Treatment with monoclonal antibodies against Clostridium difficile toxins.
New therapies are needed to manage the increasing incidence, severity, and high rate of recurrence of Clostridium difficile infection. We performed a randomized, double-blind, placebo-controlled study of two neutralizing, fully human monoclonal antibodies against C. difficile toxins A (CDA1) and B (CDB1). The antibodies were administered together as a single infusion, each at a dose of 10 mg per kilogram of body weight, in patients with symptomatic C. difficile infection who were receiving either metronidazole or vancomycin. The primary outcome was laboratory-documented recurrence of infection during the 84 days after the administration of monoclonal antibodies or placebo. Among the 200 patients who were enrolled (101 in the antibody group and 99 in the placebo group), the rate of recurrence of C. difficile infection was lower among patients treated with monoclonal antibodies (7% vs. 25%; 95% confidence interval, 7 to 29; P<0.001). The recurrence rates among patients with the epidemic BI/NAP1/027 strain were 8% for the antibody group and 32% for the placebo group (P=0.06); among patients with more than one previous episode of C. difficile infection, recurrence rates were 7% and 38%, respectively (P=0.006). The mean duration of the initial hospitalization for inpatients did not differ significantly between the antibody and placebo groups (9.5 and 9.4 days, respectively). At least one serious adverse event was reported by 18 patients in the antibody group and by 28 patients in the placebo group (P=0.09). The addition of monoclonal antibodies against C. difficile toxins to antibiotic agents significantly reduced the recurrence of C. difficile infection. (ClinicalTrials.gov number, NCT00350298.)


"Sometimes you wonder, is this really true?": Clinician assessment of patients' subjective experience of pain.
Pain is a subjective experience that must be translated by clinicians into an objective assessment to guide intervention. To understand how patients' subjective experience of pain is translated by primary care clinicians into an objective clinical assessment of pain to effectively guide intervention. We conducted nine multidisciplinary focus groups with a combined total of 60 Veteran affair (VA) primary care providers and staff from two large VA medical centers in California and Oregon. We used content analysis methods to identify key themes pertaining to clinical assessment of a subjective experience. We present four emergent themes. Theme 1: Pain is a highly individualized and subjective experience not adequately captured by a simple numeric scale; Theme 2: Conflict commonly exists between the patient's reported experience of pain and the clinician's observations and expectations of pain; Theme 3: Providers attempt to recalibrate the patient's reported experience to reflect their own understanding of pain; and Theme 4: Providers perceive that some patients may overreport their pain because they do not know how to standardize their subjective experience. A persistent challenge to pain assessment and management is how clinicians reconcile a patient's subjective self-reported experience with their own clinical assessment and personal biases. Future work should explore these themes from the patient perspective.


Perinatal and long-term outcome of fetal intracranial hemorrhage: systematic review and meta-analysis.
Fetal intracranial hemorrhage (ICH) is associated with an increased risk of perinatal mortality and morbidity. Healthcare professionals often find it challenging to counsel parents due to its rarity and diverse presentation. The aim of this systematic review and meta-analysis was to investigate the perinatal outcome of fetuses with ICH. MEDLINE, EMBASE, ClinicalTrials.gov and The Cochrane Library databases were searched. Inclusion criteria were studies reporting the outcome of fetuses, newborns and infants diagnosed with ICH. The primary outcome was perinatal death (PND), defined as the sum of intrauterine (IUD) and neonatal death (NND). The secondary outcomes were stillbirth, NND, IUD, termination of pregnancy, need for surgery/shunting at birth, cerebral palsy (defined according to the European Cerebral Palsy Network and classified as diplegia, hemiplegia, quadriplegia, dyskinetic or mixed), neurodevelopmental delay and intact survival. All outcomes were explored in the included fetuses with ICH. A subgroup analysis according to the location of the hemorrhage (intra-axial and extra-axial) was also planned. Meta-analysis of proportions was used to combine data, and pooled proportions and their 95% CI were reported. Sixteen studies (193 fetuses) were included in the meta-analysis. PND occurred in 14.6% (95% CI, 7.3-24.0%) of fetuses with ICH. Among liveborn cases, 27.6% (95% CI, 12.5-45.9%) required shunt placement or surgery after birth and 32.0% (95% CI, 22.2-42.6%) had cerebral palsy. Furthermore, 16.7% (95% CI, 8.4-27.2%) of cases had mild neurodevelopmental delay, while 31.1% (95% CI, 19.0-44.7%) experienced severe adverse neurodevelopmental outcome. Normal neurodevelopmental outcome was reported in 53.6% of fetuses. Subgroup analysis according to the location of ICH showed that PND occurred in 13.3% (95% CI, 5.7-23.4%) of fetuses with intra-axial bleeding and 26.7% (95% CI, 5.3-56.8%) of those with extra-axial bleeding. In fetuses with intra-axial hemorrhage, 25.2% (95% CI, 11.0-42.9%) required shunt placement or surgery after birth and 25.5% (95% CI, 15.3-37.2%) experienced cerebral palsy. In fetuses with intra-axial hemorrhage, mild and severe neurodevelopmental delay was observed in 14.9% (95% CI, 12.0-27.0%) and 32.8% (95% CI, 19.8-47.4%) of cases, respectively, while 53.2% (95% CI, 37.0-69.1%) experienced normal neurodevelopmental outcome. The incidence of mortality and postnatal neurodevelopmental outcome in fetuses with extra-axial hemorrhage could not be estimated reliably due to the small number of cases. Fetuses with a prenatal diagnosis of ICH are at high risk of perinatal mortality and adverse neurodevelopmental outcome. Postnatal shunt placement or surgery was required in 28% of cases and cerebral palsy was diagnosed in approximately one-third of infants. Due to the rarity of ICH, multicenter prospective registries are warranted to collect high-quality data. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.


Hyaluronan regulates cell behavior: a potential niche matrix for stem cells.
Hyaluronan is a linear glycosaminoglycan that has received special attention in the last few decades due to its extraordinary physiological functions. This highly viscous polysaccharide is not only a lubricator, but also a significant regulator of cellular behaviors during embryogenesis, morphogenesis, migration, proliferation, and drug resistance in many cell types, including stem cells. Most hyaluronan functions require binding to its cellular receptors CD44, LYVE-1, HARE, layilin, and RHAMM. After binding, proteins are recruited and messages are sent to alter cellular activities. When low concentrations of hyaluronan are applied to stem cells, the proliferative activity is enhanced. However, at high concentrations, stem cells acquire a dormant state and induce a multidrug resistance phenotype. Due to the influence of hyaluronan on cells and tissue morphogenesis, with regards to cardiogenesis, chondrogenesis, osteogenesis, and neurogenesis, it is now been utilized as a biomaterial for tissue regeneration. This paper summarizes the most important and recent findings regarding the regulation of hyaluronan in cells.


Time-dependent Degradation Pattern of Cardiac Troponin T in Cases of Death by Burn.
Death due to burn occurs frequently. This study investigated time-dependent alterations in cardiac troponin T (cTnT) associated with fatal burns. Cardiac tissue samples were collected from 10 medico-legal autopsies after informed consent from the relatives and post-mortem degradation by incubation of the cardiac tissue was studied at room temperature for different time periods. The cases included in this study were the subjects of burns without any prior history of disease who died in the hospital and their exact time of death was known. An efficient extraction protocol to analyse the banding pattern of cTnT in post-mortem tissue was developed. The data show a distinct time-dependent profile corresponding to the degradation of cTnT by proteases found in cardiac muscle. Both post-mortem interval and cardiac tissue of burned corpses had a statistically significant effect where the greatest amount of protein breakdown was observed within the first 41.20 hours, after which intact protein slowly disappears. The average molecular weight of all fragments showed intact cTnT to be rapidly degraded into smaller fragments. In cases of burns, such knowledge will assist in knowing if there were previous scars that might have mimicked a burn and also help to properly evaluate the real cause of death.


The sensory symptoms of diabetic polyneuropathy are improved with alpha-lipoic acid: the SYDNEY trial.
Because alpha-lipoic acid (ALA), a potent antioxidant, prevents or improves nerve conduction attributes, endoneurial blood flow, and nerve (Na(+) K(+) ATPase activity in experimental diabetes and in humans and may improve positive neuropathic sensory symptoms, in this report we further assess the safety and efficacy of ALA on the Total Symptom Score (TSS), a measure of positive neuropathic sensory symptoms. Metabolically stable diabetic patients with symptomatic (stage 2) diabetic sensorimotor polyneuropathy (DSPN) were randomized to a parallel, double-blind study of ALA (600 mg) (n = 60) or placebo (n = 60) infused daily intravenously for 5 days/week for 14 treatments. The primary end point was change of the sum score of daily assessments of severity and duration of TSS. Secondary end points were sum scores of neuropathy signs (NIS), symptoms (NSC), attributes of nerve conduction, quantitative sensation tests (QSTs), and an autonomic test. At randomization, the groups were not significantly different by the criteria of metabolic control or neuropathic end points. After 14 treatments, the TSS of the ALA group had improved from baseline by an average of 5.7 points and the placebo group by an average of 1.8 points (P < 0.001). Statistically significant improvement from baseline of the ALA, as compared with the placebo group, was also found for each item of the TSS (lancinating and burning pain, asleep numbness and prickling), NIS, one attribute of nerve conduction, and global assessment of efficacy. Intravenous racemic ALA, a potent antioxidant, rapidly and to a significant and meaningful degree, improved such positive neuropathic sensory symptoms as pain and several other neuropathic end points. This improvement of symptoms was attributed to improved nerve pathophysiology, not to increased nerve fiber degeneration. Because of its safety profile and its effect on positive neuropathic sensory symptoms and other neuropathic end points, this drug appears to be a useful ancillary treatment for the symptoms of diabetic polyneuropathy.


Sebaceous carcinoma of the skin of the breast: a case report.
Sebaceous gland tumours are rare and their presence should be considered as a marker for Muir-Torre Syndrome, alerting to search for an occult malignancy. A 43-year-old Caucasian female patient underwent excision of a sebaceous cyst. Histopathology confirmed a sebaceous carcinoma. Further investigations revealed multiple intra-abdominal malignancies. She has been under regular follow-up in the relevant clinics. Sebaceous carcinoma should be excised completely and followed-up for the detection of possible metastases. Surgical removal of primary or metastatic cancers may be curative and should be attempted wherever possible. It is very important for clinicians not to miss such skin lesions as they may precede the presentation of internal malignancies.


