcutaneously to mice bearing subcutaneous and intracranial xenografts, MnTnHex-2-PyP(5+) caused a significant (P ≤ 0.001) growth delay in D 245 MG, D-256 MG, D-341 MED, and D-456 MG tumors. Growth delay for mice bearing subcutaneous xenografts ranged from 3 days in D-54 MG to 34 days in D-341 MED. With mice bearing intracranial xenografts, MnTnHex-2-PyP(5+) increases median survival by 33% in adult glioblastoma multiforme (D-256 MG; p≤ 0.001) and 173% in pediatric medulloblastoma (D-341 MED, <0.001). The beneficial effects of MnTnHex-2-PyP(5+) are presumably achieved either (1) indirectly via elimination of signaling reactive oxygen and nitrogen species (in particular superoxide and peroxynitrite) which in turn would prevent activation of transcription factors; or (2) directly by coupling with cellular reductants and redox-sensitive signaling proteins. The former action is antioxidative while the latter action is presumably pro-oxidative in nature. Our findings suggest that the use of Mn porphyrin-based SOD mimics, and in particular lipophilic analogues such as MnTnHex-2-PyP(5+), is a promising approach for brain tumor therapy.


FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath.
Spirochetes have periplasmic flagella composed of a core surrounded by a sheath. The pathogen Leptospira interrogans has four flaB (proposed core subunit) and two flaA (proposed sheath subunit) genes. The flaA genes are organized in a locus with flaA2 immediately upstream of flaA1. In this study, flaA1 and flaA2 mutants were constructed by transposon mutagenesis. Both mutants still produced periplasmic flagella. The flaA1 mutant did not produce FlaA1 but continued to produce FlaA2 and retained normal morphology and virulence in a hamster model of infection but had reduced motility. The flaA2 mutant did not produce either the FlaA1 or the FlaA2 protein. Cells of the flaA2 mutant lacked the distinctive hook-shaped ends associated with L. interrogans and lacked translational motility in liquid and semisolid media. These observations were confirmed with a second, independent flaA2 mutant. The flaA2 mutant failed to cause disease in animal models of acute infection. Despite lacking FlaA proteins, the flagella of the flaA2 mutant were of the same thickness as wild-type flagella, as measured by electron microscopy, and exhibited a normal flagellum sheath, indicating that FlaA proteins are not essential for the synthesis of the flagellum sheath, as observed for other spirochetes. This study shows that FlaA subunits contribute to leptospiral translational motility, cellular shape, and virulence.


Pooled deep sequencing of Plasmodium falciparum isolates: an efficient and scalable tool to quantify prevailing malaria drug-resistance genotypes.
Molecular surveillance for drug-resistant malaria parasites requires reliable, timely, and scalable methods. These data may be efficiently produced by genotyping parasite populations using second-generation sequencing (SGS). We designed and validated a SGS protocol to quantify mutant allele frequencies in the Plasmodium falciparum genes dhfr and dhps in mixed isolates. We applied this new protocol to field isolates from children and compared it to standard genotyping using Sanger sequencing. The SGS protocol accurately quantified dhfr and dhps allele frequencies in a mixture of parasite strains. Using SGS of DNA that was extracted and then pooled from individual isolates, we estimated mutant allele frequencies that were closely correlated to those estimated by Sanger sequencing (correlations, >0.98). The SGS protocol obviated most molecular steps in conventional methods and is cost saving for parasite populations >50. This SGS genotyping method efficiently and reproducibly estimates parasite allele frequencies within populations of P. falciparum for molecular epidemiologic studies.


