Proinflammatory cytokine responses in patients with psoriasis.
Psoriasis is one of the most common, immune-mediated, chronic inflammatory skin diseases. Proinflammatory cytokines play an important pathogenetic role at a local level. To assess whether the proinflammatory cytokines IL-1β, IL-6, IL-17, IL-22 and TNF-α are released systemically during psoriasis. Peripheral blood mononuclear cells (PBMCs) were isolated from 30 patients with psoriasis and 30 healthy volunteers. Cytokine production was assessed in supernatants using an enzyme immunoassay after stimulation of PBMCs with microbial stimuli. In addition, flow cytometry was used to determine the subsets of monocytes involved and the intracellular TNF-α production in monocytes. IL-17 levels were significantly higher in the supernatants of PBMCs from psoriatic patients after stimulation with phytohemagglutinin. TNF-α production was also significantly higher in cells from psoriatic patients after stimulation with all stimuli, as compared with health volunteers. Similar changes were not found for the other cytokines. A statistically significant difference was observed between patients and controls for inflammatory CD14(+)/CD16(+) monocytes (p<0.0001) and patrolling CD14-/CD16(+) monocytes. Hyper-production of TNF-α is documented in psoriasis. These results support the concept that there is a systemic, proinflammatory component in psoriasis.


HLA antibody detection with solid phase assays: great expectations or expectations too great?
Alloantibodies directed against HLA antigens, are a barrier to long-term solid organ allograft survival. The clinical impact of preformed, donor-directed HLA alloantibodies range from acceptable risk to unequivocal contraindication for organ transplantation. HLA antibodies are key factors that limit patient access to donor organs. Serological methods were once the only approach to identify HLA antigens and antibodies. Limitations in these technologies led to the development of solid phase approaches. In the early 1990s, the development of the polymerase chain reaction enabled DNA-based HLA antigen testing to be performed. By the mid-1990s, microparticle-based technology that utilized flow cytometry for analysis was developed to detect both classes I and II HLA antibodies. These methodologies revolutionized clinical histocompatibility testing. The strengths and weaknesses of these assays are described in detail in this review.


An integrated approach to processing WHO-2016 verbal autopsy data: the InterVA-5 model.
Verbal autopsy is an increasingly important methodology for assigning causes to otherwise uncertified deaths, which amount to around 50% of global mortality and cause much uncertainty for health planning. The World Health Organization sets international standards for the structure of verbal autopsy interviews and for cause categories that can reasonably be derived from verbal autopsy data. In addition, computer models are needed to efficiently process large quantities of verbal autopsy interviews to assign causes of death in a standardised manner. Here, we present the InterVA-5 model, developed to align with the WHO-2016 verbal autopsy standard. This is a harmonising model that can process input data from WHO-2016, as well as earlier WHO-2012 and Tariff-2 formats, to generate standardised cause-specific mortality profiles for diverse contexts. The software development involved building on the earlier InterVA-4 model, and the expanded knowledge base required for InterVA-5 was informed by analyses from a training dataset drawn from the Population Health Metrics Research Collaboration verbal autopsy reference dataset, as well as expert input. The new model was evaluated against a test dataset of 6130 cases from the Population Health Metrics Research Collaboration and 4009 cases from the Afghanistan National Mortality Survey dataset. Both of these sources contained around three quarters of the input items from the WHO-2016, WHO-2012 and Tariff-2 formats. Cause-specific mortality fractions across all applicable WHO cause categories were compared between causes assigned in participating tertiary hospitals and InterVA-5 in the test dataset, with concordance correlation coefficients of 0.92 for children and 0.86 for adults. The InterVA-5 model's capacity to handle different input formats was evaluated in the Afghanistan dataset, with concordance correlation coefficients of 0.97 and 0.96 between the WHO-2016 and the WHO-2012 format for children and adults respectively, and 0.92 and 0.87 between the WHO-2016 and the Tariff-2 format respectively. Despite the inherent difficulties of determining "truth" in assigning cause of death, these findings suggest that the InterVA-5 model performs well and succeeds in harmonising across a range of input formats. As more primary data collected under WHO-2016 become available, it is likely that InterVA-5 will undergo minor re-versioning in the light of practical experience. The model is an important resource for measuring and evaluating cause-specific mortality globally.