Longevity genes, cardiac ageing, and the pathogenesis of cardiomyopathy: implications for understanding the effects of current and future treatments for heart failure.
The two primary molecular regulators of lifespan are sirtuin-1 (SIRT1) and mammalian target of rapamycin complex 1 (mTORC1). Each plays a central role in two highly interconnected pathways that modulate the balance between cellular growth and survival. The activation of SIRT1 [along with peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α) and adenosine monophosphate-activated protein kinase (AMPK)] and the suppression of mTORC1 (along with its upstream regulator, Akt) act to prolong organismal longevity and retard cardiac ageing. Both activation of SIRT1/PGC-1α and inhibition of mTORC1 shifts the balance of cellular priorities so as to promote cardiomyocyte survival over growth, leading to cardioprotective effects in experimental models. These benefits may be related to direct actions to modulate oxidative stress, organellar function, proinflammatory pathways, and maladaptive hypertrophy. In addition, a primary shared benefit of both SIRT1/PGC-1α/AMPK activation and Akt/mTORC1 inhibition is the enhancement of autophagy, a lysosome-dependent degradative pathway, which clears the cytosol of dysfunctional organelles and misfolded proteins that drive the ageing process by increasing oxidative and endoplasmic reticulum stress. Autophagy underlies the ability of SIRT1/PGC-1α/AMPK activation and Akt/mTORC1 suppression to extend lifespan, mitigate cardiac ageing, alleviate cellular stress, and ameliorate the development and progression of cardiomyopathy; silencing of autophagy genes abolishes these benefits. Loss of SIRT1/PGC-1α/AMPK function or hyperactivation of Akt/mTORC1 is a consistent feature of experimental cardiomyopathy, and reversal of these abnormalities mitigates the development of heart failure. Interestingly, most treatments that have been shown to be clinically effective in the treatment of chronic heart failure with a reduced ejection fraction have been reported experimentally to exert favourable effects to activate SIRT1/PGC-1α/AMPK and/or suppress Akt/mTORC1, and thereby, to promote autophagic flux. Therefore, the impairment of autophagy resulting from derangements in longevity gene signalling is likely to represent a seminal event in the evolution and progression of cardiomyopathy.


The link between homocysteine, folic acid and vitamin B12 in chronic kidney disease.
Patients with chronic kidney disease or end-stage renal disease experience tremendous cardiovascular risk. Cardiovascular events are the leading causes of death in these patient populations, thus the interest in non-traditional risk factors such as hyperhomocysteinemia, folic acid and vitamin B12 metabolism is growing. Hyperhomocysteinemia is commonly found in CKD patients because of impaired renal metabolism and reduced renal excretion. Folic acid, the synthetic form of vitamin B9, is critical in the conversion of homocysteine to methionine like vitamin B12. Folic acid has also been shown to improve endothelial function without lowering homocysteine, suggesting an alternative explanation for the effect of folic acid on endothelial function. Whether hyperhomocysteinemia represents a reliable marker of cardiovascular risk and cardiovascular mortality or a therapeutic target in this population remains unclear. However, it is reasonable to consider folic acid with or without methylcobalamin supplementation as appropriate adjunctive therapy in patients with CKD. The purpose of this review is to summarize the characteristics of homocysteine, folic acid, and vitamin B12 metabolism, the mechanism of vascular damage, and the outcome of vitamin supplementation on hyperhomocysteinemia in patients with CKD, ESRD, dialysis treatment, and in kidney transplant recipients.


Cardiovascular risk stratification by coronary computed tomography angiography imaging: current state-of-the-art.
Current cardiovascular risk stratification by use of clinical risk score systems or plasma biomarkers is good but less than satisfactory in identifying patients at residual risk for coronary events. Recent clinical evidence puts now further emphasis on the role of coronary anatomy assessment by coronary computed tomography angiography (CCTA) for the management of patients with stable ischaemic heart disease. Available computed tomography (CT) technology allows the quantification of plaque burden, identification of high-risk plaques, or the functional assessment of coronary lesions for ischaemia detection and revascularization for refractory angina symptoms. The current CT armamentum is also further enhanced by perivascular Fat Attenuation Index (FAI), a non-invasive metric of coronary inflammation, which allows for the first time the direct quantification of the residual vascular inflammatory burden. Machine learning and radiomic features' extraction and spectral CT for tissue characterization are also expected to maximize the diagnostic and prognostic yield of CCTA. The combination of anatomical, functional, and biological information on coronary circulation by CCTA offers a unique toolkit for the risk stratification of patients, and patient selection for targeted aggressive prevention strategies. We hereby provide a review of the current state-of-the art in the field and discuss how integrating the full capacities of CCTA into clinical care pathways opens new opportunities for the tailored management of coronary artery disease.


Innovations in collaboration for the public's health through the Turning Point Initiative: the W.K. Kellogg Foundation perspective.
The need for a more integrated public health system led the W. K. Kellogg Foundation (WKKF) to establish Turning Point (TP): Collaborating for a New Century in Public Health, with a goal of transforming and strengthening the current public health infrastructure. WKKF partners with the Robert Wood Johnson Foundation (RWJF), the National Association of County and City Health Officials, the University of Washington, the Lewin Group, and the New York Academy of Medicine to support activities of 41 community and tribal jurisdiction grantee partnerships in 14 states. Preliminary evaluation of partnerships participation indicate: (1) most have accomplished breadth; (2) many did not achieve depth prior to the end of initial recruitment and (3) when the mission was internalized, penetration occurred. Strengthening public health systems requires redevelopment of human, organizational, financial, educational and information resources, and as in the past, WKKF will work to this end.


Automated quantification of coronary plaque with computed tomography: comparison with intravascular ultrasound using a dedicated registration algorithm for fusion-based quantification.
Previous studies have used semi-automated approaches for coronary plaque quantification on multi-detector row computed tomography (CT), while an automated quantitative approach using a dedicated registration algorithm is currently lacking. Accordingly, the study aimed to demonstrate the feasibility and accuracy of automated coronary plaque quantification on cardiac CT using dedicated software with a novel 3D coregistration algorithm of CT and intravascular ultrasound (IVUS) data sets. Patients who had undergone CT and IVUS were enrolled. Automated lumen and vessel wall contour detection was performed for both imaging modalities. Dedicated automated quantitative software (QCT) with a unique registration algorithm was used to fuse a complete IVUS run with a CT angiography volume using true anatomical markers. At the level of the minimal lumen area (MLA), percentage lumen area stenosis, plaque burden, and degree of remodelling were obtained on CT. Additionally, mean plaque burden was assessed for the whole coronary plaque. At the identical level within the coronary artery, the same variables were derived from IVUS. Fifty-one patients (40 men, 58 ± 11 years, 103 coronary arteries) with 146 lesions were evaluated. Quantitative computed tomography and IVUS showed good correlation for MLA (n = 146, r = 0.75, P < 0.001). At the level of the MLA, both techniques were well-correlated for lumen area stenosis (n = 146, r = 0.79, P < 0.001) and plaque burden (n = 146, r = 0.70, P < 0.001). Mean plaque burden (n = 146, r = 0.64, P < 0.001) and remodelling index (n = 146, r = 0.56, P < 0.001) showed significant correlations between QCT and IVUS. Automated quantification of coronary plaque on CT is feasible using dedicated quantitative software with a novel 3D registration algorithm.


Epidemiological review of gastric cancer in India.
Stomach cancer is the one of the leading cause of cancer in southern region of India. Its incidence is decreasing worldwide yet on global scale stomach cancer remains one of the most common causes of cancer death. Etiology of gastric cancer includes Helicobacter pylori infection, diet and lifestyle, tobacco, alcohol and genetic susceptibility. In this review, we tried to find the contribution of Indian scientist in understanding the descriptive and observational epidemiology of stomach cancer. PubMed was used as a search platform using key words such as "stomach cancer, treatment, clinical characteristics, stomach cancer outcome, epidemiology, etiological factor and their corresponding Mesh terms were used in combination with Boolean operators OR, AND". Most of the reported studies on gastric cancer from India are case report or case series and few are case-control studies. Indian studies on this topic are limited and have observed H. pylori infection, salted tea, pickled food, rice intake, spicy food, soda (additive of food), tobacco and alcohol as risk factors for gastric cancer. More research is required to understand the etiology, develop suitable screening test, to demarcate high-risk population and to develop and evaluate the effect of primary prevention programs.


Improving Recognition and Reporting of Adverse Drug Reactions in the NICU: A Quality Improvement Project.
Adverse drug reactions (ADRs) are under-recognized and under-reported in the Neonatal Intensive Care Unit (NICU) population, with up to 95% of all ADRs not reported. Compared with non-elderly adults, pediatric patients are 3 times more likely to experience an ADR, with varying rates from 0.6% to 16.8%. The Children's Mercy NICU has an ADR rate of 0.29% (2015). This high rate presents an opportunity to increase recognition and reporting, and improve characterization of ADRs in the NICU. The primary aim of this quality improvement project was for 70% of patients who received specified medications (indomethacin, dexmedetomidine, fentanyl, lorazepam, dexamethasone, or hydrocortisone) in the first 3 months of age to be assessed daily for ADRs. We selected these medications due to the frequency of use and well-understood ADR associations. For each ADR recognized, the Naranjo score, was calculated and compared with the neonatal-specific Du score to assess the effectiveness of ADR characterization. Implementation occurred on May 15, 2017. We completed 3 PDSA cycles over 1 year. The bedside monitoring tool was utilized 83% of the time. Twenty-eight potential ADRs were identified, far exceeding the number reported before implementation. The Du score appeared to better characterize ADRs compared with the Naranjo score. Use of a bedside monitoring tool improves ADR detection. We experienced challenges with consistently identifying patients on target drugs and getting the tool to the bedside. Application of the Du score for ADR classification in neonates appears to be more appropriate than the use of the Naranjo.