Prevalence of Antimicrobial Resistance and Hemolytic Phenotypes in Culturable Arctic Bacteria.
Many Arctic biomes, which are populated with abundant and diverse microbial life, are under threat: climate change and warming temperatures have raised concerns about diversity loss and possible emergence of pathogenic microorganisms. At present, there is little information on the occurrence of Arctic virulence-associated phenotypes. In this study we worked with 118 strains of bacteria (from 10 sampling sites in the Arctic region, located in Greenland and the Svalbard Archipelago) isolated using R2A medium. These strains belong to 4 phyla and represent 36 different bacterial genera. Phenotypic resistance to 8 clinically important antimicrobials (ampicillin, chloramphenicol, ciprofloxacin, cefotaxime, erythromycin, imipenem, kanamycin, and tetracycline) and thermotolerance range were determined. In addition, a screening of all isolates on blood agar media and erythrocytes suspension of bovine and sheep erythrocytes for virulence-linked hemolytic activity was performed. Although antimicrobial resistance profiles varied among the isolates, they were consistent within bacterial families and genera. Interestingly, a high number of isolates (83/104) were resistant to the tested concentration of imipenem (4 mg/L). In addition, one third of the isolates showed hemolytic activity on blood agar, however, in only 5% of the isolates hemolytic activity was also observed in the cell extracts when added to erythrocyte suspensions for 60 min. The observed microbial phenotypes contribute to our understanding of the presence of virulence-associated factors in the Arctic environments, while highlighting the potential risks associated with changes in the polar areas in the light of climate change.


Evolutionary movement of centromeres in horse, donkey, and zebra.
Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing considerably, we investigated the role of CR in the karyotype evolution of the genus Equus. Using appropriate panels of BAC clones in FISH experiments, we compared the centromere position and marker order arrangement among orthologous chromosomes of Burchelli's zebra (Equus burchelli), donkey (Equus asinus), and horse (Equus caballus). Surprisingly, at least eight CRs took place during the evolution of this genus. Even more surprisingly, five cases of CR have occurred in the donkey after its divergence from zebra, that is, in a very short evolutionary time (approximately 1 million years). These findings suggest that in some species the CR phenomenon could have played an important role in karyotype shaping, with potential consequences on population dynamics and speciation.


Continuum percolation of polydisperse hyperspheres in infinite dimensions.
We analyze the critical connectivity of systems of penetrable d-dimensional spheres having size distributions in terms of weighed random geometrical graphs, in which vertex coordinates correspond to random positions of the sphere centers, and edges are formed between any two overlapping spheres. Edge weights naturally arise from the different radii of two overlapping spheres. For the case in which the spheres have bounded size distributions, we show that clusters of connected spheres are treelike for d→∞ and they contain no closed loops. In this case, we find that the mean cluster size diverges at the percolation threshold density η(c)→2(-d), independently of the particular size distribution. We also show that the mean number of overlaps for a particle at criticality z(c) is smaller than unity, while z(c)→1 only for spheres with fixed radii. We explain these features by showing that in the large dimensionality limit, the critical connectivity is dominated by the spheres with the largest size. Assuming that closed loops can be neglected also for unbounded radii distributions, we find that the asymptotic critical threshold for systems of spheres with radii following a log-normal distribution is no longer universal, and that it can be smaller than 2(-d) for d→∞.


Selection of medical diagnostic codes for analysis of electronic patient records. Application to stroke in a primary care database.
Electronic patient records from primary care databases are increasingly used in public health and health services research but methods used to identify cases with disease are not well described. This study aimed to evaluate the relevance of different codes for the identification of acute stroke in a primary care database, and to evaluate trends in the use of different codes over time. Data were obtained from the General Practice Research Database from 1997 to 2006. All subjects had a minimum of 24 months of up-to-standard record before the first recorded stroke diagnosis. Initially, we identified stroke cases using a supplemented version of the set of codes for prevalent stroke used by the Office for National Statistics in Key health statistics from general practice 1998 (ONS codes). The ONS codes were then independently reviewed by four raters and a restricted set of 121 codes for 'acute stroke' was identified but the kappa statistic was low at 0.23. Initial extraction of data using the ONS codes gave 48,239 cases of stroke from 1997 to 2006. Application of the restricted set of codes reduced this to 39,424 cases. There were 2,288 cases whose index medical codes were for 'stroke annual review' and 3,112 for 'stroke monitoring'. The frequency of stroke review and monitoring codes as index codes increased from 9 per year in 1997 to 1,612 in 2004, 1,530 in 2005 and 1,424 in 2006. The one year mortality of cases with the restricted set of codes was 29.1% but for 'stroke annual review,' 4.6% and for 'stroke monitoring codes', 5.7%. In the analysis of electronic patient records, different medical codes for a single condition may have varying clinical and prognostic significance; utilisation of different medical codes may change over time; researchers with differing clinical or epidemiological experience may have differing interpretations of the relevance of particular codes. There is a need for greater transparency in the selection of sets of codes for different conditions, for the reporting of sensitivity analyses using different sets of codes, as well as sharing of code sets among researchers.