Omega-3 fatty acids and cardiovascular disease.
Cardioceuticals are nutritional supplements that contain all the essential nutrients including vitamins, minerals, omega-3-fatty acids and other antioxidants like a-lipoic acid and coenzyme Q10 in the right proportion that provide all round protection to the heart by reducing the most common risks associated with the cardiovascular disease including high low-density lipoprotein cholesterol and triglyceride levels and factors that contribute to coagulation of blood. Omega-3 fatty acids have been shown to significantly reduce the risk for sudden death caused by cardiac arrhythmias and all-cause mortality in patients with known coronary heart disease. Omega-3 fatty acids are also used to treat hyperlipidemia and hypertension. There are no significant drug interactions with omega-3 fatty acids. The American Heart Association recommends consumption of two servings of fish per week for persons with no history of coronary heart disease and at least one serving of fish daily for those with known coronary heart disease. Approximately 1 g/day of eicosapentaenoic acid plus docosahexaenoic acid is recommended for cardio protection. Higher dosages of omega-3 fatty acids are required to reduce elevated triglyceride levels (2-4 g/day). Modest decreases in blood pressure occur with significantly higher dosages of omega-3 fatty acids.


Unique roles of nanotechnology in medicine and cancer-II.
Applications of nanotechnology in medicine and cancer are becoming increasingly popular. Common nanomaterials and devices applicable in cancer medicine are classifiable as liposomes, polymeric-micelles, dendrimers, nano-cantilevers, carbon nanotubes, quantum dots, magnetic-nanoparticles, gold nanoparticles (AuNPs) and certain miscellaneous nanoparticles. Here, we present review of the structure, function and utilities of the various approved, under trial and pretrial nanodevices applicable in the cancer care and medicine. The liposomes are phospholipid-vesicles made use in carrying drugs to the target site minimizing the bio-distribution toxicity and a number of such theranostics have been approved for clinical practice. Newly worked out liposomes and polymeric micelles are under the trail phases for nano-therapeutic utility. A multifunctional dendrimer conjugate with imaging, targeting and drug molecules of paclitaxel has been recently synthesized for cancer theranostic applications. Nano-cantilever based assays are likely going to replace the conventions methods of chemical pathological investigations. Carbon nanotubes are emerging for utility in regenerative and cancer medicine. Quantum dots hold great promise for the micro-metastasis and intra-operative tumor imaging. Important applications of magnetic nanoparticles are in the cardiac stents, photodynamic therapy and liver metastasis imaging. The AuNPs have been employed for cell imaging, computed tomography and cancer therapy. Besides these categories, miscellaneous other nanoparticles are being discovered for utility in the cancer diagnosis and disease management. However, the use of nanoparticles should be cautious since the toxic effects of nanoparticles are not well-known. The use of nanoparticles in the clinical practice and their toxicity profile require further extensive research.


Immunohistochemical characteristics of regenerated bone after surgical therapy of advanced ligature-induced peri-implantitis defects.
This study seeks to assess and compare immunohistochemical characteristics of regenerated and pristine bone areas following surgical therapy of advanced peri-implantitis. At ligature-induced peri-implantitis defects, the intrabony component was filled with a natural bone mineral (NBM), and the supracrestal component was treated by either an equine bone block (EB) or implantoplasty. NBM and EB were soak-loaded with rhBMP-2 or sterile saline. Membrane (i.e., native collagen) protected sites were submerged for 12 weeks. Osteocalcin (OC) and transglutaminase 2 (TG2; angiogenesis) antigen reactivity was assessed within the augmented-(AA) and pristine bone (PB) areas at non-exposed sites (n = 39 defects). In all groups investigated, mean OC (AA, 0.5 ± 0.4 to 1.9 ± 2.9 %/PB, 1.7 ± 2.6 to 3.5 ± 6.5 %) and TG2 (AA, 0.6 ± 0.5 to 1.3 ± 1.5 %/PB, 0.5 ± 0.5 to 1.6 ± 1.9 %) values within AA did not significantly differ from those values assessed within PB (P > 0.05, respectively). AA formed in different treatment groups may not be considered as qualitatively (i.e., OC and TG2) compromised bone.