What Should I Eat and Why? The Environmental, Genetic, and Behavioral Determinants of Food Choice: Summary from a Pennington Scientific Symposium.
This review details the proceedings of a Pennington Biomedical scientific symposium titled, "What Should I Eat and Why? The Environmental, Genetic, and Behavioral Determinants of Food Choice." The symposium was designed to review the literature about energy homeostasis, particularly related to food choice and feeding behaviors, from psychology to physiology. This review discusses the intrinsic determinants of food choice, including biological mechanisms (genetics), peripheral and central signals, brain correlates, and the potential role of the microbiome. This review also address the extrinsic determinants (environment) of food choice within our physical and social environments. Finally, this review reports the current treatment practices for the clinical management of eating-induced overweight and obesity. An improved understanding of these determinants will inform best practices for the clinical treatment and prevention of obesity. Strategies paired with systemic shifts in our public health policies and changes in our "obesogenic" environment will be most effective at attenuating the obesity epidemic.


Changes in physical symptoms during the menopause transition.
This article analyzes physical symptoms experienced by mid-age Australian women in different stages of the menopause transition. A total of 8,623 women, aged 45 to 50 years in 1996, who participated the mid-age cohort of the Australian Longitudinal Study on Women's Health, completed Survey 1 in 1996 and Survey 2 in 1998. Women were assigned to 1 of 6 menopause groups according to their menopausal status at Surveys 1 and 2, and compared on symptoms experienced at Surveys 1 and 2, adjusted for lifestyle, behavioral and demographic factors. At Survey 1, the most commonly reported symptoms were headaches, back pain, stiff joints, tiredness, and difficulty sleeping. Perimenopausal women were more likely than premenopausal or postmenopausal women to report these symptoms. Hot flushes and night sweats were more common among postmenopausal women. Compared with those who remained premenopausal, women who were in the early stages of menopause or perimenopausal were more likely to report tiredness, stiff joints, difficulty sleeping, and hot flushes at Survey 2. Women who remained perimenopausal were also more likely to report back pain and leaking urine. Compared with premenopausal women, odds ratios for night sweats increased for women in consecutive stages of the menopause transition and remained high in the postmenopausal women.


Rating the strength of scientific evidence: relevance for quality improvement programs.
To summarize an extensive review of systems for grading the quality of research articles and rating the strength of bodies of evidence, and to highlight for health professionals and decision-makers concerned with quality measurement and improvement the available "best practices" tools by which these steps can be accomplished. Drawing on an extensive review of checklists, questionnaires, and other tools in the field of evidence-based practice, this paper discusses clinical, management, and policy rationales for rating strength of evidence in a quality improvement context, and documents best practices methods for these tasks. After review of 121 systems for grading the quality of articles, 19 systems, mostly specific, met a priori scientific standards for grading systematic reviews, randomized controlled trials, observational studies, and diagnostic tests; eight systems (of 40 reviewed) met similar standards for rating the overall strength of evidence. All can be used as is or adapted for particular types of evidence reports or systematic reviews. Formally grading study quality and rating overall strength of evidence, using sound instruments and procedures, can produce reasonable levels of confidence about the science base for parts of quality improvement programs. With such information, health care professionals and administrators concerned with quality improvement can understand better the level of science (versus only clinical consensus or opinion) that supports practice guidelines, review criteria, and assessments that feed into quality assurance and improvement programs. New systems are appearing and research is needed to confirm the conceptual and practical underpinnings of these grading and rating systems, but the need for those developing systematic reviews, practice guidelines, and quality or audit criteria to understand and undertake these steps is becoming increasingly clear.


Genetically dictated change in host mucus carbohydrate landscape exerts a diet-dependent effect on the gut microbiota.
We investigate how host mucus glycan composition interacts with dietary carbohydrate content to influence the composition and expressed functions of a human gut community. The humanized gnotobiotic mice mimic humans with a nonsecretor phenotype due to knockout of their α1-2 fucosyltransferase (Fut2) gene. The fecal microbiota of Fut2(-) mice that lack fucosylated host glycans show decreased alpha diversity relative to Fut2(+) mice and exhibit significant differences in community composition. A glucose-rich plant polysaccharide-deficient (PD) diet exerted a strong effect on the microbiota membership but eliminated the effect of Fut2 genotype. Additionally fecal metabolites predicted host genotype in mice on a polysaccharide-rich standard diet but not on a PD diet. A more detailed mechanistic analysis of these interactions involved colonization of gnotobiotic Fut2(+) and Fut2(-) mice with Bacteroides thetaiotaomicron, a prominent member of the human gut microbiota known to adaptively forage host mucosal glycans when dietary polysaccharides are absent. Within Fut2(-) mice, the B. thetaiotaomicron fucose catabolic pathway was markedly down-regulated, whereas BT4241-4247, an operon responsive to terminal β-galactose, the precursor that accumulates in the Fut2(-) mice, was significantly up-regulated. These changes in B. thetaiotaomicron gene expression were only evident in mice fed a PD diet, wherein B. thetaiotaomicron relies on host mucus consumption. Furthermore, up-regulation of the BT4241-4247 operon was also seen in humanized Fut2(-) mice. Together, these data demonstrate that differences in host genotype that affect the carbohydrate landscape of the distal gut interact with diet to alter the composition and function of resident microbes in a diet-dependent manner.


How Athila retrotransposons survive in the Arabidopsis genome.
Transposable elements are selfish genetic sequences which only occasionally provide useful functions to their host species. In addition, models of mobile element evolution assume a second type of selfishness: elements of different families do not cooperate, but they independently fight for their survival in the host genome. We show that recombination events among distantly related Athila retrotransposons have led to the generation of new Athila lineages. Their pattern of diversification suggests that Athila elements survive in Arabidopsis by a combination of selfish replication and of amplification of highly diverged copies with coding potential. Many Athila elements are non-autonomous but still conserve intact open reading frames which are under the effect of negative, purifying natural selection. The evolution of these mobile elements is far more complex than hitherto assumed. Strict selfish replication does not explain all the patterns observed.


The pea aphid uses a version of the terminal system during oviparous, but not viviparous, development.
In most species of aphid, female nymphs develop into either sexual or asexual adults depending on the length of the photoperiod to which their mothers were exposed. The progeny of these sexual and asexual females, in turn, develop in dramatically different ways. The fertilized oocytes of sexual females begin embryogenesis after being deposited on leaves (oviparous development) while the oocytes of asexual females complete embryogenesis within the mother (viviparous development). Compared with oviparous development, viviparous development involves a smaller transient oocyte surrounded by fewer somatic epithelial cells and a smaller early embryo that comprises fewer cells. To investigate whether patterning mechanisms differ between the earliest stages of the oviparous and viviparous modes of pea aphid development, we examined the expression of pea aphid orthologs of genes known to specify embryonic termini in other insects. Here we show that pea aphid oviparous ovaries express torso-like in somatic posterior follicle cells and activate ERK MAP kinase at the posterior of the oocyte. In addition to suggesting that some posterior features of the terminal system are evolutionarily conserved, our detection of activated ERK in the oocyte, rather than in the embryo, suggests that pea aphids may transduce the terminal signal using a mechanism distinct from the one used in Drosophila. In contrast with oviparous development, the pea aphid version of the terminal system does not appear to be used during viviparous development, since we did not detect expression of torso-like in the somatic epithelial cells that surround either the oocyte or the blastoderm embryo and we did not observe restricted activated ERK in the oocyte. We suggest that while oviparous oocytes and embryos may specify posterior fate through an aphid terminal system, viviparous oocytes and embryos employ a different mechanism, perhaps one that does not rely on an interaction between the oocyte and surrounding somatic cells. Together, these observations provide a striking example of a difference in the fundamental events of early development that is both environmentally induced and encoded by the same genome.


Direct comparison of shot-to-shot noise performance of all normal dispersion and anomalous dispersion supercontinuum pumped with sub-picosecond pulse fiber-based laser.
Coherence of supercontinuum sources is critical for applications involving characterization of ultrafast or rarely occurring phenomena. With the demonstrated spectral coverage of supercontinuum extending from near-infrared to over 10 μm in a single nonlinear fiber, there has been a clear push for the bandwidth rather than for attempting to optimize the dynamic properties of the generated spectrum. In this work we provide an experimental assessment of the shot-to-shot noise performance of supercontinuum generation in two types of soft glass photonic crystal fibers. Phase coherence and intensity fluctuations are compared for the cases of an anomalous dispersion-pumped fiber and an all-normal dispersion fiber. With the use of the dispersive Fourier transformation method, we demonstrate that a factor of 100 improvement in signal-to-noise ratio is achieved in the normal-dispersion over anomalous dispersion-pumped fiber for 390 fs long pump pulses. A double-clad design of the photonic lattice of the fiber is further postulated to enable a pump-related seeding mechanism of normal-dispersion supercontinuum broadening under sub-picosecond pumping, which is otherwise known for similar noise characteristics as modulation instability driven, soliton-based spectra.


Retrospective estimation of exposure to benzene in a leukaemia case-control study of petroleum marketing and distribution workers in the United Kingdom.
To provide quantitative estimates of exposure to benzene for cases and controls in an epidemiology study to investigate the risk of leukaemia in petroleum distribution workers. Work histories were obtained for cases and controls together with detailed information on the distribution sites. For each job in the work history, an estimate of exposure (parts per million (ppm)) was obtained by multiplying a measure derived from exposure data by modifying factors to reflect the differences between the conditions that existed at the time of measurement and those at the time of interest. The modifying factors used related to job activity, the number of road tankers loaded, the benzene content of the gasoline, the mixture of products handled, temperature, and loading technology. Cumulative exposures for each case and control were obtained by multiplying the exposure estimates for each job by the duration of time in the respective jobs, and summing these over all jobs in the work history. Peak exposure and exposure through dermal contact were quantitatively classified for each job. Measured exposures were obtained for 30 job categories, and ranged from 0.003 to 8.20 ppm. 40% of work histories were assigned background exposures, with a further 34% assigned the exposure estimate for a driver carrying out top submerged loading of motor fuel into road tankers. Cumulative exposures ranged from < 1 to > 200 ppm-years, although 81% were < 5 ppm-years. Comparison of the exposure estimates for selected jobs with data from sources not used in the study showed similar results. The estimates of exposure to benzene in this study provide a sound basis for the epidemiological analyses.