Flow diverters for ruptured posterior circulation perforator aneurysms: multicenter experience and literature review.
The management of ruptured posterior circulation perforator aneurysms (rPCPAs) remains unclear. We present our experience in treating rPCPAs with flow diverter stents (FDs) and evaluate their safety and efficacy at mid- to long-term follow-up. A diagnostic and therapeutic algorithm for rPCPAs is also proposed. We retrospectively analyzed data from all consecutive patients with rPCPAs treated with FDs at our institutions between January 2013 and July 2019. Clinical presentations, time of treatments, intra- and perioperative complications, and clinical and angiographic outcomes were recorded, with a mid- to long-term follow-up. A systematic review of the literature on rPCPAs treated with FDs was also performed. Seven patients with seven rPCPAs were treated with FDs. All patients presented with an atypical subarachnoid hemorrhage distribution and a low to medium Hunt-Hess grade. In 29% of cases rPCPAs were identified on the initial angiogram. In 57% of cases, FDs were inserted within 2 days of the diagnosis. Immediate aneurysm occlusion was observed in 14% of the cases and in 71% at the first follow-up (mean 2.4 months). At mean follow-up of 33 months (range 3-72 months) one case of delayed ischemic complication occurred. Six patients had a modified Rankin Scale (mRS) score of 0 and one patient had an mRS score of 4 at the latest follow-up. The best management for rPCPAs remains unclear, but FDs seem to have lower complication rates than other treatment options. Further studies with larger series are needed to confirm the role of FDs in rPCPA.


Issues of medical management in adults with spina bifida.
The objective of this study was to establish the range of medical issues among those attending a clinic for adults with spina bifida (SB) and hydrocephalus (CASBAH). Owing to improvements in medical care in the past 3-4 decades, an increasing number of SB patients go on to prosper in adult life. Since 1990 there has been a CASBAH service in Belfast serving Northern Ireland on a regional basis. There are now 237 patients with SB remaining on the live register at CASBAH. All records were reviewed with regard to site of lesion, ambulatory ability, shunt placement and regularity of attendance. The records of 193 were also reviewed with reference to musculoskeletal problems, bladder function, bowel function, renal impairment and hypertension, skin breakdown, epilepsy and the incidence of clinically significant Chiari/hydrosyringomyelia. Of the patients on the register, 36% are wheelchair dependent, 8% have some ambulatory capacity but are largely wheelchair dependent, 22% are ambulatory with aid and 34% are independently ambulatory. Bladder function is normal in 8%, whilst 32% describe normal bowel function. Renal impairment is present in 48% of patients, and 15% are on anti-hypertensive therapy. Epilepsy is an active issue in 9%, and intraventricular shunts are in situ in 37% of patients. Scoliosis is present in 50% and 70% have joint deformities or contractures. Five patients have become symptomatic from the Chiari/hydrosyringomyelia complex. These data reflect the considerable range of disability in adult SB patients, the challenges presented in long-term management and the need for organised follow-up.