Dynamics of human teeth in function by means of double pulsed holography; an experimental investigation.
Investigation in vivo of small deformation and mobility processes in the masticatory system of man has been until now a very intricate problem. Mechanical as well as noncontact methods have been utilized earlier in order to record the mobility pattern of teeth and prosthodontic appliances. In this paper holographic interferometry will be presented as a solution of some odontological measurement problems. The method was first tested in a simulator arrangement and then used in a number of clinical experiments. A special, totally reflecting paint was used for surface preparation prior to holography. A Q-switched double-pulsed ruby laser was combined with an electronic subminiature force sensor for pulse triggering, which was actuated by the masticatory force of the patient. Force increases and pulse positions were registered synchronously on the screen of an oscilloscope. The applied force exerted by the patient's masticatory muscles could thus be defined according to its point of application, direction, amplitude, and duration. The corresponding surface deformation was evaluated by means of a synchronized, double-exposed hologram. Conclusions could be drawn regarding the relative and absolute mobility of the teeth and related structures of the holographed jaw section.


Household presentation of acute gastroenteritis in a primary care sentinel network: retrospective database studies.
Acute gastroenteritis (AGE) is a highly transmissible condition spreading rapidly between individuals and within households. Rotavirus vaccination was introduced in the UK in 2013. The study objectives were to investigate how acute gastroenteritis incidence changed over 25 years and household incidence of AGE since 2013. Repeated cross-sectional study of Royal College of General Practitioners Research and Surveillance Centre network. We used a negative binomial model to report incidence rate ratio (IRR) using the last 5 years data. We also conducted a retrospective cohort analysis, using a shared gamma frailty model (2013-2017). We explored the impact of child under 5- years, household size, socioeconomic status quintile, and rurality. In the cross-sectional analysis, the IRR of AGE in households with a child of under 5 years was 12.20 (95%CI 11.08-13.45-, p < 0.001) compared with households without; the IRR fell across IMD quintiles, for example there is a 37% decrease in incidence comparing IMD quintile 1 to quintile 5 (95%CI -0.52-0.76, p < 0.001), The cohort study revealed that the presence of an under 5 in the household was associated with a higher risk of household presentation (HR = 6.29, 95% CI 5.61-7.06, p < 0.001). In addition, we observe a reduction in risk of presentation from the most to the least deprived socioeconomic quintile (second quintile: HR = 0.74 (95%CI 0.59-0.92), to least deprived quintile, HR = 0.55 (95%CI 0.41-0.74). We saw a lower association with male gender, white ethnicity and living outside London, but an increased association with increasing household size. The incidence of AGE has changed over time: pre-school children, larger households, and living in London were associated with higher rates, and male gender and higher economic status associated with lower rates.


Procalcitonin-guided diagnosis and antibiotic stewardship revisited.
Several controlled clinical studies have evaluated the potential of the infection biomarker procalcitonin (PCT) to improve the diagnostic work-up of patients with bacterial infections and its influence on decisions regarding antibiotic therapy. Most research has focused on lower respiratory tract infections and critically ill sepsis patients. A clinical utility for PCT has also been found for patients with urinary tract infections, postoperative infections, meningitis, and patients with acute heart failure with possible superinfection (i.e., pneumonia). In these indications, PCT levels measured on hospital admission were found to substantially reduce the initiation of antibiotic treatment in low-risk situations (i.e., bronchitis, chronic obstructive pulmonary disease exacerbation). For more severe infections (i.e., pneumonia, sepsis), antibiotic stewardship by monitoring of PCT kinetics resulted in shorter antibiotic treatment durations with early cessation of therapy. Importantly, these strategies appear to be safe without increasing the risk for mortality, recurrent infections, or treatment failures. PCT kinetics also proved to have prognostic value correlating with disease severity (i.e., pancreatitis, abdominal infection) and resolution of illness (i.e., sepsis). Although promising findings have been published in these different types of infections, there are a number of limitations regarding PCT, including suboptimal sensitivity and/or specificity, which makes a careful interpretation of PCT in the clinical context mandatory. This narrative review aims to update clinicians on the strengths and limitations of PCT for patient management, focusing on research conducted within the last 4 years.


