[Two cases of familial amyotrophic lateral sclerosis with a SOD1L126S mutation showing high age at onset and slow progression].
An 80-year-old man (patient 1) was admitted to our hospital with numbness of the right leg and weakness of the lower extremities, predominantly in the right leg, for 1 year previously. Neurological examination revealed moderate weakness without atrophy, and fasciculation in the bilateral lower extremities. No hyperreflexia was noted, and the plantar response was flexor. Neither bulbar palsy nor sensory disturbance was observed. Electromyography (EMG) showed a chronic neurogenic pattern, including giant motor unit potentials and reduced interference in all four limb muscles. MRI images of the cervical and lumbar spines showed severe age-related spondylosis. The clinical and laboratory findings led to a diagnosis of spinal progressive muscular atrophy. Motor paralysis progressed slowly for the following four years, culminating in respiratory failure. A 79-year-man, the younger brother of patient 1 (patient 2), suffered from gait disturbance for 3 years before the admission to our hospital. During the following 3 years, bilateral leg weakness developed, causing difficulty walking. Neurological examination revealed a diffuse mild weakness with atrophy and fasciculation in the bilateral lower extremities; the right deltoid muscle was also mildly weak. Mild hyperreflexia was also noted on the left side, and the plantar response was extensor on the left. EMG showed acute and chronic neurogenic patterns in the four limb muscles. Because the missense mutation c.377 T > C; p.L126S was found on exon 5 of the superoxide dismutase (SOD) 1 gene in this patient, a diagnosis of familial ALS was made. Eight patients reported as familial ALS with the SOD1L126S mutation, including the present cases, all developed an onset of weakness in the lower extremities, but showed few upper motor neuron signs. It is important to consider the possibility of this type of familial ALS in a case of spinal progressive muscular atrophy with late-onset and mild progression, if family history is positive.


Outcomes of fetal listed patients awaiting heart transplantation.
HTx in neonates is mainstay therapy for those with severe cardiomyopathies and congenital heart disease. Fetal listing for HTx has been proposed as a way to increase the potential window for a donor with outcomes predicted to be similar to the neonatal population. Data from the PHTS, a prospective multicenter study, were used to examine the outcomes of fetuses listed between 1993 and 2009. Four thousand three hundred and sixty-five children were listed for HTx during this period. Fetuses comprised 1% and neonates 19.8% of listed patients. In those patients listed as fetus and transplanted, the median wait time from listing to HTx was 55 days (range 4-255), with a median of 25 days (range 0-233) after birth. By six months post-listing, a higher proportion of fetal listed patients had undergone HTx with a lower waitlist mortality when compared with neonate. There was no significant difference in survival following HTx between the two group (p = 0.4). While the results of this study may be less applicable to current practice due to changes in referrals for fetal listing, they do indicate that fetal listing can be a reasonable option. These results are of particular interest at the present time given the ongoing public discourse on the proposed elimination of fetal listing within UNOS.


Nanoparticle technology: amplifying the effective sensitivity of biomarker detection to create a urine test for hGH.
Several clinical-grade immunoassays exist for the specific measurement of hGH or its isoforms in blood but there is an urgent need to apply these same reliable assays to the measurement of hGH in urine as a preferred 'non-invasive' biofluid. Unfortunately, conventional hGH immunoassays cannot attain the sensitivity required to detect the low concentrations of hGH in urine. The lowest limit of sensitivity for existing hGH immunoassays is >50 pg/mL, while the estimated concentration of urinary hGH is about 1 pg/m-50 times lower than the sensitivity threshold. We have created novel N-isopropylacrylamide (NIPAm)-based hydrogel nanoparticles functionalized with an affinity bait. When introduced into an analyte-containing solution, the nanoparticles can perform, in one step, (1) complete harvesting of all solution phase target analytes, (2) full protection of the captured analyte from degradation and (3) sequestration of the analyte, effectively increasing the analyte concentration up to a hundredfold. N-isopropylacrylamide nanoparticles functionalized with Cibacron Blue F3GA bait have been applied to raise the concentration of urinary hGH into the linear range of clinical grade immunoassays. This technology now provides an opportunity to evaluate the concentration of hGH in urine with high precision and accuracy.


Learning curve for single-incision laparoscopic resection of right-sided colon cancer by complete mesocolic excision.
Single-incision laparoscopic surgery is cosmetically beneficial, but technically challenging. In this study, the learning curve (LC) for single-incision laparoscopic right hemicolectomy (SILRC), incorporating complete mesocolic excision to resect right-sided colon cancer, was investigated through multidimensional techniques. Between December 2009 and May 2015, 64 patients each underwent SILRC of right-sided colon cancer at Severance Hospital, performed in all instances by the same surgeon. Moving average and cumulative sum control chart (CUSUM) were used for LC analyses retrospectively. Surgical failure was defined as conversion to conventional laparoscopic surgery, postsurgical morbidity within 30 days, harvested lymph node count <12, or local tumor recurrence. Both moving average and CUSUM graphics of operative time registered nadirs at the 24th patient, with slight ascent thereafter, reaching a plateau at the 40th patient. The CUSUM for surgical success peaked at the 23rd patient. Operative time for 23 patients in phase 1 (1-23) and for 41 patients in phase 2 (24-64) of the LC did not differ significantly. By comparison, significant differences in patients of phase 2 included larger tumor size, higher harvested lymph node counts, longer proximal resection margins, and more advanced disease. As indicated by multidimensional statistical analyses, the LC for SILRC of right-sided colon cancer was 23 patients. In terms of operative time and surgical success, SILRC is feasible for surgeons experienced in LS, but may prove more challenging for novices, given the fundamental technical difficulties of this procedure.


Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells.
We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.


Intolerance of cow's milk and chronic constipation in children.
Chronic diarrhea is the most common gastrointestinal symptom of intolerance of cow's milk among children. On the basis of a prior open study, we hypothesized that intolerance of cow's milk can also cause severe perianal lesions with pain on defecation and consequent constipation in young children. We performed a double-blind, crossover study comparing cow's milk with soy milk in 65 children (age range, 11 to 72 months) with chronic constipation (defined as having one bowel movement every 3 to 15 days). All had been referred to a pediatric gastroenterology clinic and had previously been treated with laxatives without success; 49 had anal fissures and perianal erythema or edema. After 15 days of observation, the patients received cow's milk or soy milk for two weeks. After a one-week washout period, the feedings were reversed. A response was defined as eight or more bowel movements during a treatment period. Forty-four of the 65 children (68 percent) had a response while receiving soy milk. Anal fissures and pain with defecation resolved. None of the children who received cow's milk had a response. In all 44 children with a response, the response was confirmed with a double-blind challenge with cow's milk. Children with a response had a higher frequency of coexistent rhinitis, dermatitis, or bronchospasm than those with no response (11 of 44 children vs. 1 of 21, P=0.05); they were also more likely to have anal fissures and erythema or edema at base line (40 of 44 vs. 9 of 21, P<0.001), evidence of inflammation of the rectal mucosa on biopsy (26 of 44 vs. 5 of 21, P=0.008), and signs of hypersensitivity, such as specific IgE antibodies to cow's-milk antigens (31 of 44 vs. 4 of 21, P<0.001). In young children, chronic constipation can be a manifestation of intolerance of cow's milk.


The PDZ ligand domain of the human papillomavirus type 16 E6 protein is required for E6's induction of epithelial hyperplasia in vivo.
Human papillomaviruses (HPVs) are the causative agent of warts. Infections with high-risk HPVs are associated with anogenital and head and neck cancers. One of the viral genes responsible for HPV's oncogenic activity is E6. Mice expressing the HPV-16 E6 protein in their epidermis (K14E6(WT)) develop epithelial hyperplasia and squamous carcinomas. Numerous cellular proteins interact with E6, some of which can be grouped based on common amino acid motifs in their E6-binding domains. One such group, the PDZ partners, including hDLG, hSCRIBBLE, MUPP1, and MAGI, bind to the carboxy-terminal four amino acids of E6 through their PDZ domains. E6's interaction with the PDZ partners leads to their degradation. Additionally, E6's binding to PDZ proteins has been correlated with its ability to transform baby rat kidney cells in tissue culture and to confer tumorigenicity onto cells in xenograft experiments. To address whether the ability of E6 to bind PDZ domain partners is necessary for E6 to confer epithelial hyperproliferation in vivo, we generated transgenic mice that express in stratified squamous epithelia a mutant of E6 lacking the last six amino acids at its carboxyl terminus, E6(Delta 146-151), from the human keratin 14 (K14) promoter. The K14E6(Delta 146-151) mice exhibit a radiation response similar to that of the K14E6(WT) mice, demonstrating that this protein, as predicted, retains an ability to inactivate p53. However, the K14E6(Delta 146-151) mice fail to display epithelial hyperplasia. These results indicate that an interaction of E6 with PDZ partners is necessary for its induction of epithelial hyperplasia.


A qualitative exploration of patients' attitudes towards the 'Participate Inform Notice Know' (PINK) patient safety video.
To explore patients' attitudes towards the PINK video, a patient education video aimed at encouraging hospital patients' involvement in safety-relevant behaviours. Qualitative semi-structured interviews. Detailed field notes were taken during the interviews which were analysed using content analysis. One National Health System (NHS) teaching hospital based in London, UK. Thirty-six in-patients aged between 20 and 79 years, 18 of them males. The PINK video is a short animated educational video aimed at encouraging patients to be involved in the safety of their care during hospitalization. Patients' perceptions of how informative, relevant and acceptable the video is; attitudes towards participating in the recommended safety-related behaviours and; potential negative side effects of watching the video. Overall the video was received favourably among the interviewees. Commonly cited benefits included raising awareness and facilitating patients to be involved in their care during their hospital stay. More variability was found in participants' views with regard to the video's role as a patient safety enhancement tool. A number of suggestions for improvement of the video were provided relating to tailoring its content and design to meet the needs of individual patients and their circumstances. Educational videos such as PINK have significant potential to empower patients in the safety and quality of their care. However, efforts to implement patient safety educational videos in practice need to consider different patient groups' needs and characteristics instead of trying to adopt 'a one size fits all' approach.