Biological variation of hematology tests based on the 1999-2002 National Health and Nutrition Examination Survey.
Biological variation consists of between-person (BP) and within-person (WP) variation. Estimates of WP coefficients of variation (CVw) and BP coefficients of variation (CVg) for hematology laboratory tests were estimated from the 1999-2002 National Health and Nutrition Examination Survey (NHANES). NHANES is a survey of the civilian noninstitutionalized U.S. population that uses a stratified, multistage probability design. Between- and within-person variations were estimated for 18 hematology tests. For WP variation, a nonrandom sample was obtained with a median of 17 days between two test measurements. Between-person variation was estimated from the WP sample and additional participants were matched for age group, gender, and race and ethnicity to the WP sample. The BP and WP variations were estimated on as many as 2496 and 852 sample participants, respectively. Mean corpuscular hemoglobin concentration had the lowest CVg (2.25% for men and 2.40% for women), and mean corpuscular volume had the lowest CVw (0.31% for men and 0.37% for women). The index of individuality (CVw/CVg) ranged from 0.06 for mean corpuscular volume for men and women to 0.62 for segmented neutrophil number for men, and 0.55 for segmented neutrophil percent for women. Women had higher CVw compared with men for hematocrit, hemoglobin, mean corpuscular volume, red blood cell count, and red blood cell distribution width. Several hematology tests' CVw also differed by age group, including mean corpuscular volume; eosinophil, lymphocyte and segmented neutrophil percent; monocyte and segmented neutrophil number; white blood cell count; and red blood cell distribution width.


Identification of γ-synuclein as a stage-specific marker in bladder cancer by immunohistochemistry.
Previous studies have shown that the expression level of γ-synuclein (SNCG) is associated with progression of many different malignant tumors. In this study, we discuss and assess the prognostic ability of SNCG in bladder cancer. Medical records (2005-2013) were retrospectively reviewed for the population of interest. SNCG expression was identified immunohistochemically from bladder cancer tissues of 113 bladder cancer patients. The survival rate was calculated by the Kaplan-Meier method. Cox proportional hazard regression model was used for analysis of predictors of bladder cancer. SNCG was overexpressed in bladder cancer tissues compared with the normal bladder tissues (p<0.0001). SNCG expression in bladder cancer tissue was strongly related to tumor stage. However, SNCG level was not a prognostic factor of survival. Our results demonstrate that SNCG is highly expressed in bladder cancer tissue and its expression is stage-specific, but it is not helpful for predicting outcome in bladder cancer patients.


Neuron-derived orphan receptor-1 modulates cardiac gene expression and exacerbates angiotensin II-induced cardiac hypertrophy.
Hypertensive cardiac hypertrophy (HCH) is a common cause of heart failure (HF), a major public health problem worldwide. However, the molecular bases of HCH have not been completely elucidated. Neuron-derived orphan receptor-1 (NOR-1) is a nuclear receptor whose role in cardiac remodelling is poorly understood. The aim of the present study was to generate a transgenic mouse over-expressing NOR-1 in the heart (TgNOR-1) and assess the impact of this gain-of-function on HCH. The CAG promoter-driven transgenesis led to viable animals that over-expressed NOR-1 in the heart, mainly in cardiomyocytes and also in cardiofibroblasts. Cardiomyocytes from TgNOR-1 exhibited an enhanced cell surface area and myosin heavy chain 7 (Myh7)/Myh6 expression ratio, and increased cell shortening elicited by electric field stimulation. TgNOR-1 cardiofibroblasts expressed higher levels of myofibroblast markers than wild-type (WT) cells (α 1 skeletal muscle actin (Acta1), transgelin (Sm22α)) and were more prone to synthesise collagen and migrate. TgNOR-1 mice experienced an age-associated remodelling of the left ventricle (LV). Angiotensin II (AngII) induced the cardiac expression of NOR-1, and NOR-1 transgenesis exacerbated AngII-induced cardiac hypertrophy and fibrosis. This effect was associated with the up-regulation of hypertrophic (brain natriuretic peptide (Bnp), Acta1 and Myh7) and fibrotic markers (collagen type I α 1 chain (Col1a1), Pai-1 and lysyl oxidase-like 2 (Loxl2)). NOR-1 transgenesis up-regulated two key genes involved in cardiac hypertrophy (Myh7, encoding for β-myosin heavy chain (β-MHC)) and fibrosis (Loxl2, encoding for the extracellular matrix (ECM) modifying enzyme, Loxl2). Interestigly, in transient transfection assays, NOR-1 drove the transcription of Myh7 and Loxl2 promoters. Our findings suggest that NOR-1 is involved in the transcriptional programme leading to HCH.