Which behavioral, emotional and school problems in middle-childhood predict early sexual behavior?
Mental health and school adjustment problems are thought to distinguish early sexual behavior from normative timing (16-18 years), but little is known about how early sexual behavior originates from these problems in middle-childhood. Existing studies do not allow for co-occurring problems, differences in onset and persistence, and there is no information on middle-childhood school adjustment in relationship to early sexual activity. This study examined associations between several middle-childhood problems and early sexual behavior, using a subsample (N = 4,739, 53 % female, 98 % white, mean age 15 years 6 months) from a birth cohort study, the Avon Longitudinal Study of Parents and Children. Adolescents provided information at age 15 on early sexual behavior (oral sex and/or intercourse) and sexual risk-taking, and at age 13 on prior risk involvement (sexual behavior, antisocial behavior and substance use). Information on hyperactivity/inattention, conduct problems, depressive symptoms, peer relationship problems, school dislike and school performance was collected in middle-childhood at Time 1 (6-8 years) and Time 2 (10-11 years). In agreement with previous research, conduct problems predicted early sexual behavior, although this was found only for persistent early problems. In addition, Time 2 school dislike predicted early sexual behavior, while peer relationship problems were protective. Persistent early school dislike further characterized higher-risk groups (early sexual behavior preceded by age 13 risk, or accompanied by higher sexual risk-taking). The study establishes middle-childhood school dislike as a novel risk factor for early sexual behavior and higher-risk groups, and the importance of persistent conduct problems. Implications for the identification of children at risk and targeted intervention are discussed, as well as suggestions for further research.


Experiencing Financial Strain and Clinically Assessed Caries Experience in Dentate Adults Aged 25-44 Years: An Exploration of Potential Pathways.
Large socioeconomic inequalities still exist in oral health. It is already known that oral health-related behaviour may contribute to these inequalities, but why people with a lower socioeconomic position behave less healthily is not easily understood. A possible explanation that integrates insights on health behaviour, stress, and financial resources is the pathway of behavioural responses to financial strain. The aim of this study was to assess to what extent financial strain is associated with clinically assessed caries experience in a population-based study of dentate adults, independently of other socioeconomic indicators. Furthermore, the potential mediating pathways of oral health-related behaviours (oral hygiene, dietary habits, preventive dental visits) were explored. Dentate participants, aged 25-44 years, taking part in a survey on oral health and preventive behaviour in the Netherlands in 2013 were clinically examined on - among others - caries experience (DMFS index) and level of oral hygiene (OHI-s index). Financial strain, frequency of tooth brushing, dietary habits, attendance of (preventive) dental visits in the past year, and demographic variables were assessed via questionnaires. Negative binomial hurdle models were used to study the association between financial strain and DMFS and between oral health behavioural indicators and DMFS. Although it was observed that experiencing financial strain did not seem to affect whether there is any caries experience or not, among those having any caries (DMFS >0) suffering from financial strain was associated with a higher caries prevalence, independent of educational level and income. None of the studied potential mediators could explain this association.


Exercise response in Parkinson's disease: insights from a cross-sectional comparison with sedentary controls and a per-protocol analysis of a randomised controlled trial.
To investigate the acute and adaptation cardiovascular and metabolic training responses in people with Parkinson's disease (pwP). (1) A cross-sectional study of exercise response of pwP compared with sedentary controls and (2) an interventional study of exercise training in pwP. Community leisure facilities. pwP (n=83) and sedentary controls (n=55). Study 1 included participants from a two-arm-parallel single-blind phase II randomised controlled trial (RCT), that undertook a baseline maximal incremental exercise test and study 2 included those randomised to the exercise group in the RCT, who completed a 6-month weekly exercise programme (n=37). The intervention study 2 was a prescribed exercise program consisting of sessions lasting 60 min, two times a week over a 6-month period. The control group followed the same protocol which derived the same cardiorespiratory parameters, except that they were instructed to aim for a cadence of ~60 revolutions per minute and the unloaded phase lasted 3 min with an initial step of 25 W. Stepwise incremental exercise test to volitional exhaustion was the primary outcome measure. Study 1 showed higher maximum values for heart rate (HR), VO2 L/min, VCO2 L/min and ventilation L/min for the control group; respiratory exchange ratio (RER), perceived exertion and O2 pulse (VO2 L/min/HR) did not differ between groups. In study 2, for pwP who adhered to training (n=37), RER increased significantly and although there was no significant change in aerobic capacity or HR response, reduced blood pressure was found. An abnormal cardiovascular response to exercise was observed in pwP compared to controls. After the exercise programme, metabolic deficiencies remained for pwP. These observations add to the pathogenic understanding of PD, acknowledge an underling metabolic contribution and support that certain cardiovascular symptoms may improve as a result of this type of exercise.