Biotransformation of D(-)-ephedrine and L(+)-ephedrine in the rabbit, in vivo and in vitro.
Investigations were carried out with radiolabeled D(-)-ephedrine and L(+)-ephedrine to establish whether differences exist in their metabolic fate in the rabbit, in vivo and in vitro. In liver microsomal preparations, a) D(-)-ephedrine was metabolized at a faster rate than L(+)-ephedrine, b) benzoic acid was formed from D(-)ephedrine at a rate about three times greater than from the L(+)-isomer, and c) the relative amounts of norephedrine and 1-phenyl-1,2-propranediol formed from both ephedrine isomers were nearly identical throughout the entire incubation period. In vivo, both ephedrine isomers were extensively metabolized and the majority of total radioactivity (71-91%) was excreted within 24 hr. A greater 14C-excretion rate was observed for L(+)-ephedrine. From an analysis of 0- to 24-hr urine, it was found that a) 47-50% of the urinary 14C was attributable to acidic metabolites (hippuric acid and benzoic acid) from L(+)- and D(-)-ephedrine, b) from 4 to 16% of the total 14C obtained with both isomers was accountable as 1-phenyl-1,2-propanediol, either free or as a glucuronide conjugate, c) no appreciable quantities of sulfate or glucuronide conjugates of p-hydroxylated metabolites of ephedrine or norephedrine was detectable, and d) small amounts (less than 4% of metabolites corresponding to unchanged ephedrine, norephedrine, or 1-hydroxy-1-phenyl-2-propanone were found in urine of animals given either isomer. These experiments indicate that the major pathway for the biotransformation of D(-)-ephedrine and L(+)-ephedrine involves N-demethylation and oxidative deamination of the side chain.


Defining stem cell dynamics in models of intestinal tumor initiation.
Cancer is a disease in which cells accumulate genetic aberrations that are believed to confer a clonal advantage over cells in the surrounding tissue. However, the quantitative benefit of frequently occurring mutations during tumor development remains unknown. We quantified the competitive advantage of Apc loss, Kras activation, and P53 mutations in the mouse intestine. Our findings indicate that the fate conferred by these mutations is not deterministic, and many mutated stem cells are replaced by wild-type stem cells after biased, but still stochastic events. Furthermore, P53 mutations display a condition-dependent advantage, and especially in colitis-affected intestines, clones harboring mutations in this gene are favored. Our work confirms the previously theoretical notion that the tissue architecture of the intestine suppresses the accumulation of mutated lineages.


BARgaining membranes for autophagosome formation: Regulation of autophagy and tumorigenesis by Bif-1/Endophilin B1.
Autophagy is an intracellular system for the bulk degradation of cytoplasmic components enclosed within double-membrane structures known as autophagosomes. To date, many autophagy-related (Atg) genes have been identified by independent genetic screens for autophagy-defective mutants in yeast; however, the molecular machinery required for the biogenesis of autophagosomes in mammalian systems has yet to be determined.(1,2) Recently, we have reported that Bif-1 interacts with Beclin 1 through UVRAG and promotes the activation of class III phosphatidylinositol 3-kinase (PI3KC3) and the formation of autophagosomes.(3) Moreover, we have found that loss of Bif-1 promotes starvation-induced caspase activation, but prolongs cell survival by suppressing autophagydependent cell death, and enhances spontaneous tumorigenesis in mice. Bif-1 is a member of the endophilin family, which possesses membrane binding and liposome tubulation activities.(4) During nutrient deprivation, Bif-1 accumulates in punctate foci where it co-localizes with LC3, Atg5 and Atg9. Time-lapse microscopy analyses reveal that Bif-1-positive small vesicles expand by recruiting and fusing with Atg9-positive small membranes to form autophagosomes. Taken together, our findings highlight Bif-1 as a potential regulator of autophagosome biogenesis and as a tumor suppressor.


Radiosurgery for glomus jugulare tumors.
Results for treating glomus jugulare tumors with radiosurgery have been limited by short follow-up and small number of patients. We report our experience using LINAC or CyberKnife in 21 tumors with a median follow-up of 66 months (Mean follow-up of 60 months). In addition, we have a subset of eight patients that were followed out for more than 10 years. Patients were treated with doses ranging from 1400 cGy to 2700 cGy. We retrospectively assessed patients for efficacy and post treatment side effects. All patients had stable neurological symptoms, and two patients experienced transient ipsilateral tongue weakness and hearing loss, both of which subsequently resolved. One patient experienced transient ipsilateral vocal cord paresis; however, this patient received previous external beam radiotherapy. All tumors remained stable or decreased in size by MRI exam. Our results support radiosurgery as an effective and safe method of treatment for glomus jugulare tumors with low morbidity as evidenced by a larger number of patients and long term follow-up.


Sympathetic modulation by carvedilol and losartan reduces angiotensin II-mediated lipolysis in subcutaneous and visceral fat.
Advanced heart failure is characterized by increased activation of the renin-angiotensin system and the development of cachexia. Angiotensin II (Ang II) has been proposed as a lipid metabolism regulator. The effects of exogenous Ang II (osmotic minipump, 525 ng/kg/min for 12 d) on interstitial sc glycerol and norepinephrine levels, indexes of lipolysis, and sympathetic activation, respectively, were measured in Sprague Dawley rats by consecutive microdialysis performed in vivo in white adipose tissue. Higher sustained interstitial glycerol and norepinephrine levels were found after 7 and 12 d of Ang II infusion. Triglyceride to DNA content ratio and adipocyte diameter were reduced in sc and visceral (retroperitoneal and epididymal) fat tissues of Ang II-infused rats, whose body weight was lower and blood pressure higher. Losartan, an Ang II receptor 1 blocker, and carvedilol, an alpha1-nonselective-beta1,2,3-adrenergic blocker, but not doxazosin, an alpha1-selective-adrenergic blocker, lowered glycerol and norepinephrine levels, preventing lipolysis and weight loss. Our results indicate that Ang II stimulates lipolysis in sc and visceral adipocytes by sympathetic activation and beta-adrenergic-receptor stimulation. Nonselective-beta-adrenergic and Ang II-receptor1 blockade markedly attenuated the rise of norepinephrine, preventing catabolic effects. The metabolic benefits of carvedilol and losartan, in addition to recognized protective cardiovascular effects, may be relevant in cachectic patients with advanced heart failure.


Appraisal of levels and patterns of occupational exposure to 1,3-butadiene.
Objectives 1,3-butadiene is classified as carcinogenic to human by inhalation and the association with leukemia has been observed in several epidemiological studies. The aim of this study was to evaluate data about occupational exposure levels to 1,3-butadiene in the Italian working force. Methods Airborne concentrations of 1,3-butadiene were extracted from the Italian database on occupational exposure to carcinogens in the period 1996-2015. Descriptive statistics were calculated for exposure-related variables. An analysis through linear mixed model was performed to determine factors influencing the exposure level. The probability of exceeding the exposure limit was predicted using a mixed-effects logistic model. Concurrent exposures with other occupational carcinogens were investigated using the two-step cluster analysis. Results The total number of exposure measurements selected was 23 885, with an overall arithmetic mean of 0.12 mg/m3. The economic sector with the highest number of measurements was manufacturing of chemicals (18 744). The most predictive variables of the exposure level resulted to be the occupational group and its interaction with the measurement year. The highest likelihood of exceeding the exposure limit was found in the manufacture of coke and refined petroleum products. Concurrent exposures were frequently detected, mainly with benzene, acrylonitrile and ethylene dichloride, and three main clusters were identified. Conclusions Exposure to 1,3-butadiene occurs in a wide variety of activity sectors and occupational groups. The use of several statistical analysis methods applied to occupational exposure databases can help to identify exposure situations at high risk for workers' health and better target preventive interventions and research projects.


Terpene synthases from Cannabis sativa.
Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.


Prospective study with specific Re-Assessment time points to determine time to recovery following a Sports-Related Concussion in university-aged student-athletes.
Time to recovery for symptom burden and neurocognition following a Sports-Related Concussion (SRC) has previously been determined by consolidating varying re-assessment time points into a singular point, and has not been established for Vestibular-Ocular-Motor (VOM) function or academic ability. Establish when recovery of symptom burden, neurocognition, VOM function, and academic ability occurs in university-aged student-athletes. Student-athletes completed an assessment battery (Post-Concussion Symptom Scale (PCSS), Immediate Post-Concussion Assessment and Cognitive Test (ImPACT), Vestibular Ocular-Motor Screening (VOMS), Perceived Academic Impairment Tool (PAIT)) during pre-season (n = 140), within 48 hours, 4, 8 and 14 days post-SRC and prior to Return To Play (RTP) and were managed according to the Rugby Football Union' community pathway (n = 42). Student-athletes were deemed recovered or impaired according to Reliable Change Index' (RCI) or compared to their individual baseline. Symptom burden recovers by four days post-SRC on RCI and to baseline by eight days. VOM function and academic ability recovers by 8 days. Some student-athletes demonstrated worse performance at RTP on all tests by RCI and to baseline, except for on VOMS score and near point convergence by RCI change. Variation in individual university-aged student-athletes requires a multi-faceted approach to establish what dysfunctions post-SRC exist and when recovery occurs.