Inhibitory effect of mixture herbs/spices on formation of heterocyclic amines and mutagenic activity of grilled beef.
Natural antioxidants in spices and herbs have attracted considerable attention as potential inhibitors against the formation of mutagenic heterocyclic amines (HCAs) in heat-processed meat. In this study, the inhibitory activity of four spices/herbs and their mixtures on HCAs formation in grilled beef were examined. A simplex centroid mixture design with four components comprising turmeric, curry leaf, torch ginger and lemon grass in 19 different proportions were applied on beef samples before grilling at 240 ºC for 10 min. The HCAs were extracted from the samples using solid phase extraction (SPE) method and analysed using Liquid chromatography mass spectrometry LC-MS/MS. All spices/herbs in single or mixture forms were found to reduce total HCA concentrations in marinated grilled beef ranging from 21.2% for beef marinated with curry leaf to 94.7% for the combination of turmeric and lemon grass (50:50 w/w). At the optimum marinade formula (turmeric: lemon grass 52.4%: 47.6%), concentration of 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), Harman, Norharman and AαC were 2.2, 1.4, 0.5, 2.8 and 1.2 ng/g, respectively. The results of the mutagenic activity demonstrated that this optimised marinade formula significantly (p < 0.05) diminished mutagenicity of marinated grilled beef in bacterial Ames test.


Insertional inactivation of the downless gene in a family of transgenic mice.
The mouse downless (dl) gene is a morphogenetic gene that plays a role in dermal-epidermal interaction and regulation of hair follicle induction during fetal development. We report here the identification of a transgenic mouse line with an insertional mutation in the dl gene. The genomic sequences flanking the transgenic insert have been cloned and used as hybridization probes to confirm that the mutant transgenic mice are homozygous for the transgenic insert and that the site of integration lies on mouse chromosome 10. Genomic probes that are close to or within the downless gene are now available and should permit the characterization of a gene that is involved in induction of a specific type of epithelial morphogenesis.


Early-life or lifetime sun exposure, sun reaction, and the risk of squamous cell carcinoma in an Asian population.
It has been widely accepted that sun exposure is a risk factor of squamous cell carcinoma (SCC) among fair-skinned populations. However, sun exposure and sun reaction have not been explored in Asians and no gender-specific data were available. In a case-control study, 176 incident skin cancer cases were recruited from National Cheng-Kung University Medical Center from 1996 to 1999. Controls included 216 age-, gender-, and residency-matched subjects from the southwestern Taiwan. A questionnaire was administered to collect information on life style and other risk factors. Logistic regression analysis was performed to evaluate the association between sun exposure or sun reaction and the risk of SCC by gender. Early-age (age 15 to 24) and lifetime sun exposure were significantly associated with increased risk of SCC in a dose-response pattern [odds ratio (OR) = 1.49-3.08, trend p = 0.009 and 0.0007, respectively]. After stratified by gender, the third tertile of early-age sun exposure was significantly associated with the SCC risk among men (OR = 3.08). The second and third tertiles of lifetime sun exposure was significantly associated with SCC risk among women (OR = 3.78 and 4.53, respectively). Skin reaction after 2-h sun exposure during childhood and adolescence was not significantly associated with the risk of SCC. Lifetime sun exposure was more related to SCC risk in women, while early-age sun exposure was more relevant to men's SCC risk. This may be attributable to different lifestyle between men and women.