Frequency-dependent interaural delays in the medial superior olive: implications for interaural cochlear delays.
Neurons in the medial superior olive (MSO) are tuned to the interaural time difference (ITD) of sound arriving at the two ears. MSO neurons evoke a strongest response at their best delay (BD), at which the internal delay between bilateral inputs to MSO matches the external ITD. We performed extracellular recordings in the superior olivary complex of the anesthetized gerbil and found a majority of single units localized to the MSO to exhibit BDs that shifted with tone frequency. The relation of best interaural phase difference to tone frequency revealed nonlinearities in some MSO units and others with linear relations with characteristic phase between 0.4 and 0.6 cycles. The latter is usually associated with the interaction of ipsilateral excitation and contralateral inhibition, as in the lateral superior olive, yet all MSO units exhibited evidence of bilateral excitation. Interaural cochlear delays and phase-locked contralateral inhibition are two mechanisms of internal delay that have been suggested to create frequency-dependent delays. Best interaural phase-frequency relations were compared with a cross-correlation model of MSO that incorporated interaural cochlear delays and an additional frequency-independent delay component. The model with interaural cochlear delay fit phase-frequency relations exhibiting frequency-dependent delays with precision. Another model of MSO incorporating inhibition based on realistic biophysical parameters could not reproduce observed frequency-dependent delays.


Improved measurement for mothers, newborns and children in the era of the Sustainable Development Goals.
An urgent priority in maternal, newborn and child health is to accelerate the scale-up of cost-effective essential interventions, especially during labor, the immediate postnatal period and for the treatment of serious infectious diseases and acute malnutrition. Tracking intervention coverage is a key activity to support scale-up and in this paper we examine priorities in coverage measurement, distinguishing between essential interventions that can be measured now and those that require methodological development. We conceptualized a typology of indicators related to intervention coverage that distinguishes access to care from receipt of an intervention by the population in need. We then built on documented evidence on coverage measurement to determine the status of indicators for essential interventions and to identify areas for development. Contact indicators from pregnancy to childhood were identified as current indicators for immediate use, but indicators reflecting the quality of care provided during these contacts need development. At each contact point, some essential interventions can be measured now, but the need for development of indicators predominates around interventions at the time of birth and interventions to treat infections. Addressing this need requires improvements in routine facility based data capture, methods for linking provider and community-based data, and improved guidance for effective coverage measurement that reflects the provision of high-quality care. Coverage indicators for some essential interventions can be measured accurately through household surveys and be used to track progress in maternal, newborn and child health. Other essential interventions currently rely on contact indicators as proxies for coverage but urgent attention is needed to identify new measurement approaches that directly and reliably measure their effective coverage.


Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae.
Resistance to copper (Cu) toxicity in the respiratory pathogen Streptococcus pneumoniae is regulated by the Cu-specific metallosensor CopY. CopY is structurally related to the antibiotic-resistance regulatory proteins MecI and BlaI from Staphylococcus aureus, but is otherwise poorly characterized. Here we employ a multi-pronged experimental strategy to define the Spn CopY coordination chemistry and the unique mechanism of allosteric activation by Zn(ii) and allosteric inhibition by Cu(i) of cop promoter DNA binding. We show that Zn(ii) is coordinated by a subunit-bridging 3S 1H2O complex formed by the same residues that coordinate Cu(i), as determined by X-ray absorption spectroscopy and ratiometric pulsed alkylation-mass spectrometry (rPA-MS). Apo- and Zn-bound CopY are homodimers by small angle X-ray scattering (SAXS); however, Zn stabilizes the dimer, narrows the conformational ensemble of the apo-state as revealed by ion mobility-mass spectroscopy (IM-MS), and activates DNA binding in vitro and in cells. In contrast, Cu(i) employs the same Cys pair to form a subunit-bridging, kinetically stable, multi-metallic Cu·S cluster (KCu ≈ 1016 M-1) that induces oligomerization beyond the dimer as revealed by SAXS, rPA-MS and NMR spectroscopy, leading to inhibition of DNA binding. These studies suggest that CopY employs conformational selection to drive Zn-activation of DNA binding, and a novel Cu(i)-mediated assembly mechanism that dissociates CopY from the DNA via ligand exchange-catalyzed metal substitution, leading to expression of Cu resistance genes. Mechanistic parallels to antibiotic resistance repressors MecI and BlaI are discussed.