Function and X-ray crystal structure of Escherichia coli YfdE.
Many food plants accumulate oxalate, which humans absorb but do not metabolize, leading to the formation of urinary stones. The commensal bacterium Oxalobacter formigenes consumes oxalate by converting it to oxalyl-CoA, which is decarboxylated by oxalyl-CoA decarboxylase (OXC). OXC and the class III CoA-transferase formyl-CoA:oxalate CoA-transferase (FCOCT) are widespread among bacteria, including many that have no apparent ability to degrade or to resist external oxalate. The EvgA acid response regulator activates transcription of the Escherichia coli yfdXWUVE operon encoding YfdW (FCOCT), YfdU (OXC), and YfdE, a class III CoA-transferase that is ~30% identical to YfdW. YfdW and YfdU are necessary and sufficient for oxalate-induced protection against a subsequent acid challenge; neither of the other genes has a known function. We report the purification, in vitro characterization, 2.1-Å crystal structure, and functional assignment of YfdE. YfdE and UctC, an orthologue from the obligate aerobe Acetobacter aceti, perform the reversible conversion of acetyl-CoA and oxalate to oxalyl-CoA and acetate. The annotation of YfdE as acetyl-CoA:oxalate CoA-transferase (ACOCT) expands the scope of metabolic pathways linked to oxalate catabolism and the oxalate-induced acid tolerance response. FCOCT and ACOCT active sites contain distinctive, conserved active site loops (the glycine-rich loop and the GNxH loop, respectively) that appear to encode substrate specificity.


Geographic and ecologic heterogeneity in elimination thresholds for the major vector-borne helminthic disease, lymphatic filariasis.
Large-scale intervention programmes to control or eliminate several infectious diseases are currently underway worldwide. However, a major unresolved question remains: what are reasonable stopping points for these programmes? Recent theoretical work has highlighted how the ecological complexity and heterogeneity inherent in the transmission dynamics of macroparasites can result in elimination thresholds that vary between local communities. Here, we examine the empirical evidence for this hypothesis and its implications for the global elimination of the major macroparasitic disease, lymphatic filariasis, by applying a novel Bayesian computer simulation procedure to fit a dynamic model of the transmission of this parasitic disease to field data from nine villages with different ecological and geographical characteristics. Baseline lymphatic filariasis microfilarial age-prevalence data from three geographically distinct endemic regions, across which the major vector populations implicated in parasite transmission also differed, were used to fit and calibrate the relevant vector-specific filariasis transmission models. Ensembles of parasite elimination thresholds, generated using the Bayesian fitting procedure, were then examined in order to evaluate site-specific heterogeneity in the values of these thresholds and investigate the ecological factors that may underlie such variability We show that parameters of density-dependent functions relating to immunity, parasite establishment, as well as parasite aggregation, varied significantly between the nine different settings, contributing to locally varying filarial elimination thresholds. Parasite elimination thresholds predicted for the settings in which the mosquito vector is anopheline were, however, found to be higher than those in which the mosquito is culicine, substantiating our previous theoretical findings. The results also indicate that the probability that the parasite will be eliminated following six rounds of Mass Drug Administration with diethylcarbamazine and albendazole decreases markedly but non-linearly as the annual biting rate and parasite reproduction number increases. This paper shows that specific ecological conditions in a community can lead to significant local differences in population dynamics and, consequently, elimination threshold estimates for lymphatic filariasis. These findings, and the difficulty of measuring the key local parameters (infection aggregation and acquired immunity) governing differences in transmission thresholds between communities, mean that it is necessary for us to rethink the utility of the current anticipatory approaches for achieving the elimination of filariasis both locally and globally.


Near fatal ingestion of oil of cloves.
A case of ingestion of oil of cloves is presented, which resulted in coma, fits, a coagulopathy, and acute liver damage. This is not unlike the syndrome produced in the late stages of a substantial paracetamol overdose, and a similar treatment regimen is proposed.


Temporal embryonic expression of the sea urchin early H1 gene is controlled by sequences immediately upstream and downstream of the TATA element.
The sea urchin early histone H1 gene is expressed only during a very short interval in the life cycle of the organism. In addition to a pool of stored maternal transcripts, the gene is active from the 16-cell stage to the early blastula stage of development, at which time the gene is transcriptionally repressed. We have defined the minimal sequences required for the proper temporal expression of this gene during early embryogenesis. These sequences from -65 to +39 include the TATA element, a conserved sequence CCCACGTACGCAA (part of USE 0) just upstream of the TATA element, an Inr element at the transcription start site, and an internal sequence in the leader region of the H1 transcript. Surprisingly, we could find no role for two highly conserved elements, a GC box (USE I) and an H1-specific element (USE II), that are important in the expression of vertebrate H1 genes. The internal element functions as a positive regulator when three copies are ectopically placed upstream of an early histone H3 TATA box. A temporal regulator is localized within the USE 0 sequence CCCACGTACGCAA, and it apparently plays a role in activation and probably repression of the gene.


Self-supervised recurrent depth estimation with attention mechanisms.
Depth estimation has been an essential task for many computer vision applications, especially in autonomous driving, where safety is paramount. Depth can be estimated not only with traditional supervised learning but also via a self-supervised approach that relies on camera motion and does not require ground truth depth maps. Recently, major improvements have been introduced to make self-supervised depth prediction more precise. However, most existing approaches still focus on single-frame depth estimation, even in the self-supervised setting. Since most methods can operate with frame sequences, we believe that the quality of current models can be significantly improved with the help of information about previous frames. In this work, we study different ways of integrating recurrent blocks and attention mechanisms into a common self-supervised depth estimation pipeline. We propose a set of modifications that utilize temporal information from previous frames and provide new neural network architectures for monocular depth estimation in a self-supervised manner. Our experiments on the KITTI dataset show that proposed modifications can be an effective tool for exploiting temporal information in a depth prediction pipeline.


[Intensive care medicine on medical undergraduation: student's perspective].
There are deficiencies on Intensive Medicine (IM) teaching in most of medical undergraduate schools. Those deficiencies may imply damages on their clinical competence. The objective of this study was to analyze current status of IM teaching and the medical undergraduate student interest in this speciality. A cross-sectional study was performed in 2005. We applied a self-reported questionnaire to enrolled students between the sixth and the last semesters of two medical schools from Salvador-Bahia. The questionnaire contained questions about students' interest and knowledge on IM, and opinion on IM teaching in their schools. We studied 570 students. Most of them (57.5%) had never realized a clerkship in intensive care unit (ICU) despite classifying its usefulness as high (mean of 4.14 ± 1.05, in a scale from 1 to 5). IM interest was high or very high in 53.7% of sample. Almost all students (97%) thought that IM topics should be more explored at their curriculum. Only 42.1% reported to be able to assess a critical care patient and this assurance was higher among students with previous clerkship in ICU (p < 0.001). Shock, cardiopulmonary resuscitation and sepsis were the most interesting topics in ICU for students' opinion. This study revealed a high interest in IM among medical undergraduate students. However, most had never practice a clerkship in ICU, demonstrating to be an important factor on undergraduate student performance faced to a critical care patient.


Associations between night work and BMI, alcohol, smoking, caffeine and exercise--a cross-sectional study.
Shift work is associated with negative health effects. Increased prevalence of several cardiovascular risk factors among shift workers/night workers compared with day workers have been shown resulting in increased risk of cardiovascular events among shift workers and night workers. Previous studies have taken a dichotomous approach to the comparison between day and night workers. The present study uses a continuous approach and provides such a new perspective to the negative effects of night work load as a possible risk factor for undesirable health effects. This cross sectional study (The SUrvey of Shift work, Sleep and Health (SUSSH)) uses data collected from December 2008 to March 2009. The study population consists of Norwegian nurses. The study collected information about demographic and lifestyle factors: Body Mass Index (BMI), smoking habits, alcohol consumption, caffeine consumption and exercise habits. The lifestyle parameters were evaluated using multiple hierarchical regression and binary logistic regression. Number of night shifts worked last year (NNL) was used as operationalization of night work load. Adjustment for possible confounders were made. Obesity was defined as BMI > 30. Alcohol Consumption was evaluated using the short form of the Alcohol Use Disorders Identification Test Consumption (AUDIT-C). Data were analyzed using SPSS version 22. We had data from 2059 nurses. NNL was significantly and positively associated with BMI, both when evaluated against BMI as a continuous parameter (Beta = .055, p < .05), and against obesity (OR = 1.01, 95 % CI = 1.00-1.01). The AUDIT-C score was significantly and positively associated with hours worked per week (OR = 1.03, 95 % CI = 1.01-1.05). We found a positive significant association between night work load and BMI. This suggests that workers with a heavy night work load might need special attention and frequent health checks due to higher risk of undesirable health effects.


Control of graft-versus-host disease and graft rejection by T cell depletion of donor and recipient with Campath-1 antibodies. Results of matched sibling transplants for malignant diseases.
Campath-1 (CDw52) antibodies (IgM and IgG2b) have been used in vitro and in vivo for control of GVHD and prevention of rejection following bone marrow transplantation. Results of 951 patients with malignant disease transplanted from HLA-matched siblings are reported. Both Campath-1M and Campath-1G are shown to be effective when used in vitro for prevention of graft-versus-host disease (GVHD). Graft failure was reduced by addition of cyclosporin A (CsA) post-transplant and possibly also by total lymphoid irradiation (TLI) pre-transplant. However, treatment of the recipient with Campath-1G to deplete residual lymphocytes was more effective, reducing the incidence of graft failure from 21% to 9% (in the absence of CsA). GVHD was virtually eliminated and leukaemia-free survival was improved. However, the risk of relapse was increased by T cell depletion, certainly in CML and to a lesser extent in AML. Addition of donor T cells to the depleted bone marrow or early post-transplant restored the risks of GVHD, graft failure and relapse to much the same as without T cell depletion. One problem associated with the use of Campath-1G in vivo was a significant delay (by up to 7 days) in neutrophil engraftment. This was unlikely to be caused by toxicity to progenitor cells and we argue that small numbers of lymphocytes may be required to assist early engraftment, possibly by cytokine production. If this problem can be overcome, T cell depletion of donor and recipient may be a good alternative to conventional GVHD prophylaxis for matched sibling transplants, resulting in a superior quality of life for the survivors. It is also likely to be particularly beneficial in transplants for non-malignant diseases and transplants from unrelated donors.