Window screening, ceilings and closed eaves as sustainable ways to control malaria in Dar es Salaam, Tanzania.
Malaria transmission in Africa occurs predominantly inside houses where the primary vectors prefer to feed. Human preference and investment in blocking of specific entry points for mosquitoes into houses was evaluated and compared with known entry point preferences of the mosquitoes themselves. Cross-sectional household surveys were conducted in urban Dar es Salaam, Tanzania to estimate usage levels of available options for house proofing against mosquito entry, namely window screens, ceilings and blocking of eaves. These surveys also enabled evaluation of household expenditure on screens and ceilings and the motivation behind their installation. Over three quarters (82.8%) of the 579 houses surveyed in Dar es Salaam had window screens, while almost half (48.9%) had ceilings. Prevention of mosquito entry was cited as a reason for installation of window screens and ceilings by 91.4% (394/431) and 55.7% (127/228) of respondents, respectively, but prevention of malaria was rarely cited (4.3%, 22/508). The median cost of window screens was between US $ 21-30 while that of ceilings was between US $301-400. The market value of insecticide-treated nets, window screening and ceilings currently in use in the city was estimated as 2, 5 and 42 million US$. More than three quarters of the respondents that lacked them said it was too expensive to install ceilings (82.2%) or window screens (75.5%). High coverage and spending on screens and ceilings implies that these techniques are highly acceptable and excellent uptake can be achieved in urban settings like Dar es Salaam. Effective models for promotion and subsidization should be developed and evaluated, particularly for installation of ceilings that prevent entry via the eaves, which are the most important entry point for mosquitoes that cause malaria, a variety of neglected tropical diseases and the nuisance which motivates uptake.


The projected timing of climate departure from recent variability.
Ecological and societal disruptions by modern climate change are critically determined by the time frame over which climates shift beyond historical analogues. Here we present a new index of the year when the projected mean climate of a given location moves to a state continuously outside the bounds of historical variability under alternative greenhouse gas emissions scenarios. Using 1860 to 2005 as the historical period, this index has a global mean of 2069 (±18 years s.d.) for near-surface air temperature under an emissions stabilization scenario and 2047 (±14 years s.d.) under a 'business-as-usual' scenario. Unprecedented climates will occur earliest in the tropics and among low-income countries, highlighting the vulnerability of global biodiversity and the limited governmental capacity to respond to the impacts of climate change. Our findings shed light on the urgency of mitigating greenhouse gas emissions if climates potentially harmful to biodiversity and society are to be prevented.


Selective internal radiation therapy with SIR-Spheres in hepatocellular carcinoma and cholangiocarcinoma.
Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) often present at stages where patients have limited treatment options. Use of selective internal radiation therapy (SIRT) with yttrium-90 (Y-90) resin microspheres has progressed as data increasingly speak to its utility in patients with both intermediate and late stage disease in these cancers. In anticipation of the pending completion of several prospective randomized controlled multicenter studies exploring the use of Y-90 resin microspheres in primary liver cancers, this article outlines mechanisms involved in SIRT administration and reviews key efficacy and safety data that are currently available in the literature involving use of this therapy in both HCC and ICC.


Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study.
The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targets mapA and ceuE for the detection of Campylobacter jejuni and Campylobacter coli, leading global causes of bacterial gastroenteritis. In silico analyses of mapA and ceuE primer and probe sequences from 1,713 genetically diverse C. jejuni and C. coli genomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of the mapA-ceuE assay was the result of interspecies diversity and intraspecies conservation of the target genes in C. jejuni and C. coli Rare instances of a lack of specificity among C. coli isolates were due to introgression in mapA or sequence diversity in ceuE The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software.


Rapid Mass Spectrometry Imaging to Assess the Biochemical Profile of Pituitary Tissue for Potential Intraoperative Usage.
Pituitary adenomas are relatively common intracranial neoplasms that are frequently treated with surgical resection. Rapid visualization of pituitary tissue remains a challenge as current techniques either produce little to no information on hormone-secreting function or are too slow to practically aid in intraoperative or even perioperative decision-making. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) represents a powerful method by which molecular maps of tissue samples can be created, yielding a two-dimensional representation of the expression patterns of small molecules and proteins from biologic samples. In this chapter, we review the use of MALDI MSI, its application to the characterization of the pituitary gland, and its potential applications for guiding the management of pituitary adenomas.


Randomised controlled trial to assess the impact of a lifestyle intervention (ActWELL) in women invited to NHS breast screening.
In Scotland, the incidence of breast cancer is predicted to rise significantly in the next few decades and while there are measures to support reductions in morbidity and mortality, the breast cancer community is currently exploring preventative opportunities including supporting weight management programmes in postmenopausal women. This study aims to assess the effectiveness and cost-effectiveness of a theory-based, community delivered, minimal contact, weight management (diet, physical activity and behaviour change techniques) programme (ActWELL) in women with a body mass index (BMI) >25 kg/m2 attending routine breast cancer screening appointments. The study will be a four-centre, 1:1 parallel group randomised controlled trial of a 12-month weight management intervention initiated in breast cancer screening centres, delivered by trained Breast Cancer Now lifestyle coaches in community settings. The intervention programme involves two intervention meetings with coaches plus (up to) nine telephone contacts over 12 months. The programme will focus on personalised diet (including alcoholic and sugary drinks) and physical activity habits. Behaviour change techniques include self-monitoring, goal setting, implementation intentions, action and coping plans. The study has a sample size of 414 women with a BMI >25 kg/m2 attending routine National Health Service breast cancer screening appointments. Measures will be taken at baseline, 12 weeks and at 12-month follow-up, complemented by qualitative interviews exploring perceived acceptability and impact on habitual behaviours. The two co-primary outcomes are mean change in measured body weight and change in physical activity between groups to 12 months. Secondary outcomes are changes in eating habits, alcohol intake, sedentary time, quality of life, waist circumference, lipid, haemoglobin A1c and insulin profiles, blood pressure and cost-effectiveness of the intervention. The protocol has been approved by East of Scotland Research Ethics Committee (17/ES/0073). All participants provide written informed consent. Dissemination will be through peer-reviewed publication and conference presentations. ISRCTN11057518; Pre-results.


A role for VAV1 in experimental autoimmune encephalomyelitis and multiple sclerosis.
Multiple sclerosis, the most common cause of progressive neurological disability in young adults, is a chronic inflammatory disease. There is solid evidence for a genetic influence in multiple sclerosis, and deciphering the causative genes could reveal key pathways influencing the disease. A genome region on rat chromosome 9 regulates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Using interval-specific congenic rat lines and association of single-nucleotide polymorphisms with inflammatory phenotypes, we localized the gene of influence to Vav1, which codes for a signal-transducing protein in leukocytes. Analysis of seven human cohorts (12,735 individuals) demonstrated an association of rs2546133-rs2617822 haplotypes in the first VAV1 intron with multiple sclerosis (CA: odds ratio, 1.18; CG: odds ratio, 0.86; TG: odds ratio, 0.90). The risk CA haplotype also predisposed for higher VAV1 messenger RNA expression. VAV1 expression was increased in individuals with multiple sclerosis and correlated with tumor necrosis factor and interferon-gamma expression in peripheral blood and cerebrospinal fluid cells. We conclude that VAV1 plays a central role in controlling central nervous system immune-mediated disease and proinflammatory cytokine production critical for disease pathogenesis.


Functional evaluation indicates physical losses after hematopoietic stem cell transplantation.
To perform a function evaluation of patients before and after hematopoietic stem cell transplantation. From November 2008 to November 2010, 29 female (58%) and 21 male patients (42%) with median age of 48 years (range: 24-67) were enrolled in this study. Data collection was performed before and after autologous or allogeneic hematopoietic stem cell transplantation. Evaluation instruments included the 2-minute walking test to evaluate gait performance with assessment of the oxygen saturation, heart rate and Borg Scale before and after the test; grip strength for strength evaluation, Schober Test for spine mobility testing and maximum and adapted activity scores of the Human Activity Profile questionnaire to test functionality in daily activities. Fifty patients were evaluated at baseline; six did not undergo hematopoietic stem cell transplantation (three died, one refused and two were excluded). Thus 44/50 (88% - 21 allogeneic and 23 autologous) transplantations were performed. Only 33 of the 44 patients (75%) performed evaluations after transplantation (nine died and two were excluded). Of the patients who performed both evaluations, significantly lower values were found in the evaluation after transplantation for the 2-minute walking test (p-value = 0.004), grip strength of both right and left hands (p-value = 0.004 and p-value < 0.0001, respectively), the Schober Test, and maximum and adapted activity scores (p-value < 0.0001). The heart rate was higher (p-value = 0.01) before the 2-minute walking test and oxygen saturation was higher (p-value = 0.02) after. Statistical differences indicate functional impairment after transplantation showing physical losses in this population.