Improving Students' Learning With Effective Learning Techniques: Promising Directions From Cognitive and Educational Psychology.
Many students are being left behind by an educational system that some people believe is in crisis. Improving educational outcomes will require efforts on many fronts, but a central premise of this monograph is that one part of a solution involves helping students to better regulate their learning through the use of effective learning techniques. Fortunately, cognitive and educational psychologists have been developing and evaluating easy-to-use learning techniques that could help students achieve their learning goals. In this monograph, we discuss 10 learning techniques in detail and offer recommendations about their relative utility. We selected techniques that were expected to be relatively easy to use and hence could be adopted by many students. Also, some techniques (e.g., highlighting and rereading) were selected because students report relying heavily on them, which makes it especially important to examine how well they work. The techniques include elaborative interrogation, self-explanation, summarization, highlighting (or underlining), the keyword mnemonic, imagery use for text learning, rereading, practice testing, distributed practice, and interleaved practice. To offer recommendations about the relative utility of these techniques, we evaluated whether their benefits generalize across four categories of variables: learning conditions, student characteristics, materials, and criterion tasks. Learning conditions include aspects of the learning environment in which the technique is implemented, such as whether a student studies alone or with a group. Student characteristics include variables such as age, ability, and level of prior knowledge. Materials vary from simple concepts to mathematical problems to complicated science texts. Criterion tasks include different outcome measures that are relevant to student achievement, such as those tapping memory, problem solving, and comprehension. We attempted to provide thorough reviews for each technique, so this monograph is rather lengthy. However, we also wrote the monograph in a modular fashion, so it is easy to use. In particular, each review is divided into the following sections: General description of the technique and why it should work How general are the effects of this technique? 2a. Learning conditions 2b. Student characteristics 2c. Materials 2d. Criterion tasks Effects in representative educational contexts Issues for implementation Overall assessment The review for each technique can be read independently of the others, and particular variables of interest can be easily compared across techniques. To foreshadow our final recommendations, the techniques vary widely with respect to their generalizability and promise for improving student learning. Practice testing and distributed practice received high utility assessments because they benefit learners of different ages and abilities and have been shown to boost students' performance across many criterion tasks and even in educational contexts. Elaborative interrogation, self-explanation, and interleaved practice received moderate utility assessments. The benefits of these techniques do generalize across some variables, yet despite their promise, they fell short of a high utility assessment because the evidence for their efficacy is limited. For instance, elaborative interrogation and self-explanation have not been adequately evaluated in educational contexts, and the benefits of interleaving have just begun to be systematically explored, so the ultimate effectiveness of these techniques is currently unknown. Nevertheless, the techniques that received moderate-utility ratings show enough promise for us to recommend their use in appropriate situations, which we describe in detail within the review of each technique. Five techniques received a low utility assessment: summarization, highlighting, the keyword mnemonic, imagery use for text learning, and rereading. These techniques were rated as low utility for numerous reasons. Summarization and imagery use for text learning have been shown to help some students on some criterion tasks, yet the conditions under which these techniques produce benefits are limited, and much research is still needed to fully explore their overall effectiveness. The keyword mnemonic is difficult to implement in some contexts, and it appears to benefit students for a limited number of materials and for short retention intervals. Most students report rereading and highlighting, yet these techniques do not consistently boost students' performance, so other techniques should be used in their place (e.g., practice testing instead of rereading). Our hope is that this monograph will foster improvements in student learning, not only by showcasing which learning techniques are likely to have the most generalizable effects but also by encouraging researchers to continue investigating the most promising techniques. Accordingly, in our closing remarks, we discuss some issues for how these techniques could be implemented by teachers and students, and we highlight directions for future research.


Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.
Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane.