The protein C activator AB002 rapidly interrupts thrombus development in baboons.
Although thrombin is a key enzyme in the coagulation cascade and is required for both normal hemostasis and pathologic thrombogenesis, it also participates in its own negative feedback via activation of protein C, which downregulates thrombin generation by enzymatically inactivating factors Va and VIIIa. Our group and others have previously shown that thrombin's procoagulant and anticoagulant activities can be effectively disassociated to varying extents through site-directed mutagenesis. The thrombin mutant W215A/E217A (WE thrombin) has been one of the best characterized constructs with selective activity toward protein C. Although animal studies have demonstrated that WE thrombin acts as an anticoagulant through activated protein C (APC) generation, the observed limited systemic anticoagulation does not fully explain the antithrombotic potency of this or other thrombin mutants. AB002 (E-WE thrombin) is an investigational protein C activator thrombin analog in phase 2 clinical development (clinicaltrials.gov NCT03963895). Here, we demonstrate that this molecule is a potent enzyme that is able to rapidly interrupt arterial-type thrombus propagation at exceedingly low doses (<2 µg/kg, IV), yet without substantial systemic anticoagulation in baboons. We demonstrate that AB002 produces APC on platelet aggregates and competitively inhibits thrombin-activatable fibrinolysis inhibitor (carboxypeptidase B2) activation in vitro, which may contribute to the observed in vivo efficacy. We also describe its safety and activity in a phase 1 first-in-human clinical trial. Together, these results support further clinical evaluation of AB002 as a potentially safe and effective new approach for treating or preventing acute thrombotic and thromboembolic conditions. This trial was registered at www.clinicaltrials.gov as #NCT03453060.


Vascular access: anatomy, examination, management.
A systematic approach to managing vascular access problems is the key to reducing current high rates of access thrombosis and failure. This approach begins with a thorough knowledge of vascular access anatomy that, when combined with the physical examination, can help optimize access planning and maintenance. Because of the high complication rate of synthetic grafts, there has been increased emphasis on creating autogenous arteriovenous (AV) fistulae, which, once established, are more trouble-free. The benefit of increased fistula creation will not be realized, however, until the high rate of early fistula failure is reduced. It is widely recommended that graft surveillance programs be implemented and that stenosis be corrected when accompanied by graft dysfunction. Graft blood flow (Q(a)) is the preferred surveillance method, but has a poor accuracy in predicting thrombosis. Most studies that have evaluated the benefit of Q(a) surveillance have used historical control groups, or have been retrospective or nonrandomized. Consequently, we believe it is not currently possible to make definitive, evidence-based recommendations concerning Q(a) surveillance. The most important factor in access survival may be a team approach with an organized commitment to access planning followed by recognition and treatment of access problems.


Raman microspectroscopy of human aortic valves: investigation of the local and global biochemical changes associated with calcification in aortic stenosis.
Raman microimaging was applied to study the biochemical composition in the aortic valves obtained from patients with calcific aortic stenosis. This progressive disease affects an increasing number of elderly patients with hyperlipidemia and hypercholesterolemia. Lipid accumulation in the tissue is associated with pathogenesis and progression of cardiac valve calcification. This is in line with our finding that lipid deposits, predominantly composed of cholesterol and its esters, are frequently co-localized with calcium salt deposits, even at an early stage of their development. Overall changes in the biochemical composition of the tissue upon pathology progression are less obvious. Globally, although the cholesterol level rises, the relative lipid-to-protein content decreases. The results broaden the knowledge of biochemical alterations in dysfunctional human aortic valves and may be helpful in designing lipid lowering therapies.


Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers.
Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species.


Oblique rotation of factors: a novel pattern recognition strategy to classify fluorescence excitation-emission matrices of human blood plasma for early diagnosis of colorectal cancer.
Colorectal cancer (CRC) ranks high in both men and women, accounting for about 13% of all cancers. In this study, a novel pattern recognition strategy is proposed to improve early diagnosis of CRC through visualizing the relationship between different spectral patterns in a case-control research. Partial least squares-discriminant analysis (PLS-DA) and supervised Kohonen network (SKN) were used to classify the fluorescence excitation-emission matrices (EEMs) from 289 human blood plasma samples containing CRC patients, adenomas tumor, other non-malignant findings and healthy individuals. To obtain optimal factors, oblique rotation (OR) and genetic algorithm (GA) were used to rotate the factors by optimizing transformation matrix elements. Transformed factors were introduced to SKN to build a classification model and the model performance was examined via comparison with a common classifier; PLS-DA. Classification models were built for CRC-healthy and adenomas-healthy samples and the best results were obtained through applying GA-OR on PLS factors and introducing them to the classifiers. Non-error rates for SKN and PLS-DA models assisted with GA (for selecting more informative PLS factors) and OR were equal to 0.97 and 0.95 in cross validation and 0.93 and 0.90 for prediction of the external test set, respectively. Moreover, according to the acceptable results for adenomas-healthy cases using optimal factors, CRC can be diagnosed in early stages. Combining classifiers and optimal factors proved to be efficient for distinguishing healthy and malignant samples, and OR can significantly improve performance of the classification model.