SU-E-J-60: Comparison of CT-On-Rails and a 3D Surface Imaging System for Image Guided Pelvic Radiation Therapy for Both Supine and Prone Patient Positions.
We investigate the feasibility of using AlignRT for pelvic radiation image guidance. The uniqueness of our study is that all patients have multiple CT-on-rails (CTOR) scans to compare corresponding AlignRT images to. Ten patients receiving pelvic radiation were enrolled in this study. Two simulation CT scans were performed in supine and prone positions for each patient. Body surface contours were generated in treatment planning system and exported to AlignRT to serve as reference images. The patient was aligned to treatment isocenter with room lasers, and then scanned with both CTOR and AlignRT in both supine and prone positions. Image guidance shifts were calculated for both modalities by comparison to the simulation CT and the differences between them were analyzed. These procedures were performed for each patient once per week for five weeks. The average and maximum difference of displacement between AlignRT and CTOR were calculated for each patient. For supine position, there are 4 patients who had the average difference of displacement between AlignRT and CTOR along any direction (vertical, longitudinal, and lateral) greater than 0.5cm, and 1 patient greater than 1cm. For prone position, there are 7 patients who had the average difference greater than 0.5cm, and 3 patients greater than 1cm. For supine position, there are 4 patients who had the maximum difference great than 1cm. For prone position, there are 9 patients who had the maximum difference greater than 1 cm. The difference of displacement between AlignRT and CTOR is greater for prone position than for supine position. AlignRT does not appear to be an advisable image guidance approach for pelvic radiation therapy for patients with either supine or prone position. There appears to be a potential for large alignment discrepancies (up to 2.25 cm) between AlignRT and CTOR.


Is it always me first? Effects of self-tagging on third-person perspective-taking.
Self-relevant information is associated with facilitation of perceptual and memory processes. In 2 experiments, participants verified the number of dots within a virtual room that were visible to a given perspective, corresponding to participants' own first-person perspectives or the third-person perspectives for self- and other-associated avatars. Perspectives were either congruent or incongruent with respect to the number of dots visible to each. In Experiment 1, we examined perspective taking for self- and other-associated avatars relative to one another; both avatars appeared simultaneously in the virtual room, and participants made judgments based on the prompted avatar's perspective. In Experiment 2, we examined perspective taking for each avatar relative to the first-person perspective; only 1 avatar was visible in the virtual room (Self or Other, varying by trial), and participants made judgments based on their first-person view or the avatar's perspective. Experiment 2 also included a replication of the third-person paradigm used in Experiment 1. Results from Experiment 1 (replicated in Experiment 2) demonstrated an advantage for judgments of the Self (vs. Other) avatar's perspective; both avatars elicited reliable interference effects of similar magnitude. Results from Experiment 2 further demonstrated that participants prioritized the first-person (vs. third-person) perspective, and that the presence of the Self (vs. Other) avatar improved performance for the first- and third-person perspectives when those perspectives were congruent. Taken together, these findings suggest that self-relevant perspectives are prioritized when they are actively engaged and when they can be subsumed within the first-person view. Such prioritization appears to occur by strategic means.


Fetal hippocampal grafts containing CA3 cells restore host hippocampal glutamate decarboxylase-positive interneuron numbers in a rat model of temporal lobe epilepsy.
Degeneration of CA3-pyramidal neurons in hippocampus after intracerebroventricular kainic acid (KA) administration, a model of temporal lobe epilepsy, results in hyperexcitability within both dentate gyrus and the CA1 subfield. It also leads to persistent reductions in hippocampal glutamate decarboxylase (GAD) interneuron numbers without diminution in Nissl-stained interneuron numbers, indicating loss of GAD expression in a majority of interneurons. We hypothesize that enduring loss of GAD expression in hippocampal interneurons after intracerebroventricular KA is attributable to degeneration of their CA3 afferent input; therefore, fetal CA3 grafts can restore GAD interneuron numbers through graft axon reinnervation of the host. We analyzed GAD interneuron density in the adult rat hippocampus at 6 months after KA administration after grafting of fetal mixed hippocampal, CA3 or CA1 cells into the CA3 region at 45 d after lesion, in comparison with "lesion-only" and intact hippocampus. In dentate and CA1 regions of the lesioned hippocampus receiving grafts of either mixed hippocampal or CA3 cells, GAD interneuron density was both significantly greater than lesion-only hippocampus and comparable with the intact hippocampus. In the CA3 region, GAD interneuron density was significantly greater than lesion-only hippocampus but less than the intact hippocampus. Collectively, the overall GAD interneuron density in the lesioned hippocampus receiving either mixed hippocampal or CA3 grafts was restored to that in the intact hippocampus. In contrast, GADinterneuron density in the lesioned hippocampus receiving CA1 grafts remained comparable with lesion-only hippocampus. Thus, grafts containing CA3 cells restore CA3 lesion-induced depletions in hippocampal GAD interneurons, likely by reinnervation of GAD-deficient interneurons. This specific graft-mediated effect is beneficial because reactivation of interneurons could ameliorate both loss of functional inhibition and hyperexcitability in CA3-lesioned hippocampus.


Extensive metabolic cross-talk in melon fruit revealed by spatial and developmental combinatorial metabolomics.
• Variations in tissue development and spatial composition have a major impact on the nutritional and organoleptic qualities of ripe fleshy fruit, including melon (Cucumis melo). To gain a deeper insight into the mechanisms involved in these changes, we identified key metabolites for rational food quality design. • The metabolome, volatiles and mineral elements were profiled employing an unprecedented range of complementary analytical technologies. Fruits were followed at a number of time points during the final ripening process and tissues were collected across the fruit flesh from rind to seed cavity. Approximately 2000 metabolite signatures and 15 mineral elements were determined in an assessment of temporal and spatial melon fruit development. • This study design enabled the identification of: coregulated hubs (including aspartic acid, 2-isopropylmalic acid, β-carotene, phytoene and dihydropseudoionone) in metabolic association networks; global patterns of coordinated compositional changes; and links of primary and secondary metabolism to key mineral and volatile fruit complements. • The results reveal the extent of metabolic interactions relevant to ripe fruit quality and thus have enabled the identification of essential candidate metabolites for the high-throughput screening of melon breeding populations for targeted breeding programmes aimed at nutrition and flavour improvement.


Attitudes towards long-acting depot antipsychotics: a survey of patients, relatives and psychiatrists.
In many countries fewer than 20% of individuals with schizophrenia receive depot antipsychotic medication. Frequently stated reasons are psychiatrist's, patient's and relative's objections to depot treatment. This is the first study that directly compares the attitudes to depot antipsychotics of psychiatrists, patients and relatives. A semi-structured questionnaire about their attitudes towards depot antipsychotics was completed by 255 participants (83 patients diagnosed with schizophrenic disorder, 81 psychiatrists in private practice and 91 relatives, not directly related to the patients). Patients were more negative towards depot injections than psychiatrists and relatives. They particularly fear to be constricted in their autonomy when treated with depot antipsychotics and that injections might be painful. About 67% of all patients in our sample did not receive information about depot antipsychotics from their psychiatrist. Less than 10% of psychiatrists offer depot treatment after a first psychotic episode. Psychiatrists use depot antipsychotics in a conservative way, although they attribute positive traits to the method. Patients' negative attitudes might relate to the low level of information. To enhance the use of depot antipsychotics, information practices of psychiatrists should be improved. Patients should be informed about different forms of treatment during early stages of the illness.


Nuclear dynamics of topoisomerase IIβ reflects its catalytic activity that is regulated by binding of RNA to the C-terminal domain.
DNA topoisomerase II (topo II) changes DNA topology by cleavage/re-ligation cycle(s) and thus contributes to various nuclear DNA transactions. It is largely unknown how the enzyme is controlled in a nuclear context. Several studies have suggested that its C-terminal domain (CTD), which is dispensable for basal relaxation activity, has some regulatory influence. In this work, we examined the impact of nuclear localization on regulation of activity in nuclei. Specifically, human cells were transfected with wild-type and mutant topo IIβ tagged with EGFP. Activity attenuation experiments and nuclear localization data reveal that the endogenous activity of topo IIβ is correlated with its subnuclear distribution. The enzyme shuttles between an active form in the nucleoplasm and a quiescent form in the nucleolus in a dynamic equilibrium. Mechanistically, the process involves a tethering event with RNA. Isolated RNA inhibits the catalytic activity of topo IIβ in vitro through the interaction with a specific 50-residue region of the CTD (termed the CRD). Taken together, these results suggest that both the subnuclear distribution and activity regulation of topo IIβ are mediated by the interplay between cellular RNA and the CRD.


ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression.
Recurrent somatic ASXL1 mutations occur in patients with myelodysplastic syndrome, myeloproliferative neoplasms, and acute myeloid leukemia, and are associated with adverse outcome. Despite the genetic and clinical data implicating ASXL1 mutations in myeloid malignancies, the mechanisms of transformation by ASXL1 mutations are not understood. Here, we identify that ASXL1 mutations result in loss of polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) tri-methylation. Through integration of microarray data with genome-wide histone modification ChIP-Seq data, we identify targets of ASXL1 repression, including the posterior HOXA cluster that is known to contribute to myeloid transformation. We demonstrate that ASXL1 associates with the PRC2, and that loss of ASXL1 in vivo collaborates with NRASG12D to promote myeloid leukemogenesis.


Red blood cell lithium-sodium countertransport in the tecumseh blood pressure study.
Human essential hypertension has more than one cause, but to dissect out subtypes, markers are required. The maximal activity of red blood cell lithium-sodium countertransport has been shown to be increased in hypertensive patients in case-control and population-based studies; in the latter, its distribution is a mixture of two overlapping but distinguishable subpopulations. In the present study, we classified 705 participants in the Tecumseh Blood Pressure Study as having either normal (mean, 0.234 mmol/l cells/hr; n = 614) or high (mean, 0.463 mmol/l cells/hr; n = 91) red blood cell lithium-sodium countertransport to determine if the red blood cell marker is associated with distinctive physiological characteristics. We found that subjects with elevated lithium-sodium countertransport have higher average blood pressure and a greater prevalence of hypertension than those with normal countertransport and that elevated blood pressure had been present since youth. Hemodynamically, the high countertransport group is characterized by elevated vascular resistance, whereas sympathetic nervous system activity appears to be slightly depressed. Subjects with increased lithium-sodium countertransport, compared with those with normal countertransport, have significantly lower average left ventricular mass index and only very infrequently demonstrate left ventricular hypertrophy. Our results support the usefulness of measurements of the maximal activity of red blood cell lithium-sodium countertransport as a way of distinguishing subgroups in the population. Our data are consistent with the idea that subjects with an elevated maximal activity for red blood cell lithium-sodium countertransport are a subset of the population with a genetic lesion that predisposes them to the development of essential hypertension.