Latency-related gene encoded by bovine herpesvirus 1 promotes virus growth and reactivation from latency in tonsils of infected calves.
Infection of calves with bovine herpesvirus 1 (BHV-1) results in transient immunosuppression that may lead to bacterium-induced pneumonia and, occasionally, death. Although sensory neurons in the trigeminal ganglia (TG) are the primary site of BHV-1 latency, viral genomes are detected in the tonsils of latently infected calves. Dexamethasone (DEX) consistently induces reactivation from latency, and viral gene expression is detected in TG and tonsils. In sensory neurons of latently infected calves, the latency-related (LR) gene is abundantly expressed and is required for reactivation from latency. In the present study, we compared the abilities of wild-type (wt) BHV-1 and a strain with a mutation in the LR gene (the LR mutant strain) to grow in the tonsils of infected calves and reactivate from latency. Lower levels of the LR mutant virus were detected in the tonsils of acutely infected calves. LR mutant viral DNA was consistently detected by PCR in the tonsils of latently infected calves, suggesting that the establishment of a latent or persistent infection occurred. Although the LR mutant did not reactivate from latency in vivo after DEX treatment, explantation of tonsil tissue from calves latently infected with the LR mutant yielded infectious virus. Relative to wt BHV-1, the LR mutant did not induce explant-induced reactivation as efficiently. These studies indicate that the LR gene promotes virus shedding from tonsil tissue during acute infection and reactivation from latency in tonsil tissue in vivo. We suggest that incorporation of the LR gene mutation into existing modified live vaccines would prevent reactivation from latency in neural and nonneural sites and would thus prevent transmission to other animals.


Diagnosis and management of narcolepsy in the Indian scenario.
The diagnosis and management of narcolepsy in the Indian context needs to be revisited especially in the wake of concerns raised by sleep medicine experts that the entity could be formidably underdiagnosed, as well as undertreated in our setting. The history, clinical records, polysomnographic/multiple sleep latency test data, and treatment records of five hundred consecutive patients attending a dedicated sleep clinic between the years 2013 and 2016 were retrospectively analyzed. The response to treatment measures and improvement in daytime functioning were periodically assessed by personal/telephonic interview and E-mail communication. Thirteen patients were diagnosed with narcolepsy based on the standard criteria of which three had cataplexy. The mean age of presentation was 23.23 years and the male:female ratio was 2.25:1. The mean duration from the onset of symptoms to diagnosis was 4.2 years. Two patients responded to nonpharmacological interventions alone, and six to modafinil, while two patients remained symptomatic and required treatment with methylphenidate. One patient was lost to follow-up, while two others are due for their first follow-up. A refurbished outlook of the diagnostic methodology and treatment paths tailored to our clinical scenario can potentially impact the future of narcolepsy management and research in our country.


Transgenic plant production of Cyanovirin-N, an HIV microbicide.
Cyanovirin-N (CV-N) is a microbicide candidate that inactivates a wide range of HIV strains by binding to gp120. Production of CV-N, or any protein microbicide, needs to be at extremely high levels and low cost to have an impact on global health. Thus, it is unlikely that fermentor-based systems will be suitable, including recombinant E. coli, where CV-N aggregates and dimers have consistently been found. Transgenic plants may provide a suitable expression system for protein microbicides, as production can be easily and economically scaled up. Here, Nicotiana tabacum was transformed with a gene encoding CV-N to explore proof of concept for the production of CV-N in transgenic plants. Plant-derived rCV-N was recoverable at levels of 130 ng/mg of fresh leaf tissue, or at least 0.85% of total soluble plant protein. Western blot analysis demonstrated that virtually all of the rCV-N was expressed in the desired monomeric form. Functionality was demonstrated by specific binding to gp120 and protection of T-cells from in vitro HIV infection. Hydroponic culturing of transgenic plants demonstrated CV-N rhizosecretion at levels of 0.64 mug/ml hydroponic media after 24 days. Therefore, we suggest that transgenic plants have the potential to provide strategies for large-scale protein microbicide production.