Diversity of methanogenic archaea in freshwater sediments of lacustrine ecosystems.
About half of the global methane (CH4 ) emission is contributed by the methanogenic archaeal communities leading to a significant increase in global warming. This unprecedented situation has increased the ever growing necessity of evaluating the control measures for limiting CH4 emission to the atmosphere. Unfortunately, research endeavors on the diversity and functional interactions of methanogens are not extensive till date. We anticipate that the study of the diversity of methanogenic community is paramount for understanding the metabolic processes in freshwater lake ecosystems. Although there are several disadvantages of conventional culture-based methods for determining the diversity of methanogenic archaeal communities, in order to understand their ecological roles in natural environments it is required to culture the microbes. Recently different molecular techniques have been developed for determining the structure of methanogenic archaeal communities thriving in freshwater lake ecosystem. The two gene based cloning techniques required for this purpose are 16S rRNA and methyl coenzyme M reductase (mcrA) in addition to the recently developed metagenomics approaches and high throughput next generation sequencing efforts. This review discusses the various methods of culture-dependent and -independent measures of determining the diversity of methanogen communities in lake sediments in lieu of the different molecular approaches and inter-relationships of diversity of methanogenic archaea.


Financial Burden for Patients With Chronic Myeloid Leukemia Enrolled in Medicare Part D Taking Targeted Oral Anticancer Medications.
The number of targeted oral anticancer medications (TOAMs) has grown rapidly in the past decade. The high cost of TOAMs raises concerns about the financial aspect of treatment, especially for patients enrolled in Medicare Part D plans because of the coverage gap. We identified patients with chronic myeloid leukemia (CML) who were new TOAM users from the SEER registry data linked with Medicare Part D data, from years 2007 to 2012. We followed these patients throughout the calendar year when they started taking the TOAMs and examined their out-of-pocket (OOP) payments and gross drug costs, taking into account their benefit phase, plan type, and cost share group. We found that 726 (81%) of the 898 patients with CML who received TOAMs had reached the catastrophic phase of their Medicare Part D benefit within the year of medication initiation, with a large majority of patients reaching this phase in less than a month. Patients without subsidies showed a clear pattern of a spike in OOP payments when they began treatment with TOAMs. The OOP payment for patients with subsidies was substantially lower. The monthly gross drug costs were similar between patients with and without subsidies. Patients experience quick entry and exit from the coverage gap (also called the donut hole) as a result of the high price of TOAMs. Closing the donut hole will provide financial relief during the initial month(s) of treatment but will not completely eliminate the financial burden.


Past and present variability in leaf length of evergreen members of Nothofagus subgenus Lophozonia related to ecology and population dynamics.
The lengths of fossil leaves of Nothofogus cunninghamii, and of closely related fossil species from sediments older than the Middle Pleistocene showed greater variation than fossil leaves from younger sediments and extant leaves from modern sediment samples and forest floor litter. Leaf sizes of samples of the other two extant evergreen species of Nothofagus subgenus Lophazonia, N. moorei and N.menziesii, were no more variable than modern N.cunninhamii. Leaf lengths of modern and Middle-Late Pleistocene N. cunninhamii and of modern N. menziesii were more or less unimodal in distribution, whereas the leaves of N. moorei follow bimodal distributions, and leaves from the Early Pleistocene Regatta Point and the Oligocene Link- Rapid River sediments tended to be bimodal and highly variable. Leaf lengths from the Oligo-Miocene Monpeelyata sediments were also highly variable, but the sample size was too small to determine if they follow bimodal distributions. The bimodality and high variability in the older sites are unlikely to have been due to taphonomic processes. Thus, within population variability appears to have declined during the Late Tertiary-Early Pleistocene, although the presence of cryptic species in the fossil record could also explain the results. Both a decline in variability within species, and the extinction of Nothofugus species, are consistent with a well documented Late Cainozoic decline in rainforest diversity in Tasmania. Log transformed leaf lengths of modern forest floor litter samples of N. cunninhamii were Strongly correlated with climatic parameters, particularly summer temperature, suggesting that this parameter is a strong determinant of leaf size in N. cunninhamii Changes in N. cunninhamii leaf lengths within Middle Pleistocene sediments were associated with floristic changes interpreted as transitions in glacial/interglacial cycles but these changes were small compared with changes predicted Iron climatic correlation of extant samples, probably because of limited Emetic variability in local populations at the time of deposition.


Modelling of human Wiskott-Aldrich syndrome protein mutants in zebrafish larvae using in vivo live imaging.
Wiskott-Aldrich syndrome (WAS) and X-linked neutropenia (XLN) are immunodeficiencies in which the function of several haematopoietic cell lineages is perturbed as a result of mutations in the actin regulator WASp. From in vitro cell biology experiments, and biochemical and structural approaches, we know much about the functional domains of WASp and how WASp might regulate the dynamic actin cytoskeleton downstream of activators such as Cdc42, but in vivo experiments are much more challenging. In patients, there is a correlation between clinical disease and genotype, with severe reductions in WASp expression or function associating with complex multilineage immunodeficiency, whereas specific mutations that cause constitutive activation of WASp result in congenital neutropenia. Here, we take advantage of the genetic tractability and translucency of zebrafish larvae to first characterise how a null mutant in zfWASp influences the behaviour of neutrophils and macrophages in response to tissue damage and to clearance of infections. We then use this mutant background to study how leukocyte lineage-specific transgenic replacement with human WASp variants (including normal wild type and point mutations that either fail to bind Cdc42 or cannot be phosphorylated, and a constitutively active mutant equivalent to that seen in XLN patients) alter the capacity for generation of neutrophils, their chemotactic response to wounds and the phagocytic clearance capacity of macrophages. This model provides a unique insight into WASp-related immunodeficiency at both a cellular and whole organism level.


Medium and long-term risks of specific cardiovascular diseases in survivors of 20 adult cancers: a population-based cohort study using multiple linked UK electronic health records databases.
The past few decades have seen substantial improvements in cancer survival, but concerns exist about long-term cardiovascular disease risk in survivors. Evidence is scarce on the risks of specific cardiovascular diseases in survivors of a wide range of cancers to inform prevention and management. In this study, we used large-scale electronic health records data from multiple linked UK databases to address these evidence gaps. For this population-based cohort study, we used linked primary care, hospital, and cancer registry data from the UK Clinical Practice Research Datalink to identify cohorts of survivors of the 20 most common cancers who were 18 years or older and alive 12 months after diagnosis and controls without history of cancer, matched for age, sex, and general practice. We compared risks for a range of cardiovascular disease outcomes using crude and adjusted Cox models. We fitted interactions to investigate effect modification, and flexible parametric survival models to estimate absolute excess risks over time. Between Jan 1, 1990, and Dec 31, 2015, 126 120 individuals with a diagnosis of a cancer of interest still being followed up at least 1 year later were identified and matched to 630 144 controls. After exclusions, 108 215 cancer survivors and 523 541 controls were included in the main analyses. Venous thromboembolism risk was elevated in survivors of 18 of 20 site-specific cancers compared with that of controls; adjusted hazard ratios (HRs) ranged from 1·72 (95% CI 1·57-1·89) in patients after prostate cancer to 9·72 (5·50-17·18) after pancreatic cancer. HRs decreased over time, but remained elevated more than 5 years after diagnosis. We observed increased risks of heart failure or cardiomyopathy in patients after ten of 20 cancers, including haematological (adjusted HR 1·94, 1·66-2·25, with non-Hodgkin lymphoma; 1·77, 1·50-2·09, with leukaemia; and 3·29, 2·59-4·18, with multiple myeloma), oesophageal (1·96, 1·46-2·64), lung (1·82, 1·52-2·17) kidney (1·73, 1·38-2·17) and ovarian (1·59, 1·19-2·12). Elevated risks of arrhythmia, pericarditis, coronary artery disease, stroke, and valvular heart disease were also observed for multiple cancers, including haematological malignancies. HRs for heart failure or cardiomyopathy and venous thromboembolism were greater in patients without previous cardiovascular disease and in younger patients. However, absolute excess risks were generally greater with increasing age. Increased risks of these outcomes seemed most pronounced in patients who had received chemotherapy. Survivors of most site-specific cancers had increased medium-term to long-term risk for one or more cardiovascular diseases compared with that for the general population, with substantial variations between cancer sites. Wellcome Trust and Royal Society.


Social impact of HIV/AIDS on clients attending a teaching hospital in Southern Nigeria.
People living with human immunodeficiency virus and acquired immune deficiency syndrome (PLWHA) face numerous social challenges. The objectives of this study were to assess the level of self-disclosure of status by PLWHA, to describe the level and patterns of stigma and discrimination, if any, experienced by the PLWHA and to assess the effect of sero-positivity on the attitude of friends, family members, health workers, colleagues and community. This was a cross-sectional descriptive study carried out among PLWHA attending the University of Uyo Teaching Hospital, Uyo, Southern Nigeria. Information was obtained using an interviewer-administered semi-structured questionnaire, which was analysed using the Epi 6 software. A total of 331 respondents were interviewed. A majority, 256 (77.3%), of the respondents were within the age range of 25-44 years. A total of 121 (36.6%) PLWHA were single and 151 (46.6%) were married, while the rest were widowed, divorced or separated. A majority, 129 (85.4%), of the married respondents disclosed their status to their spouses and 65 (50.4%) were supportive. Apart from spouses, disclosure to mothers (39.9%) was highest. Most clients (57.7%) did not disclose their status to people outside their immediate families for fear of stigmatization. Up to 111 (80.4%) of the respondents working for others did not disclose their status to their employers. Among those whose status was known, discrimination was reported to be highest among friends (23.2%) and at the workplace (20.2%). Attitudes such as hostility (14.5%), withdrawal (11.7%) and neglect (6.8%) were reported from the private hospitals. Apart from disclosure to spouses, the level of disclosure to others was very low. Those whose status was known mainly received acceptance from their families but faced discriminatory attitudes such as hostility, neglect and withdrawal from friends, colleagues and hospital workers. There is a need for more enlightenment campaigns on HIV/AIDS by stakeholders to reduce stigma and discrimination and ensure adequate integration of PLWHA into the society.