Common and distinct impacts of autistic traits and alexithymia on social reward.
According to the social motivation hypothesis of autism, individuals with high levels of autistic traits experience reduced levels of reward from social interactions. However, empirical evidence to date has been mixed, with some studies reporting lower levels of social reward in individuals with Autism Spectrum Disorder (ASD), and others finding no difference when compared to typically developing controls. Alexithymia, a subclinical condition associated with the reduced ability to identify and describe one's own emotions, has been found to account for other affective difficulties observed inconsistently in individuals with ASD. The current study used a nonclinical sample (N = 472) to explore the associations between autistic traits and the value of six types of social reward, as measured by the Social Reward Questionnaire. In addition, we measured alexithymia to assess if this accounted for associations between autistic traits and social reward. There were three main findings. Firstly, higher levels of autistic traits were associated with significantly less enjoyment of admiration and sociability, and adding alexithymia to these models did not account for any additional variance. Secondly, both autistic traits and alexithymia were uniquely associated with reduced levels of enjoyment of prosocial interactions and sexual relationships. Thirdly, autistic traits were associated with higher levels of enjoyment of passivity and negative social potency, but these associations were no longer significant once alexithymia was taken into account, suggesting that co-occurring alexithymia accounted for these apparent associations. Overall, the current findings provide a novel and more nuanced picture of the relationship between autistic traits and social reward.


Distracting communications in the operating theatre.
Research suggests that there are problems of communication effectiveness in surgery. Here we describe the content, initiators and recipients of communications that intrude or interfere with individual surgical cases. We also consider the level at which the surgical team and its team members are distracted by these case-irrelevant communications (CICs). Two psychologist observers sampled 48 general surgery procedures and they recorded the initiator and the recipient of CIC events, their content and the level of observable distraction that they caused. Irrelevant comments and queries (i.e. 'small-talk') accounted for half of the observed CICs. From the remaining CICs that we observed, most were related to the organization and administration of the case-list, to operating theatre provisions and to teaching junior staff. Surgeons initiated a third of the observed CICs, while receiving two thirds of them. External staff visiting the operating theatre initiated the most distracting communications. The CICs addressed to surgeons introduced significantly less distraction to the operating theatre than those addressed to anaesthetists and nurses. Some of the observed CICs contributed to the administration of the operating theatre case-list. Nonetheless, this communication can interfere with highly sensitive work. More effectively co-ordinated communication could reduce this interference. More research should assess the communication effectiveness and the impact of CICs on task performance in the operating theatre.


A meta-analysis of blood lead levels in India and the attributable burden of disease.
Multiple studies in India have found elevated blood lead levels (BLLs) in target populations. However the data have not yet been evaluated to understand population-wide exposure levels. We used arithmetic mean blood lead data published from 2010 to 2018 on Indian populations to calculate the average BLLs for multiple subgroups. We then calculated the attributable disease burden in IQ decrement and Disability Adjusted Life Years (DALYs). Our Pubmed search yielded 1066 articles. Of these, 31 studies representing the BLLs of 5472 people in 9 states met our study criteria. Evaluating these, we found a mean BLL of 6.86 μg/dL (95% CI: 4.38-9.35) in children and 7.52 μg/dL (95% CI: 5.28-9.76) in non-occupationally exposed adults. We calculated that these exposures resulted in 4.9 million DALYs (95% CI: 3.9-5.6) in the states we evaluated. Population-wide BLLs in India remain elevated despite regulatory action to eliminate leaded petrol, the most significant historical source. The estimated attributable disease burden is larger than previously calculated, particularly with regard to associated intellectual disability outcomes in children. Larger population-wide BLL studies are required to inform future calculations. Policy responses need to be developed to mitigate the worst exposures.