Bidirectional Associations of Momentary Affect with Physical Activity and Sedentary Behaviors in Working Adults.
Affective experiences and movement-based behaviors form a system that has been shown to influence exercise adherence and mental health outcomes. Little is known about the naturalistic dynamics of the reciprocal associations in this system. We examined the time intervals at which momentary affect precedes and follows movement-based behaviors in everyday life. A community sample of working adults (n = 111) completed ecological momentary assessments (EMA) asking about current affect states (sad, happy, tired, and interested) six times a day for three consecutive days. Ratings were used to generate scores for momentary affective arousal and valence. Participants also wore an activity monitor. Total activity counts and sedentary duration in the shorter to longer time intervals (5-120 min) before or after EMA were used as indicators of movement-based behaviors. Multilevel modeling showed that current affective arousal predicted higher subsequent activity counts in the longer time intervals (120 min) and less subsequent sedentary behavior in the shorter to longer time intervals (5, 60, and 120 min). For the reversed sequence, neither movement-based behavior predicted subsequent momentary arousal or valence. Affective valence was unrelated to movement-based behaviors in either temporal direction. Some naturally occurring affective experiences (i.e., arousal) might precede, rather than follow, movement-based behaviors. Understanding affective arousal may contribute to improved management of subsequent movement-based behaviors in everyday life.


Indications, complications, and outcome of horses undergoing repeated celiotomy within 14 days after the first colic surgery: 95 cases (2005-2013).
To examine factors associated with short- and long-term prognosis for horses undergoing repeated celiotomy within 14 days after the first colic surgery. Retrospective case series. 95 horses that had undergone 2 celiotomies within a 14-day period between 2005 and 2013 at 3 equine referral hospitals. Historical, clinical, and laboratory data were compared between horses that did not survive and horses that did survive to hospital discharge (short-term survival rate) and to > 3 and > 6 months after hospital discharge (long-term survival rates). Strangulating small intestinal lesions were the most common finding during the first celiotomy (60/95 [63.2%]), and persistent gastric reflux was the most common reason for the second celiotomy (56/95 [58.9%]). Reasons for a second celiotomy were not associated with survival rate. For horses that had long-term follow-up, 22 of 92 (23.9%) survived > 6 months after hospital discharge. Two of 13 horses with intestinal resections during both surgeries survived to > 6 months after hospital discharge. Compared with horses not undergoing intestinal resection, significantly fewer horses requiring resection during 1 or both surgeries survived to hospital discharge and to > 3 and > 6 months after hospital discharge. Incisional infections occurred in 68.4% (26/38) of horses that survived to hospital discharge, and 31.6% (12/38) developed incisional hernias or dehiscence. Results indicated that the prognosis for horses undergoing repeated celiotomy is guarded, and intestinal resection negatively affects the long-term survival rate.


Validation of PHQ-2 and PHQ-9 to screen for major depression in the primary care population.
Although screening for unipolar depression is controversial, it is potentially an efficient way to find undetected cases and improve diagnostic acumen. Using a reference standard, we aimed to validate the 2- and 9-question Patient Health Questionnaires (PHQ-2 and PHQ-9) in primary care settings. The PHQ-2 comprises the first 2 questions of the PHQ-9. Consecutive adult patients attending Auckland family practices completed the PHQ-9, after which they completed the Composite International Diagnostic Interview (CIDI) depression reference standard. Sensitivities and specificities for PHQ-2 and PHQ-9 were analyzed. There were 2,642 patients who completed both the PHQ-9 and the CIDI. Sensitivity and specificity of the PHQ-2 for diagnosing major depression were 86% and 78%, respectively, with a score of 2 or higher and 61% and 92% with a score 3 or higher; for the PHQ-9, they were 74% and 91%, respectively, with a score of 10 or higher. For the PHQ-2 a score of 2 or higher detected more cases of depression than a score of 3 or higher. For the PHQ-9 a score of 10 or higher detected more cases of major depression than the PHQ determination of major depression originally described by Spitzer et al in 1999. We report the largest validation study of the PHQ-2 and PHQ-9, compared with a reference standard interview, undertaken in an exclusively primary care population. The PHQ-2 score or 2 or higher had good sensitivity but poor specificity in detecting major depression. Using a PHQ-2 threshold score of 2 or higher rather than 3 or higher resulted in more depressed patients being correctly identified. A PHQ-9 score of 10 or higher appears to detect more depressed patients than the originally described PHQ-9 scoring for major depression.


Exploiting genetic diversity from landraces in wheat breeding for adaptation to climate change.
Climate change has generated unpredictability in the timing and amount of rain, as well as extreme heat and cold spells that have affected grain yields worldwide and threaten food security. Sources of specific adaptation related to drought and heat, as well as associated breeding of genetic traits, will contribute to maintaining grain yields in dry and warm years. Increased crop photosynthesis and biomass have been achieved particularly through disease resistance and healthy leaves. Similarly, sources of drought and heat adaptation through extended photosynthesis and increased biomass would also greatly benefit crop improvement. Wheat landraces have been cultivated for thousands of years under the most extreme environmental conditions. They have also been cultivated in lower input farming systems for which adaptation traits, particularly those that increase the duration of photosynthesis, have been conserved. Landraces are a valuable source of genetic diversity and specific adaptation to local environmental conditions according to their place of origin. Evidence supports the hypothesis that landraces can provide sources of increased biomass and thousand kernel weight, both important traits for adaptation to tolerate drought and heat. Evaluation of wheat landraces stored in gene banks with highly beneficial untapped diversity and sources of stress adaptation, once characterized, should also be used for wheat improvement. Unified development of databases and promotion of data sharing among physiologists, pathologists, wheat quality scientists, national programmes, and breeders will greatly benefit wheat improvement for adaptation to climate change worldwide.


Severe impairment of patient-reported outcomes in patients with chronic hepatitis C virus infection seen in real-world practices across the world: Data from the global liver registry.
Cure of chronic hepatitis C (CHC) can lead to improvement of health-related quality of life and other patient-reported outcomes (PROs). While extensive PRO data for CHC patients who were enrolled in clinical trials are available, similar data for patients seen in real-world practices are scarce. Our aim was to assess PROs of CHC patients enrolled from real-world practices from different regions and to compare them with those enrolled in clinical trials. CHC patients seen in clinical practices and not receiving treatment were enrolled in the Global Liver Registry (GLR). Clinical and PRO (FACIT-F, CLDQ-HCV, WPAI) data were collected and compared with the baseline data from CHC patients enrolled in clinical trials. N = 12,171 CHC patients were included (GLR n = 3146, clinical trial subjects n = 9025). Patients were from 30 countries from 6 out of 7 Global Burden of Disease (GBD) super-regions. Compared with clinical trial enrollees, patients from GLR were less commonly enrolled from High-Income GBD super-region, older, more commonly female, less employed, had more type 2 diabetes, anxiety and clinically overt fatigue but less cirrhosis (all p < 0.001). Out of 15 PRO domain and summary scores, 12 were lower in GLR patients than in subjects enrolled in clinical trials (p < 0.001). In multiple regression models, anxiety, depression, and fatigue were associated with significant PRO impairment in CHC patients (p < 0.05). After adjustment for the clinico-demographic confounders, the association of PRO scores of CHC patients with enrolment settings was no longer significant (all p > 0.05). In conclusion, hepatitis C patients seen in the real-world practices have PRO impairment driven by fatigue and psychiatric comorbidities.


Rapid screening for freshwater bacterial groups by using reverse line blot hybridization.
The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the single-mismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.


The 2-monoacylglycerol moiety of dietary fat appears to be responsible for the fat-induced release of GLP-1 in humans.
Dietary triglycerides can, after digestion, stimulate the intestinal release of incretin hormones through activation of G protein-coupled receptor (GPR) 119 by 2-monoacylglycerol and by the activation of fatty acid receptors for long- and short-chain fatty acids. Medium-chain fatty acids do not stimulate the release of intestinal hormones. To dissect the mechanism of fat-induced glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) release in humans, we compared the effects of tributyrin (containing short-chain fatty acids; i.e., butyric acid), olive oil [containing long-chain fatty acids; e.g., oleic acid plus 2-oleoyl glycerol (2-OG)], and 1,3-dioctanoyl-2-oleoyl glycerol (C8-dietary oil), which is digested to form medium-chain fatty acids : i.e., octanoic acid : and 2-OG. In a randomized, single-blinded crossover study, 12 healthy white men [mean age: 24 y; BMI (in kg/m(2)): 22] were given the following 4 meals on 4 different days: 200 g carrots + 6.53 g tributyrin, 200 g carrots + 13.15 g C8-dietary oil, 200 g carrots + 19 g olive oil, or 200 g carrots. All of the lipids totaled 0.0216 mol. Main outcome measures were incremental areas under the curve for total GLP-1, GIP, and cholecystokinin (CCK) in plasma. C8-dietary oil and olive oil showed the same GLP-1 response [583 ± 101 and 538 ± 71 (pmol/L) × 120 min; P = 0.733], whereas the GIP response was higher for olive oil than for C8-dietary oil [3293 ± 404 and 1674 ± 270 (pmol/L) × 120 min; P = 0.002]. Tributyrin and carrots alone resulted in no increase in any of the measured hormones. Peptide YY (PYY) and neurotensin responses resembled those of GLP-1. Only olive oil stimulated CCK release. Under our study conditions, 2-OG and GPR119 activation can fully explain the olive oil-induced secretion of GLP-1, PYY, and neurotensin. In contrast, both oleic acid and 2-OG contributed to the GIP response. Dietary butyrate did not stimulate gut hormone secretion. Olive oil-derived oleic acid seems to be fully responsible for olive oil-induced CCK secretion. This trial was registered at clinicaltrials.gov as NCT02264951.