Creating "living" polymer surfaces to pattern biomolecules and cells on common plastics.
Creating patterns of biomolecules and cells has been applied widely in many fields associated with the life sciences, including diagnostics. In these applications it has become increasingly apparent that the spatiotemporal arrangement of biological molecules in vitro is important for the investigation of the cellular functions found in vivo. However, the cell patterning techniques often used are limited to creating 2D functional surfaces on glass and silicon. In addition, in general, these procedures are not easy to implement in conventional biological laboratories. Here, we show the formation of a living poly(ethylene glycol) (PEG) layer that can be patterned with visible light on plastic surfaces. This new and simple method can be expanded to pattern multiple types of biomolecule on either a previously formed PEG layer or a plastic substrate. Using common plastic wares (i.e., polyethylene films and polystyrene cell culture Petri-dishes), we demonstrate that these PEG-modified surfaces have a high resistance to protein adsorption and cell adhesion, while at the same time, being capable of undergoing further molecular grafting with bioactive motifs. With a photomask and a fluid delivery system, we illustrate a flexible way to immobilize biological functions with a high degree of 2D and 3D spatial control. We anticipate that our method can be easily implemented in a typical life science laboratory (without the need for specialized lithography equipment) offering the prospect of imparting desirable properties to plastic products, for example, the creation of functional microenvironments in biological studies or reducing biological adhesion to surfaces.


Cardiovascular Disease Prevention in Rheumatoid Arthritis: Compliance with Diabetes Screening Guidelines.
To evaluate compliance with diabetes screening guidelines for cardiovascular disease (CVD) prevention in rheumatoid arthritis (RA) compared to the general population. We conducted the first longitudinal study of a population-based RA cohort including all prevalent RA cases in British Columbia between 1996 and 2006 and followed until 2010, with matched general population comparators. Using administrative data, we measured compliance with general population guidelines [i.e., testing plasma glucose (PG) at least once every 3 years after age 45] after excluding individuals with previous diabetes. Followup was divided into 3-year eligibility periods. Compliance was measured as the proportion of periods with ≥ 1 PG test performed. OR (95% CI) of compliance in RA (vs general population) was calculated using generalized estimating equation models, adjusting for age and sex. Mean compliance rate per patient was also calculated and compared using the Mann-Whitney U test. Analysis included 22,624 individuals with RA, contributing 48,724 three-year eligibility periods; and 22,579 people in a general population group, contributing 51,081 three-year eligibility periods. PG was measured in 72.3% (SD 37%) of the eligible time periods in the RA sample and in 70.4% (SD 38%) for the general population (OR 1.05, 95% CI 1.02-1.09, p < 0.0001). RA individuals met recommended screening guidelines in 71.4% of their eligible periods, compared to 70.6% (p < 0.001). Screening improved over time in RA relative to the general population. Family physicians ordered nearly all the PG tests. Compliance with general population guidelines for diabetes screening in RA was suboptimal, with little difference relative to the general population, despite a higher risk of CVD and diabetes.


Analysis and functional annotation of expressed sequence tags from the Asian longhorned beetle, Anoplophora glabripennis.
The Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), is one of the most economically and ecologically devastating forest insects to invade North America in recent years. Despite its substantial impact, limited effort has been expended to define the genetic and molecular make-up of this species. Considering the significant role played by late-stadia larvae in host tree decimation, a small-scale EST sequencing project was done using a cDNA library constructed from 5(th) -instar A. glabripennis. The resultant dataset consisted of 599 high quality ESTs that, upon assembly, yielded 381 potentially unique transcripts. Each of these transcripts was catalogued as to putative molecular function, biological process, and associated cellular component according to the Gene Ontology classification system. Using this annotated dataset, a subset of assembled sequences was identified that are putatively associated with A. glabnpennis development and metamorphosis. This work will contribute to understanding of the diverse molecular mechanisms that underlie coleopteran morphogenesis and enable the future development of novel control strategies for management of this insect pest.


Assessing agreement between measurements recorded on a ratio scale in sports medicine and sports science.
The consensus of opinion suggests that when assessing measurement agreement, the most appropriate statistic to report is the "95% limits of agreement". The precise form that this interval takes depends on whether a positive relation exists between the differences in measurement methods (errors) and the size of the measurements--that is, heteroscedastic errors. If a positive and significant relation exists, the recommended procedure is to report "the ratio limits of agreement" using log transformed measurements. This study assessed the prevalence of heteroscedastic errors when investigating measurement agreement of variables recorded on a ratio scale in sports medicine and sports science. Measurement agreement (or repeatability) was assessed in 13 studies (providing 23 examples) conducted in the Centre for Sport and Exercise Sciences at Liverpool John Moores University over the past five years. The correlation between the absolute differences and the mean was positive in all 23 examples (median r = 0.37), eight being significant (P < 0.05). In 21 of 23 examples analysed, the correlation was greater than the equivalent correlation using log transformed measurements (median r = 0.01). Based on a simple meta-analysis, the assumption that no relation exists between the measurement differences and the size of measurement must be rejected (P < 0.001). When assessing measurement agreement of variables recorded on a ratio scale in sports medicine and sports science, this study (23 examples) provides strong evidence that heteroscedastic errors are the norm. If the correlation between the absolute measurement differences and the means is positive (but not necessarily significant) and greater than the equivalent correlation using log transformed measurements, the authors recommend reporting the "ratio limits of agreement".


Viral gene sequences reveal the variable history of hepatitis C virus infection among countries.
The analysis of molecular phylogenies estimated from the gene sequences of sampled viruses can provide important insights into epidemiological processes. The demographic and migration histories of the prevalent hepatitis C virus (HCV) subtypes 1a and 1b were inferred from viral gene sequences sampled in 5 countries. Estimated viral phylogenies were analyzed by use of methods based on parsimony and coalescent theory. The parsimony migration analysis suggested that the global subtype 1a and 1b epidemics are geographically structured, with asymmetrical movement of HCV strains among the sampled countries. The coalescent analysis indicated that subtype 1a infections in the United States, Brazil, and Indonesia began to increase exponentially during the 1940s and 1950s, whereas in Vietnam the increase began after the 1970s. In contrast, subtype 1b infections in these 4 countries and in Japan began to increase exponentially between 1880 and 1920, with a possible recent decrease in infection rates in Indonesia and Japan. In the United States, Brazil, and Vietnam, the epidemic growth rates for subtype 1a strains were higher than those for subtype 1b strains, whereas the growth rates were similar in Indonesia. The estimated histories of migration and population growth indicated that patterns of HCV transmission differ among countries and viral subtypes.


Raman 'optical biopsy' of human breast cancer.
Raman imaging (RI) is a novel method of medical diagnostics of human breast cancer and has a potential to become a routine optical biopsy. Up to date the present study is the most statistically reliable Raman analysis based on data of normal, benign, and cancerous breast tissues for 146 patients. This paper present the first Raman 'optical biopsy' images of the normal and cancerous breast tissue of the same patient. The results presented here demonstrate the ability of Raman spectroscopy to accurately characterize cancer tissue and distinguish between normal (noncancerous), and cancerous types. The results provide evidence that carotenoids and lipids composition of cancerous breast tissues differs significantly from that of the surrounding noncancerous breast tissue and may be a key factor responsible for mechanisms of carcinogenesis. We have found that fatty acid composition of the cancerous breast tissue is markedly different from that of the surrounding noncancerous breast tissue. The cancerous breast tissue seems to be dominated by the metabolism products of the arachidonic acid - derived cyclic eicosanoids catalyzed by cyclooxygenase, while the noncancerous breast tissue is dominated by monounsaturated oleic acid and its derivatives.


Mechanical stimulation of gap junctions in bone osteocytes is mediated by prostaglandin E2.
Gap junction-mediated intercellular communications are thought to transduce the effects of mechanical strain from osteocytes to cells on the bone surface to initiate remodeling. To determine whether gap junctions may co-ordinate the effects of mechanical loading, osteocyte-like MLO-Y4 cells were exposed to fluid flow-imposed shear stress. After exposure of MLO-Y4 to fluid flow, intercellular coupling increased in direct proportion to shear stress level. Interestingly, this stimulation is further enhanced during the post-stress period, indicating that released factor(s) is likely to be involved. The conditioned medium obtained from the fluid flow treated MLO-Y4 cells induced an increase in the number of functional gap junctions and Cx43 protein when added to non-sheer-stressed cells. Fluid flow was found to induce prostaglandin F2 (PGE2) release and increase cyclooxygenase 2 (COX-2) expression. When PGE2 was depleted from the fluid flow conditioned medium, the stimulatory effect on gap junctions was significantly decreased. Addition of the COX inhibitor indomethacin partially blocked the stimulatory effects of mechanical strain on gap junctions. Together, these studies suggest that the stimulatory effect of fluid flow on gap junctions is mediated in part by de novo synthesis and release of PGE2. Gap junctions may serve as channels for the signals generated by osteocytes in response to mechanical loading.


Protease activated receptor 2 (PAR2) activation causes migraine-like pain behaviors in mice.
Pain is the most debilitating symptom of migraine. The cause of migraine pain likely requires activation of meningeal nociceptors. Mast cell degranulation, with subsequent meningeal nociceptor activation, has been implicated in migraine pathophysiology. Degranulating mast cells release serine proteases that can cleave and activate protease activated receptors. The purpose of these studies was to investigate whether protease activated receptor 2 is a potential generator of nociceptive input from the meninges by using selective pharmacological agents and knockout mice. Ratiometric Ca++ imaging was performed on primary trigeminal and dural cell cultures after application of 2at-LIGRL-NH2, a specific protease activated receptor 2 agonist. Cutaneous hypersensitivity and facial grimace was measured in wild-type and protease activated receptor 2-/- mice after dural application of 2at-LIGRL-NH2 or compound 48-80, a mast cell degranulator. Behavioral experiments were also conducted in mice after dural application of 2at-LIGRL-NH2 (2AT) in the presence of either C391, a selective protease activated receptor 2 antagonist, or sumatriptan. 2at-LIGRL-NH2 evoked Ca2+ signaling in mouse trigeminal neurons, dural fibroblasts and in meningeal afferents. Dural application of 2at-LIGRL-NH2 or 48-80 caused dose-dependent grimace behavior and mechanical allodynia that were attenuated by either local or systemic application of C391 as well as in protease activated receptor 2-/- mice. Nociceptive behavior after dural injection of 2at-LIGRL-NH2 was also attenuated by sumatriptan. Functional protease activated receptor 2 receptors are expressed on both dural afferents and fibroblasts and activation of dural protease activated receptor 2 produces migraine-like behavioral responses. Protease activated receptor 2 may link resident immune cells to meningeal nociceptor activation, driving migraine-like pain and implicating protease activated receptor 2 as a therapeutic target for migraine in humans.