The innate antiviral immune system of the cat: molecular tools for the measurement of its state of activation.
The innate immune system plays a central role in host defence against viruses. While many studies portray mechanisms in early antiviral immune responses of humans and mice, much remains to be discovered about these mechanisms in the cat. With the objective of shedding light on early host-virus interactions in felids, we have developed 12 real-time TaqMan(®) qPCR systems for feline genes relevant to innate responses to viral infection, including those encoding for various IFNα and IFNω subtypes, IFNβ, intracellular antiviral factor Mx, NK cell stimulator IL-15 and effectors perforin and granzyme B, as well as Toll-like receptors (TLRs) 3 and 8. Using these newly developed assays and others previously described, we measured the relative expression of selected markers at early time points after viral infection in vitro and in vivo. Feline embryonic fibroblasts (FEA) inoculated with feline leukemia virus (FeLV) indicated peak levels of IFNα, IFNβ and Mx expression already 6h after infection. In contrast, Crandell-Rees feline kidney (CrFK) cells inoculated with feline herpes virus (FHV) responded to infection with high levels of IFNα and IFNβ only after 24h, and no induction of Mx could be detected. In feline PBMCs challenged in vitro with feline immunodeficiency virus (FIV), maximal expression levels of IFNα, β and ω subtype genes as well as IL-15 and TLRs 3, 7 and 8 were measured between 12 and 24h after infection, whereas expression levels of proinflammatory cytokine gene IL-6 were consistently downregulated until 48h post inoculation. A marginal upregulation of granzyme B was also observed within 3h after infection. In an in vivo experiment, cats challenged with FIV exhibited a 2.4-fold increase in IFNα expression in blood 1 week post infection. We furthermore demonstrate the possibility of stimulating feline immune cells in vitro with various immune response modifiers (IRMs) already known for their immunostimulatory properties in mice and humans, namely Poly IC, Resiquimod (R-848) and dSLIM™, a synthetic oligonucleotide containing several unmethylated CpG motifs. Stimulation of feline PBMCs with dSLIM™ and R-848 effectively enhanced expression of IFNα within 12h by factors of 6 and 12, respectively, and Poly IC induced an increase in Mx mRNA expression of 28-fold. Altogether, we describe new molecular tools and their successful use for the characterization of innate immune responses against viruses in the cat and provide evidence that feline cells can be stimulated by synthetic molecules to enhance their antiviral defence mechanisms.


Change in Renal Glomerular Collagens and Glomerular Filtration Barrier-Related Proteins in a Dextran Sulfate Sodium-Induced Colitis Mouse Model.
Renal disease is not rare among patients with inflammatory bowel disease (IBD) and is gaining interest as a target of research. However, related changes in glomerular structural have rarely been investigated. This study was aimed at clarifying the changes in collagens and glomerular filtration barrier (GFB)-related proteins of glomeruli in a dextran sulfate sodium (DSS)-induced colitis mouse model. Acute colitis was induced by administering 3.5% DSS in Slc:ICR strain mice for eight days. Histological changes to glomeruli were examined by periodic acid-Schiff (PAS) and Masson's trichrome staining. Expressions of glomerular collagens and GFB-related proteins were analyzed by immunofluorescent staining and Western blot analysis. DSS-colitis mice showed an elevated disease activity index (DAI), colon shortening, massive cellular infiltration and colon damage, confirming that DSS-colitis mice can be used as an IBD animal model. DSS-colitis mice showed increased glycoprotein and collagen deposition in glomeruli. Interestingly, we observed significant changes in glomerular collagens, including a decrease in type IV collagen, and an increment in type I and type V collagens. Moreover, declined GFB-related proteins expressions were detected, including synaptopodin, podocalyxin, nephrin and VE-cadherin. These results suggest that renal disease in DSS-colitis mice might be associated with changes in glomerular collagens and GFB-related proteins. These findings are important for further elucidation of the clinical pathological mechanisms underlying IBD-associated renal disease.