A connection between magnesium deficiency and aging: new insights from cellular studies.
Most human cells can only replicate a limited number of times in cultures before they lose the ability to divide, a phenomenon known as replicative senescence, which seems to play a role in aging at the organismal level. Recent studies have shown that culture in low magnesium (Mg) accelerates the senescence of human endothelial cells and fibroblasts. Given the numerous critical roles of Mg, it seems likely that Mg inadequacy would interfere with cellular metabolism, which could affect the senescence process. Since i) several pieces of evidence link low Mg to aging and age-related diseases and ii) the Occidental diet is relatively deficient in Mg, we propose that broadly correcting nutritional intakes of Mg might contribute to healthier aging and the prevention of age-related diseases.


Bacillary Angiomatosis and Bacteremia due to Bartonella quintana in a Patient with Chronic Lymphocytic Leukemia.
We present a 63-year-old man treated with alemtuzumab for chronic lymphocytic leukemia who developed multiple angiomatous papules and fever. Real-time polymerase chain reaction (RT-PCR) from a skin lesion and blood sample revealed Bartonella quintana as causative agent confirming the diagnosis of bacillary angiomatosis with bacteremia. Treatment with doxycycline, initially in combination with gentamicin, led to complete resolution of the lesions. This case shows the importance of considering bacillary angiomatosis as a rare differential diagnosis of angiomatous lesions in the immunocompromised patient, particularly in chronic lymphocytic leukemia and following lymphocyte depleting treatments as alemtuzumab.


Cardiovascular disease risk prevention: preliminary survey of baseline knowledge, attitude and practices of a nigerian rural community.
Knowledge and attitude are significant factors impinging on whether individuals seek healthcare service. This flows on to impact public health knowledge of prevalence of diseases, and in turn, the practice of preventive medicine. As part of the international research collaboration agenda for Prediabetes and Cardiovascular Complications Study, a preliminary survey of one of the Ndokwa communities of Nigeria has been carried out. This study was to understand the baseline knowledge, attitudes and practices of a rural community in regards to cardiovascular diseases, and behavior toward risk management. Seventy-four volunteer participants were recruited, after public lectures, through secondary school and churches in the community. The survey was done using questionnaire. The knowledge component comprised questions about educational and personal health opinion. The attitude and practice components comprised questions about exercises and visiting healthcare facilities. Occupational backgrounds were also asked. It is observed that majority of the community dwellers have (1) completed at least secondary education, (2) never attended a health check-up; and (3) do not engage in physical activity in the context of exercise. Twenty of the participants indicated not being in good health, of which only 35% have attended medical check-up for their ailment. Many of those who are yet to seek healthcare service cite affordability as their reason. With specific regards to diabetes and cardiovascular risk, over 71% of the survey participants are yet to do any blood sugar and/or lipid profile tests. This preliminary survey indicates that although the majority of respondents have secondary education and therefore are relatively literate, there is a gap between their knowledge of ill-health versus attitude and practice toward prevention; especially cardiovascular and diabetes diseases.


Perceptual skill in soccer: implications for talent identification and development.
In this review, key components of perceptual skill in soccer are identified and implications for talent identification and development highlighted. Skilled soccer players can recall and recognize patterns of play more effectively than their less skilled counterparts. This ability to encode, retrieve and recognize sport-specific information is due to complex and discriminating long-term memory structures and is crucial to anticipation in soccer. Similarly, experts use their knowledge of situational probabilities (i.e. expectations) to anticipate future events. They have a better than average idea of what is likely to happen given a particular set of circumstances. Also, proficiency-related differences in visual search strategy are observed. Skilled players use their superior knowledge to control the eye movement patterns necessary for seeking and picking up important sources of information. The nature of the task plays an important role in constraining the type of search used. Skilled soccer players use different search strategies when viewing the whole field (i.e. 11 vs 11 situations) compared with micro-states of the game (i.e. 1 vs 1, 3 vs 3 situations). Visual search behaviour also differs between defensive and offensive plays. These observations have implications for the development of perceptual training programmes and the identification of potential elite soccer players.


Decision frameworks for restoration & adaptation investment-Applying lessons from asset-intensive industries to the Great Barrier Reef.
Asset-intensive industries (including water and power utilities, mineral resources and energy) are those which require significant levels of capital investment in their assets in order to operate. These industries face challenges from uncertainty in resource availability and demand for end products, the intricate and complicated nature of their assets, and the complexity of the economic, ecological and social settings in which they operate. In these industries, the application of decision frameworks that account for this uncertainty and complexity in guiding asset investment and development is standard practice. Lessons from asset-intensive industries were applied during the concept feasibility phase of the Reef Restoration and Adaptation Program (RRAP) to establish the investment case for research and development into interventions to help the Great Barrier Reef (GBR) resist, adapt to, and recover from the impacts of climate change. The authors worked with RRAP partners to define a decision framework that included structured decision-making processes (SDM), a cost-benefit analysis (CBA), and a value of information (VoI) analysis, to establish the investment case for intervening on the GBR which led to success in securing Australian Government commitment for the next phase of the Program. With climate change expected to drive increased demand for significant levels of restoration and adaptation investment in large integrated social, ecological and economic assets (such as the GBR), the lessons from RRAP offer insights for the application of decision frameworks to inform public and private investment priorities.


Intraductal papillary mucinous neoplasm and acute pancreatitis.
Intraductal papillary mucinous neoplasms (IPMNs) are cystic pancreatic tumors that arise from the pancreatic ducts and are increasingly reported worldwide. Both benign and malignant tumors of the pancreas are thought to contribute to recurrent pancreatitis possibly by pancreatic duct obstruction, and IPMNs contribute to a major share of this burden. The rate of acute pancreatitis (AP) in IPMN patients in the largest published surgical series has varied from 12% to 67%. IPMN may be categorized into 3 forms on the basis of the areas of involvement: main pancreatic duct (MD-IPMN), side branch (SB-IPMN), or combined. Both MD-IPMN and SB-IPMN may be the cause of pancreatitis. The risk of AP seems to be similar with both main duct IPMN and SB-IPMN, although data are controversial. AP in IPMN patients is not severe and often recurs without treatment. The rate of AP does not seem to differ among benign and malignant IPMNs, and the correlation between the malignant potential and the occurrence of AP is ill defined. AP seems to occur more often in patients with IPMN that in those with usual pancreatic adenocarcinoma possibly because of obstruction of the main duct by thick, abundant mucus secretion. Although the Sendai guidelines recommend surgical resection in patients with SB-IPMN with AP, data are controversial. Moreover, in patients with an episode of pancreatitis, the finding of pancreatic cysts is often attributed to pseudocysts or fluid collections that make the diagnosis of IPMN less suspicious. Future longitudinal and prospective studies to understand the natural history of AP in patients with IPMN are required to better manage patients with recurrent AP in the setting of IPMN.


Thermostability of Well-Ordered HIV Spikes Correlates with the Elicitation of Autologous Tier 2 Neutralizing Antibodies.
In the context of HIV vaccine design and development, HIV-1 spike mimetics displaying a range of stabilities were evaluated to determine whether more stable, well-ordered trimers would more efficiently elicit neutralizing antibodies. To begin, in vitro analysis of trimers derived from the cysteine-stabilized SOSIP platform or the uncleaved, covalently linked NFL platform were evaluated. These native-like trimers, derived from HIV subtypes A, B, and C, displayed a range of thermostabilities, and were "stress-tested" at varying temperatures as a prelude to in vivo immunogenicity. Analysis was performed both in the absence and in the presence of two different adjuvants. Since partial trimer degradation was detected at 37°C before or after formulation with adjuvant, we sought to remedy such an undesirable outcome. Cross-linking (fixing) of the well-ordered trimers with glutaraldehyde increased overall thermostability, maintenance of well-ordered trimer integrity without or with adjuvant, and increased resistance to solid phase-associated trimer unfolding. Immunization of unfixed and fixed well-ordered trimers into animals revealed that the elicited tier 2 autologous neutralizing activity correlated with overall trimer thermostability, or melting temperature (Tm). Glutaraldehyde fixation also led to higher tier 2 autologous neutralization titers. These results link retention of trimer quaternary packing with elicitation of tier 2 autologous neutralizing activity, providing important insights for HIV-1 vaccine design.