Silencing of mouse Aprt is a gradual process in differentiated cells.
Mouse Aprt constructs that are highly susceptible to DNA methylation-associated inactivation in embryonal carcinoma cells were transfected into differentiated cells, where they were expressed. Construct silencing was induced by either whole-cell fusion of the expressing differentiated cells with embryonal carcinoma cells or by treatment of the differentiated cells with the DNA demethylating agent 5-aza-2'-deoxycytidine. Induction of silencing was enhanced significantly by the presence of a methylation center fragment positioned upstream of a truncated promoter comprised of two functional Sp1 binding sites. Initial silencing of the Aprt constructs was unstable, as evidenced by high spontaneous reversion frequencies ( approximately 10(-2)). Stably silenced subclones with spontaneous reversion frequencies of <10(-5) were isolated readily from the unstably silenced clones. These reversion frequencies were enhanced significantly by treatment of the cells with 5-aza-2'-deoxycytidine. A bisulfite sequence analysis demonstrated that CpG methylation initiated within the methylation center region on expressing alleles and that the induction of silencing allowed methylation to spread towards and eventually into the promoter region. Combined with the induction of revertants by 5-aza-2'-deoxycytidine, this result suggested that stabilization of silencing was due to an increased density of CpG methylation. All allelic methylation patterns were variegated, which is consistent with a gradual and evolving process. In total, our results demonstrate that silencing of mouse Aprt is a gradual process in the differentiated cells.


Early assessment and in-hospital management of patients with acute myocardial infarction at increased risk for adverse outcomes: a nationwide perspective of current clinical practice. The National Registry of Myocardial Infarction (NRMI-2) Participants.
Therapeutic decision making in critically ill patients requires both prompt and comprehensive analysis of available information. Data derived from randomized clinical trials provide a powerful tool for risk assessment in the setting of acute myocardial infarction (MI); however, timely and appropriate use of existing therapies and resources are the key determinants of outcome among high-risk patients. Demographic, procedural, and outcome data from patients with MI were collected at 1073 U.S. hospitals collaborating in the National Registry of MI (NRMI 2). Patients were classified on hospital arrival as either "low risk" or "high risk" according to a modified Thrombolysis in Myocardial Infarction II Risk Scale based on predetermined demographic, electrocardiographic, and clinical features. Among the 170,143 patients enrolled, 115,222 (67.5%) were classified as low risk and 55,521 (32.5%) as high risk for in-hospital death, recurrent ischemia, recurrent MI, congestive heart failure, and stroke. Using a composite unsatisfactory outcome measure, in-hospital adverse events were had by a greater proportion of patients initially classified as high risk compared with those classified as low risk. By multivariate analysis, age >70 years, prior MI, Killip class >1, anterior site of infarction, and the combination of hypotension and tachycardia were independent predictions of poor outcome in patients with or without ST-segment elevation on the presenting electrocardiogram. High-risk patients with ST-segment elevation were treated with thrombolytics (47.5%) or alternative forms of reperfusion therapy (9.3%) within 62 minutes and 226 minutes of hospital arrival, respectively. High-risk patients offered reperfusion therapy were also more likely to receive aspirin, beta-blockers (intravenous, oral) and angiotensin-converting enzyme inhibitors within 24 hours of infarction (all p < 0.0001), survive their event (8.4% versus 21.4% p < 0.0001), and leave the hospital sooner than those not reperfused. This large registry experience included more than 150,000 nonselected patients with MI and suggests that high-risk patients can be identified on initial hospital presentation. The current use of reperfusion and adjunctive therapies among high-risk patients is suboptimal and may directly influence outcome. Randomized trials designed to test the impact of specific management strategies on outcome according to initial risk classification are warranted.


Vaginal sonography findings and hCG dynamics of early intrauterine and tubal pregnancies.
Early intrauterine gestational sac recognition by ultrasound is useful for discriminating between intrauterine and tubal pregnancies. Transvaginal ultrasound offers improved resolution in the imaging of the uterus and adnexa. Seventy-four women with normal intrauterine pregnancies, 18 women with spontaneous abortions, and 34 women with tubal pregnancies were evaluated by transvaginal ultrasound and serial hCG determinations. Intrauterine pregnancies were followed within the first 50 days of gestation. In women with a normal uterus and singleton pregnancy, sacs were consistently recognized in association with an hCG level greater than 2004 mIU/mL (First International Reference Preparation). Two-to three-millimeter sacs were recognized in association with an hCG level of 2032 +/- 1253 on cycle day 36.1 +/- 2.0. When sacs were less than 5 mm in mean diameter, 33% exhibited a prominent echogenic rim, 38% exhibited a partial echogenic rim, and 29% exhibited no echogenic rim. Fetal cardiac activity was recognized in association with an hCG level of 27,284 +/- 11,479 on cycle day 45.0 +/- 2.9. The hCG doubling time was 1.46 +/- 1.0 days. Apparent loss of multiple gestational sacs was observed in five normal pregnancies. Apparent bleeding in the intrauterine cavity was observed in eight patients and associated with a 38% abortion rate. One false-positive sac was observed among 34 tubal pregnancies (3%). The extrauterine sonographic finding of a solid adnexal mass or cul-de-sac fluid was specific but not sensitive for the diagnosis of tubal pregnancy. The hCG doubling time in tubal pregnancies was 7.69 +/- 9.8 days. Transvaginal ultrasound and serial hCG determinations are useful for the early recognition of tubal pregnancy.


A mutation in the canalicular phospholipid transporter gene, ABCB4, is associated with cholestasis, ductopenia, and cirrhosis in adults.
Cholestatic liver disease (CLD) is a major cause of progressive liver damage and liver failure. Several forms of biliary cirrhosis are caused by mutations in specific genes. We sought to identify a genetic defect in a family with CLD impossible to assign to a distinct pathogenic entity. Clinical and histopathological characterization of the family members, microarray-based single-nucleotide polymorphism genotyping, and analysis of candidate genes were performed. Among six of 11 siblings severely affected by idiopathic CLD in a family from a population isolate in Transylvania, three died of cirrhosis (aged 5, 7, and 43 years) and three had adult-onset disease with small duct cholangiopathy, including ductopenia. Others were mildly affected and experienced intrahepatic cholestasis of pregnancy, miscarriages, or stillbirth. Pedigree studies revealed distant parental consanguinity. Genome-wide linkage analysis and autozygosity mapping yielded a single maximal lod-score of 3.88 on chromosome 7q21.1-7q22, excluding other genomic loci. Sequencing of ABCB4 at this locus revealed a novel missense mutation c.2362C>T (p.Arg788Trp) which cosegregated with severity of disease. Bile from a mutation homozygote showed a reduced phosphatidylcholine/bile acid ratio, consistent with reduced ABCB4 phosphatidylcholine transport activity. We show that a missense mutation in ABCB4 is a cause for ductopenic CLD in adulthood. Allelic status correlated with severity of liver disease ranging from intrahepatic cholestasis of pregnancy through fibrosis to cirrhosis and death in childhood and adulthood. Mutational analysis of ABCB4 should be generally considered in all patients with cholestatic liver disease of unknown etiology regardless of age and onset of disease.


Minimizing bleeding complications in spinal tumor surgery with preoperative Onyx embolization via dual-lumen balloon catheter.
Intraoperative bleeding is a significant risk in surgery for highly vascular spinal tumors, but preoperative embolization can safely decrease intraoperative blood loss in extrinsic spine tumors. Onyx, widely used for cerebrovascular embolization, has been increasingly used as an embolic agent for preoperative spinal tumor embolization. The Scepter catheter, a dual-lumen balloon catheter, may improve tumor parenchymal penetration without the danger and limitations of significant embolic reflux. This may reduce bleeding risk during spinal surgery. Eleven consecutive cases of preoperative Onyx embolization of extrinsic spinal tumors were identified, all of whom had subsequent spinal surgery. Demographic data and clinical variables were collected. Patients were divided into Scepter (n=6) and non-Scepter (n=5) groups. The Mann-Whitney U test was used to compare continuous outcome variables and the Fisher exact test was used to compare categorical variables. Estimated blood loss in the Scepter group was significantly lower than in the non-Scepter group (584±124 vs 2400±738 mL, p=0.004). The volume of intraoperative transfusion was also significantly lower (1.2±0.4 vs 5.8±1.7 units, p=0.004). There was no significant difference in the number of vessels embolized, vials of Onyx used, use of coiling adjunct, contrast load, radiation dose, or fluoroscopy time per pedicle (p>0.05). The addition of the Scepter catheter to preoperative Onyx embolization is safe and feasible. In this small series, the Scepter catheter was associated with a reduction of intraoperative bleeding by 76% and a 79% lower transfusion volume. This was not accompanied by any unwanted increase in vials of Onyx used, contrast load, radiation dose, or fluoroscopy time.


MGOS: A resource for studying Magnaporthe grisea and Oryza sativa interactions.
The MGOS (Magnaporthe grisea Oryza sativa) web-based database contains data from Oryza sativa and Magnaporthe grisea interaction experiments in which M. grisea is the fungal pathogen that causes the rice blast disease. In order to study the interactions, a consortium of fungal and rice geneticists was formed to construct a comprehensive set of experiments that would elucidate information about the gene expression of both rice and M. grisea during the infection cycle. These experiments included constructing and sequencing cDNA and robust long-serial analysis gene expression libraries from both host and pathogen during different stages of infection in both resistant and susceptible interactions, generating >50,000 M. grisea mutants and applying them to susceptible rice strains to test for pathogenicity, and constructing a dual O. sativa-M. grisea microarray. MGOS was developed as a central web-based repository for all the experimental data along with the rice and M. grisea genomic sequence. Community-based annotation is available for the M. grisea genes to aid in the study of the interactions.


The Prostate cancer Intervention Versus Observation Trial:VA/NCI/AHRQ Cooperative Studies Program #407 (PIVOT): design and baseline results of a randomized controlled trial comparing radical prostatectomy to watchful waiting for men with clinically localized prostate cancer.
Prostate cancer is the most common noncutaneous malignancy and the second leading cause of cancer death in men. Ninety percent of men with prostate cancer are over aged 60 years, diagnosed by early detection with the prostate specific antigen (PSA) blood test and have disease believed confined to the prostate gland (clinically localized). Common treatments for clinically localized prostate cancer include watchful waiting surgery to remove the prostate gland (radical prostatectomy), external beam radiation therapy and interstitial radiation therapy (brachytherapy) and androgen deprivation. Little is known about the relative effectiveness and harms of treatments due to the paucity of randomized controlled trials. The VA/NCI/AHRQ Cooperative Studies Program Study #407: Prostate cancer Intervention Versus Observation Trial (PIVOT), initiated in 1994, is a multicenter randomized controlled trial comparing radical prostatectomy to watchful waiting in men with clinically localized prostate cancer. We describe the study rationale, design, recruitment methods and baseline characteristics of PIVOT enrollees. We provide comparisons with eligible men declining enrollment and men participating in another recently reported randomized trial of radical prostatectomy versus watchful waiting conducted in Scandinavia. We screened 13,022 men with prostate cancer at 52 United States medical centers for potential enrollment. From these, 5023 met initial age, comorbidity and disease eligibility criteria and a total of 731 men agreed to participate and were randomized. The mean age of enrollees was 67 years. Nearly one-third were African-American. Approximately 85% reported they were fully active. The median prostate specific antigen (PSA) was 7.8 ng/mL (mean 10.2 ng/mL). In three-fourths of men the primary reason for biopsy leading to a diagnosis of prostate cancer was a PSA elevation or rise. Using previously developed tumor risk categorizations incorporating PSA levels, Gleason histologic grade and tumor stage, approximately 43% had low risk, 36% had medium risk and 20% had high-risk prostate cancer. Comparison to our national sample of eligible men declining PIVOT participation as well as to men enrolled in the Scandinavian trial indicated that PIVOT enrollees are representative of men being diagnosed and treated in the U.S. and quite different from men in the Scandinavian trial. PIVOT enrolled an ethnically diverse population representative of men diagnosed with prostate cancer in the United States. Results will yield important information regarding the relative effectiveness and harms of surgery compared to watchful waiting for men with predominately PSA detected clinically localized prostate cancer.


Wetting on a spherical wall: influence of liquid-gas interfacial properties.
We study the equilibrium of a liquid film on an attractive spherical substrate for an intermolecular interaction model exhibiting both fluid-fluid and fluid-wall long-range forces. We first reexamine the wetting properties of the model in the zero-curvature limit, i.e., for a planar wall, using an effective interfacial Hamiltonian approach in the framework of the well known sharp-kink approximation (SKA). We obtain very good agreement with a mean-field density functional theory (DFT), fully justifying the use of SKA in this limit. We then turn our attention to substrates of finite curvature and appropriately modify the so-called soft-interface approximation (SIA) originally formulated by Napiórkowski and Dietrich [Phys. Rev. B34, 6469 (1986)] for critical wetting on a planar wall. A detailed asymptotic analysis of SIA confirms the SKA functional form for the film growth. However, it turns out that the agreement between SKA and our DFT is only qualitative. We then show that the quantitative discrepancy between the two is due to the overestimation of the liquid-gas surface tension within SKA. On the other hand, by relaxing the assumption of a sharp interface, with, e.g., a simple "smoothing" of the density profile there, markedly improves the predictive capability of the theory, making it quantitative and showing that the liquid-gas surface tension plays a crucial role when describing wetting on a curved substrate. In addition, we show that in contrast to SKA, SIA predicts the expected mean-field critical exponent of the liquid-gas surface tension.


Phosphorylated CtIP Functions as a Co-factor of the MRE11-RAD50-NBS1 Endonuclease in DNA End Resection.
To repair a DNA double-strand break (DSB) by homologous recombination (HR), the 5'-terminated strand of the DSB must be resected. The human MRE11-RAD50-NBS1 (MRN) and CtIP proteins were implicated in the initiation of DNA end resection, but the underlying mechanism remained undefined. Here, we show that CtIP is a co-factor of the MRE11 endonuclease activity within the MRN complex. This function is absolutely dependent on CtIP phosphorylation that includes the key cyclin-dependent kinase target motif at Thr-847. Unlike in yeast, where the Xrs2/NBS1 subunit is dispensable in vitro, NBS1 is absolutely required in the human system. The MRE11 endonuclease in conjunction with RAD50, NBS1, and phosphorylated CtIP preferentially cleaves 5'-terminated DNA strands near DSBs. Our results define the initial step of HR that is particularly relevant for the processing of DSBs bearing protein blocks.


Structure induction in diagnostic causal reasoning.
Our research examines the normative and descriptive adequacy of alternative computational models of diagnostic reasoning from single effects to single causes. Many theories of diagnostic reasoning are based on the normative assumption that inferences from an effect to its cause should reflect solely the empirically observed conditional probability of cause given effect. We argue against this assumption, as it neglects alternative causal structures that may have generated the sample data. Our structure induction model of diagnostic reasoning takes into account the uncertainty regarding the underlying causal structure. A key prediction of the model is that diagnostic judgments should not only reflect the empirical probability of cause given effect but should also depend on the reasoner's beliefs about the existence and strength of the link between cause and effect. We confirmed this prediction in 2 studies and showed that our theory better accounts for human judgments than alternative theories of diagnostic reasoning. Overall, our findings support the view that in diagnostic reasoning people go "beyond the information given" and use the available data to make inferences on the (unobserved) causal rather than on the (observed) data level.


Optimization of Immunofluorescent Detection of Bone Marrow Disseminated Tumor Cells.
Cancer metastasis is the primary cause of cancer-related deaths and remains incurable. Current clinical methods for predicting metastatic recurrence are not sensitive enough to detect individual cancer cells in the body; therefore, current efforts are directed toward liquid biopsy-based assays to capture circulating and disseminated tumor cells (CTCs and DTCs) in the blood and bone marrow, respectively. The most promising strategy is fluorescence-based immunostaining using cancer cell-specific markers. However, despite recent efforts to develop robust processing and staining platforms, results from these platforms have been discordant among groups, particularly for DTC detection. While the choice of cancer cell-specific markers is a large factor in this discordance, we have found that marker-independent factors causing false signal are just as critical to consider. Bone marrow is particularly challenging to analyze by immunostaining because endogenous immune cell properties and bone marrow matrix components typically generate false staining. For immunostaining of whole tumor tissue containing ample cancer cells, this background staining can be overcome. Application of fluorescent-based staining for rare cells, however, is easily jeopardized by immune cells and autofluorescence that lead to false signal. We have specifically found two types of background staining in bone marrow samples: autofluorescence of the tissue and non-specific binding of secondary antibodies. We systematically optimized a basic immunofluorescence protocol to eliminate this background using cancer cells spiked into human bone marrow. This enhanced the specificity of automated scanning detection software. Our optimized protocol also outperformed a commercial rare cell detection protocol in detecting candidate DTCs from metastatic patient bone marrow. Robust optimization to increase the signal-to-noise ratio of immunofluorescent staining of bone marrow is required in order to achieve the necessary sensitivity and specificity for rare cell detection. Background immunofluorescent staining in bone marrow causes uncertainty and inconsistency among investigators, which can be overcome by systematically addressing each contributing source. Our optimized assay eliminates sources of background signal, and is adaptable to automated staining platforms for high throughput analysis.


A zinc selective oxytocin based biosensor.
Oxytocin is a peptide hormone with high affinity to both Zn2+ and Cu2+ ions compared to other metal ions. This affinity makes oxytocin an attractive recognition layer for monitoring the levels of these essential ions in biofluids. Native oxytocin cannot differentiate between Cu2+ and Zn2+ ions and hence it is not useful for sensing Zn2+ in the presence of Cu2+. We elucidated the effect of the terminal amine group of oxytocin on the affinity toward Cu2+ using theoretical calculations. We designed a new Zn2+ selective oxytocin-based biosensor that utilizes the terminal amine for surface anchoring, also preventing the response to Cu2+. The biosensor shows exceptional selectivity and very high sensitivity to Zn2+ in impedimetric biosensing. This study shows for the first time an oxytocin derived sensor that can be used directly for sensing Zn2+ in the presence of Cu2+.


Assessment of malaria real-time PCR methods and application with focus on low-level parasitaemia.
In epidemiological surveys and surveillance the application of molecular tools is essential in detecting submicroscopic malaria. A genus-specific conventional cytochrome b (cytb) PCR has shown high sensitivity in field studies, detecting 70% submicroscopic malaria. The main objective of this study was to assess the conversion from conventional to real-time PCR testing both SYBR and probe protocols, and including quantitative (q) PCR. The protocols were assessed applying well-defined clinical patient material consisting of 33 positive and 80 negative samples. Sequencing of positive PCR products was performed. In addition, a sensitivity comparison of real-time PCR methods was done by including five relevant assays investigating the effect of amplification target and platform. Sensitivity was further examined using field material consisting of 111 P.falciparum positive samples from Tanzanian children (< 5 years), as well as using related patient data to assess the application of q-PCR with focus on low-level parasitaemia. Both the cytb SYBR and probe PCR protocols showed as high sensitivity and specificity as their conventional counterpart, except missing one P. malariae sample. The SYBR protocol was more sensitive and specific than using probe. Overall, choice of amplification target applied is relevant for achieving ultra-sensitivity, and using intercalating fluorescence dye rather than labelled hydrolysis probes is favourable. Application of q-PCR analysis in field projects is important for the awareness and understanding of low-level parasitaemia. For use in clinical diagnosis and epidemiological studies the highly sensitive and user-friendly cytb SYBR q-PCR method is a relevant tool. The genus-specific method has the advantage that species identification by sequencing can be performed as an alternative to species-specific PCR.