Inhibitory actions of laxatives on motility and water and electrolyte transport in the gastrointestinal tract.
Castor oil and magnesium sulfate were studied for their effects on gastrointestinal contractile activity in vivo. Ricinoleic acid, the active ingredient in castor oil, magnesium sulfate and mannitol were studied and compared for their effects on net water and electrolyte absorption in vitro. Extraluminal strain gauge transducers were implanted in dogs and used to monitor the circular smooth muscle activity of the antrum, duodenum, ileum and colon after water, castor oil or a 30 percent solution of magnesium sulfate. Substances were tested during the interdigestive (fasted) and digestive (fed) states. Decreases in total activity were found for both cathartics in the antrum and ileum. Further analysis revealed that decreases in the ileum occurred primarily by a decrease in contractile rate, whereas antral decreases could be attributed primarily to a decrease in force per contraction. Proximal colonic activity tended to decrease after laxatives and feeding. The effects of ricinoleic acid, isotonic and hypertonic solutions of magnesium sulfate and mannitol on net water and electrolyte absorption were tested on everted segments of hamster jejunum. Sodium ricinoleate (2.0 mM) reduced net water transport by 48 percent (P smaller than .01). Magnesium sulfate, like mannitol, only reduced net water absorption when present as a component of a hypertonic mucosal solution. The results suggest that both inhibition of water absorption and reduced circular smooth muscle activity may be important factors in castor oil- and magnesium sulfate-induced catharsis.


Using a holistic health approach to achieve weight-loss maintenance: results from the Spirited Life intervention.
Weight-loss maintenance is essential to sustain the health benefits of weight loss. Studies with lower intensity intervention supports under real-world conditions are lacking. This study examined changes in weight and cardiometabolic biomarkers among Spirited Life participants following initial 12-month weight loss at 12-24 months and 24-42 months. A total of 719 clergy received a wellness intervention, including a 10-week online weight-loss program in the first 12 months and monthly health coaching throughout 24 months. Mean changes in weight, blood pressure, high-density lipoproteins, and triglycerides were estimated using random effects linear models, accounting for repeated measures. Weight was additionally analyzed in subsamples stratified by body mass index (BMI). At baseline, 17.1% of participants had BMI < 25 kg/m2 and 11.8% had BMI ≥ 40 kg/m2. Mean 12-month weight loss was -2.4 kg (95% CI: -2.8 kg, -2.1 kg). On average, at 42 months, participants regained weight but did not exceed baseline (-0.5 kg, 95% CI: -1.2 kg, 0.2 kg), improvements in triglycerides were completely sustained (-13.9 mg/dL, 95% CI: -18.6 mg/dL, -9.2 mg/dL), and systolic blood pressure improvements remained significant (-1.9 mmHg, 95% CI: -3.0 mmHg, -0.9 mmHg). Participants with a BMI ≥ 40 kg/m2 lost significantly more weight that was sustained at 42 months (-5.8 kg, 95% CI: -8.9 kg, -2.7 kg). The Spirited Life wellness intervention produced weight loss and, for participants with higher levels of obesity, sustained weight-loss maintenance. The intervention was effective for long-term prevention of weight gain among participants with BMI of 25 to ≤40 kg/m2, through 42 months. Wellness interventions such as Spirited Life should be considered for adoption.


The pituitary gland in patients with Langerhans cell histiocytosis: a clinical and radiological evaluation.
Langerhans cell histiocytosis (LCH) is a rare disease in which the most common endocrine manifestation is diabetes insipidus (DI). Data on anterior pituitary function in patients with LCH are limited. Thus, the present study investigated anterior pituitary function in LCH patients with DI via the evaluation of clinical and radiological findings at disease onset and during follow-up. The present study retrospectively evaluated nine patients with LCH (five males and four females). All diagnoses of LCH were made following histological and/or immunophenotypic analyses of tissue biopsies, bronchoalveolar lavage, or cerebrospinal fluid (CSF). Basal and, if necessary, dynamic pituitary function tests were used to assess anterior pituitary function, and magnetic resonance imaging (MRI) scans were used to image the pituitary. The LCH treatment modality was based on organ involvement. The mean age at onset of DI was 27.6 years (range 15-60 years). One patient (11%) exhibited single organ involvement, while eight patients (89%) displayed multisystem organ involvement. On admittance, one patient had hypogonadotropic hypogonadism, one patient exhibited panhypopituitarism [hypogonadotropic hypogonadism, central hypothyroidism, hypocortisolism, and growth hormone (GH) deficiency], and four patients (44%) displayed hyperprolactinemia. The MRI data revealed infundibular enlargement in seven patients (78%), a thalamic mass in one patient (11%), and the absence of the bright spot in all patients. A single patient (11%) showed a mass in the pons that had a partially empty sella. The patients were treated with radiation therapy (RT), chemotherapy (CT), or a combination of both (RT+CT) and were followed up for a median of 91.8 months (range 2-318 months). Seven patients were assessed during the follow-up period, of whom four patients (57.1%) developed anterior pituitary hormone deficiency, three (43%) were diagnosed with GH deficiency, and one (14%) exhibited gonadotropin deficiency. The gonadotropin deficiency in the patient, which was diagnosed on admittance, was resolved during the follow-up period. DI persisted in all patients, and the conditions of the seven patients who have remained on follow-up are stable. In the present study, patients with LCH exhibited altered function in the anterior pituitary as well as the posterior pituitary, which may be due to the natural course of the disease or the effects of treatment. The present findings indicate that anterior pituitary function should be assessed in LCH patients on admittance and during follow-up, especially in LCH patients with multisystem organ involvement.


DEAR1 is a dominant regulator of acinar morphogenesis and an independent predictor of local recurrence-free survival in early-onset breast cancer.
Breast cancer in young women tends to have a natural history of aggressive disease for which rates of recurrence are higher than in breast cancers detected later in life. Little is known about the genetic pathways that underlie early-onset breast cancer. Here we report the discovery of DEAR1 (ductal epithelium-associated RING Chromosome 1), a novel gene encoding a member of the TRIM (tripartite motif) subfamily of RING finger proteins, and provide evidence for its role as a dominant regulator of acinar morphogenesis in the mammary gland and as an independent predictor of local recurrence-free survival in early-onset breast cancer. Suppression subtractive hybridization identified DEAR1 as a novel gene mapping to a region of high-frequency loss of heterozygosity (LOH) in a number of histologically diverse human cancers within Chromosome 1p35.1. In the breast epithelium, DEAR1 expression is limited to the ductal and glandular epithelium and is down-regulated in transition to ductal carcinoma in situ (DCIS), an early histologic stage in breast tumorigenesis. DEAR1 missense mutations and homozygous deletion (HD) were discovered in breast cancer cell lines and tumor samples. Introduction of the DEAR1 wild type and not the missense mutant alleles to complement a mutation in a breast cancer cell line, derived from a 36-year-old female with invasive breast cancer, initiated acinar morphogenesis in three-dimensional (3D) basement membrane culture and restored tissue architecture reminiscent of normal acinar structures in the mammary gland in vivo. Stable knockdown of DEAR1 in immortalized human mammary epithelial cells (HMECs) recapitulated the growth in 3D culture of breast cancer cell lines containing mutated DEAR1, in that shDEAR1 clones demonstrated disruption of tissue architecture, loss of apical basal polarity, diffuse apoptosis, and failure of lumen formation. Furthermore, immunohistochemical staining of a tissue microarray from a cohort of 123 young female breast cancer patients with a 20-year follow-up indicated that in early-onset breast cancer, DEAR1 expression serves as an independent predictor of local recurrence-free survival and correlates significantly with strong family history of breast cancer and the triple-negative phenotype (ER(-), PR(-), HER-2(-)) of breast cancers with poor prognosis. Our data provide compelling evidence for the genetic alteration and loss of expression of DEAR1 in breast cancer, for the functional role of DEAR1 in the dominant regulation of acinar morphogenesis in 3D culture, and for the potential utility of an immunohistochemical assay for DEAR1 expression as an independent prognostic marker for stratification of early-onset disease.


Layer 6b Is Driven by Intracortical Long-Range Projection Neurons.
Layer 6b (L6b), the deepest neocortical layer, projects to cortical targets and higher-order thalamus and is the only layer responsive to the wake-promoting neuropeptide orexin/hypocretin. These characteristics suggest that L6b can strongly modulate brain state, but projections to L6b and their influence remain unknown. Here, we examine the inputs to L6b ex vivo in the mouse primary somatosensory cortex with rabies-based retrograde tracing and channelrhodopsin-assisted circuit mapping in brain slices. We find that L6b receives its strongest excitatory input from intracortical long-range projection neurons, including those in the contralateral hemisphere. In contrast, local intracortical input and thalamocortical input were significantly weaker. Moreover, our data suggest that L6b receives far less thalamocortical input than other cortical layers. L6b was most strongly inhibited by PV and SST interneurons. This study shows that L6b integrates long-range intracortical information and is not part of the traditional thalamocortical loop.


Automated design of probes for rRNA-targeted fluorescence in situ hybridization reveals the advantages of using dual probes for accurate identification.
Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu).


Adoptive Transfer of Phosphoantigen-Specific γδ T Cell Subset Attenuates Mycobacterium tuberculosis Infection in Nonhuman Primates.
The dominant Vγ2Vδ2 T cell subset recognizes phosphoantigen and exists only in humans and nonhuman primates. Despite the discovery of γδ T cells >30 y ago, a proof-of-concept study has not been done to prove the principle that the Vγ2Vδ2 T cell subset is protective against Mycobacterium tuberculosis and other infections. In this study, we used an adoptive cell-transfer strategy to define the protective role of Vγ2Vδ2 T cells in a primate tuberculosis (TB) model. Vγ2Vδ2 T cells for adoptive transfer displayed central/effector memory and mounted effector functions, including the production of anti-M. tuberculosis cytokines and inhibition of intracellular mycobacteria. They also expressed CXCR3/CCR5/LFA-1 trafficking/tissue-resident phenotypes and consistently trafficked to the airway, where they remained detectable from 6 h through 7 d after adoptive transfer. Interestingly, the test group of macaques receiving transfer of Vγ2Vδ2 T cells at weeks 1 and 3 after high-dose (500 CFU) M. tuberculosis infection exhibited significantly lower levels of M. tuberculosis infection burdens in lung lobes and extrapulmonary organs than did the control groups receiving PBLs or saline. Consistently, adoptive transfer of Vγ2Vδ2 T cells attenuated TB pathology and contained lesions primarily in the infection site of the right caudal lung lobe, with no or reduced TB dissemination to other lobes, spleen, or liver/kidney; in contrast, the controls showed widespread TB dissemination. The proof-of-concept finding supports the view that the dominant Vγ2Vδ2 T cell subset may be included in the rational design of a TB vaccine or host-directed therapy.


Toward Relevant and Credible Cost-Effectiveness Analyses for Value Assessment in the Decentralized U.S. Health Care System.
In the United States, there is an increased interest to understand the value of health technologies. Cost-effectiveness analysis is arguably the most appropriate framework to quantify value and to inform reimbursement decision making regarding medical interventions; however, a thorough analysis is resource intensive and complex. In many countries, the cost-effectiveness of medical interventions is evaluated by expert agencies at the national level, but in the United States, reimbursement decision making occurs at the local level. This raises the question of how we can provide a means to transparent cost-effectiveness analysis that reflects the local context and patient population and is based on the latest evidence and scientific insights. In other words, how can we maximize the relevance and credibility of cost-effectiveness evaluations in the context of a decentralized decision-making environment? Published cost-effectiveness analyses typically fail on these dimensions. Access to transparent open-source models that can be adapted to reflect the local setting in a relatively straightforward manner is an essential step toward such a goal. However, no model for cost-effectiveness analysis is ever truly "right" or "complete," and it must evolve along with clinical evidence and improvements in scientific methodology to ensure that its credibility remains. We propose a transparent approach of iterative development and collaboration between content and methodology experts to produce up-to-date, open-source consensus-based cost-effectiveness models that account for parameter and structural uncertainty to help local decision makers understand the confidence with which they might make a decision. Our proposed approach provides a way to adapt formal assessments of value-long the province of centralized health care systems-into the decentralized U.S. health care landscape. DISCLOSURES: This research was funded through the Innovation and Value Initiative, a nonprofit multistakeholder research organization. The Innovation and Value Initiative contracted with Precision Medicine Group for research activities related to this article. Jansen and Incerti are salaried employees and shareholders of Precision Medicine Group. Curtis is a paid consultant for the Innovation and Value Initiative. Curtis also reports consulting fees and grants from Amgen, AbbVie, BMS, Corrona, Janssen, Lilly, Myriad, Pfizer, Roche/Genentech, Radius, and UCB, unrelated to this article.


Mynodbcsv: lightweight zero-config database solution for handling very large CSV files.
Volumes of data used in science and industry are growing rapidly. When researchers face the challenge of analyzing them, their format is often the first obstacle. Lack of standardized ways of exploring different data layouts requires an effort each time to solve the problem from scratch. Possibility to access data in a rich, uniform manner, e.g. using Structured Query Language (SQL) would offer expressiveness and user-friendliness. Comma-separated values (CSV) are one of the most common data storage formats. Despite its simplicity, with growing file size handling it becomes non-trivial. Importing CSVs into existing databases is time-consuming and troublesome, or even impossible if its horizontal dimension reaches thousands of columns. Most databases are optimized for handling large number of rows rather than columns, therefore, performance for datasets with non-typical layouts is often unacceptable. Other challenges include schema creation, updates and repeated data imports. To address the above-mentioned problems, I present a system for accessing very large CSV-based datasets by means of SQL. It's characterized by: "no copy" approach--data stay mostly in the CSV files; "zero configuration"--no need to specify database schema; written in C++, with boost [1], SQLite [2] and Qt [3], doesn't require installation and has very small size; query rewriting, dynamic creation of indices for appropriate columns and static data retrieval directly from CSV files ensure efficient plan execution; effortless support for millions of columns; due to per-value typing, using mixed text/numbers data is easy; very simple network protocol provides efficient interface for MATLAB and reduces implementation time for other languages. The software is available as freeware along with educational videos on its website [4]. It doesn't need any prerequisites to run, as all of the libraries are included in the distribution package. I test it against existing database solutions using a battery of benchmarks and discuss the results.


Poly(ethylene glycol)-Poly(beta-amino ester)-Based Nanoparticles for Suicide Gene Therapy Enhance Brain Penetration and Extend Survival in a Preclinical Human Glioblastoma Orthotopic Xenograft Model.
Glioblastoma (GBM) is the most devastating brain cancer, and cures remain elusive with currently available neurosurgical, pharmacological, and radiation approaches. While retrovirus- and adenovirus-mediated suicide gene therapy using DNA encoding herpes simplex virus-thymidine kinase (HSV-tk) and prodrug ganciclovir has been suggested as a promising strategy, a nonviral approach for treatment in an orthotopic human primary brain tumor model has not previously been demonstrated. Delivery challenges include nanoparticle penetration through brain tumors, efficient cancer cell uptake, endosomal escape to the cytosol, and biodegradability. To meet these challenges, we synthesized poly(ethylene glycol)-modified poly(beta-amino ester) (PEG-PBAE) polymers to improve extracellular delivery and coencapsulated plasmid DNA with end-modified poly(beta-amino ester) (ePBAE) polymers to improve intracellular delivery as well. We created and evaluated a library of PEG-PBAE/ePBAE nanoparticles (NPs) for effective gene therapy against two independent primary human stem-like brain tumor initiating cells, a putative target to prevent GBM recurrence. The optimally engineered PEG-PBAE/ePBAE NP formulation demonstrated 54 and 82% transfection efficacies in GBM1A and BTIC375 cells respectively, in comparison to 37 and 66% for optimized PBAE NPs without PEG. The leading PEG-PBAE NP formulation also maintained sub-250 nm particle size up to 5 h, while PBAE NPs without PEG showed aggregation over time to micrometer-sized complexes. The comparative advantage demonstrated in vitro successfully translated into improved in vivo diffusion, with a higher amount of PEG-PBAE NPs penetrating to a distance of 2 mm from the injection site. A significant increase in median survival from 53.5 to 67 days by PEG-PBAE/pHSV-tk NP and systemic ganciclovir treatment compared to a control group in orthotopic murine model of human glioblastoma demonstrates the potential of PEG-PBAE-based NPs as an effective gene therapy platform for the treatment of human brain tumors.


Mechanical validation of a tomographic (pQCT) index for noninvasive estimation of rat femur bending strength.
Cross-sectional moment of inertia (CSMI) and volumetric cortical bone mineral density (vCtBMD) were assessed by peripheral quantitative computed tomography (pQCT) at femur midshafts from 103 Wistar female rats receiving 0 (n = 12) or 15-1000 mu g/kg/day sc of dexamethasone (n = 46) from 5 to 9 weeks of age, or 0 or 80 mg/kg 3/wk of AI(OH)(3) IP (n = 23,22) from 4 to 10 months of age. A bone strength index (BSI), calculated as the product CSMI x vCtBMD, was found to closely correlate (r = 0.94, R(2) = 0.89, p < 0.001) with the actual, mechanically tested bending breaking force of all bones. Correlation and determination coefficients obtained were higher than those usually reported employing different long-bone strength predictive formulae. The curve approached the origin and was linear throughout the wide range of CSMI, vCtBMD and BSI achieved because of age- and treatment-induced differences, showing a very low standard error of the estimate. Instead, different curve slopes and/or intercepts were found in separate analysis between data from each of the experiments when breaking force was correlated with CSMI or vCtBMD alone, or with the DEXA-assessed BMD of the mechanically assayed bone portion. Results suggest that noninvasive assessment of the BSI by means of pQCT technology provides an original tool for a precise and accurate estimation of long-bone bending strength that can be advantageously applied in crosssectional as well as longitudinal, in vivo studies employing animal models.


Laparoscopic reconstruction of ureteral strictures involving solitary renal units-1 year and 5 year outcomes.
Long-term outcome following a laparoscopic reconstruction of ureteral strictures (US) involving solitary renal units (SRU) are scarcely reported. The aim was to report short-term (1 year) and long-term (5 years) outcomes following a laparoscopic reconstruction of US in a solitary kidney. Retrospective. Records of patients operated for similar scenarios between January 2004 and January 2014 were evaluated. Clinical, biochemical and radiological profile were noted. Operative and post-operative profile were recorded. Follow-ups were scheduled at regular intervals (3 months post-procedure, 6 monthly for 2 years and yearly thereafter. Imaging was repeated at yearly intervals). Outcome was assessed by comparing pre-operative and post-operative clinical, biochemical, and radiological parameters. SAS software 9.2 version. A P < 0.05 was inferred as statistically significant. Seven patients underwent a laparoscopic reconstruction. Stricture location was upper ureter (n = 1), mid ureter (n = 2), lower ureter (n = 4). Surgeries performed were ureteroureterostomy, Boari flap ureteroneocystostomy and ureteroneocystostomy with psoas hitch. Four patients reported prior contralateral nephrectomy. Three patients underwent prior endoscopic correction. Four patients presented with elevated serum creatinine (>1.4 mg/dl). Till last follow-up, improvement in symptomatology and improvement or stabilisation of serum creatinine was perceived in all. Ureteral patency with resolution of hydronephrosis was observed in five patients at 1 year follow-up. Two patients revealed ureteral patency with persistence of hydronephrosis. Clinical, biochemical and radiological outcomes were maintained till long-term follow-up. Laparoscopic reconstruction of US in SRU offers impressive short- and long-term outcome.