Postinhibitory rebound spikes are modulated by the history of membrane hyperpolarization in the SCN.
The suprachiasmatic nucleus (SCN) of the hypothalamus regulates biological circadian time thereby directly impacting numerous physiological processes. The SCN is composed almost exclusively of gamma-aminobutyric acid (GABA)ergic neurons, many of which synapse with other GABAergic cells in the SCN to exert an inhibitory influence on their postsynaptic targets for most, if not all, phases of the circadian cycle. The overwhelmingly GABAergic nature of the SCN, along with its internal connectivity properties, provide a strong model to examine how inhibitory neurotransmission generates output signals. In the present work we show that hyperpolarizations that range from 5 to 1000 ms elicit rebound spikes in 63% of all SCN neurons tested in voltage-clamp in the SCN of adult rats and hamsters. In current-clamp recordings, hyperpolarizations led to rebound spike formation in all cells; however, low-amplitude or short-duration current injections failed to consistently activate rebound spikes. Increasing the duration of hyperpolarization from 5 to 1000 ms is strongly and positively correlated with enhanced spike probability. Additionally, the magnitude of hyperpolarization exerts a strong influence on both the amplitude of the spike, as revealed by voltage-clamp recordings, and the latency to peak current obtained in either voltage- or current-clamp mode. Our results suggest that SCN neurons may use rebound spikes as one means of producing output signals from a largely interconnected network of GABAergic neurons.


3-nitro-tyrosine as a peripheral biomarker of minimal hepatic encephalopathy in patients with liver cirrhosis.
Between 30 and 50% of the cirrhotic patients who do not show symptoms of clinical hepatic encephalopathy (HE) present minimal hepatic encephalopathy (MHE), with mild cognitive impairment. MHE impairs the quality of life, increases the risk of suffering accidents, predicts the appearance of clinical HE, and is associated with shortened lifespan. Early detection of MHE would be very useful. The "gold standard" for MHE diagnosis is the psychometric hepatic encephalopathy score (PHES). However, it is time consuming and needs adjusting for age and educational level. It would be very useful to have some blood biomarker reflecting the presence of MHE in cirrhotic patients. The aim of this work was to identify serum molecules useful as biomarkers for MHE. We measured in 63 controls, 43 cirrhotic patients without MHE, and 44 patients with MHE, from Hospital Clinico de Valencia, serum levels of different amino acids, cyclic guanosine monophosphate (cGMP), nitrites+nitrates, and 3-nitrotyrosine. We analyzed for each parameter its diagnostic accuracy as an indicator of MHE, as assessed using the PHES. These studies supported that 3-nitro-tyrosine is a good marker for MHE. To validate its utility as a biomarker for MHE, we analyzed in a second cohort of 44 cirrhotic patients without MHE and 18 patients with MHE, from Hospital Arnau de Vilanova, serum levels of 3-nitro-tyrosine, methionine, and citrulline. Citrulline (173±17%), methionine (173±16%), and 3-nitro-tyrosine (857±92%) were increased in sera from patients with MHE when compared with those without MHE. The receiver operating characteristic (ROC) curve analysis of 3-nitro-tyrosine for the diagnosis of MHE in the first cohort showed an area under the curve (AUC) value of 0.96 (95% confidence interval 0.93-0.99). At the cutoff of 14 nM, the specificity was 93%, sensitivity 89%, and positive and negative predictive values were both 91%. When the same cutoff was applied to the second cohort, the specificity was 83% and sensitivity was 94%. The positive and negative predictive values were 70 and 97%, respectively. This pilot study, to be validated in a larger cohort, shows that determination of 3-nitro-tyrosine in serum, which is easy and not time consuming, is useful to identify patients with MHE, with good sensitivity, specificity, and positive and negative predictive values.


Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells.
The interaction of lipids and proteins plays an important role in plasma membrane bioactivity, and much can be learned from their diffusion characteristics. Here we present the combination of super-resolution STED microscopy with scanning fluorescence correlation spectroscopy (scanning STED-FCS, sSTED-FCS) to characterize the spatial and temporal heterogeneity of lipid interactions. sSTED-FCS reveals transient molecular interaction hotspots for a fluorescent sphingolipid analogue. The interaction sites are smaller than 80 nm in diameter and lipids are transiently trapped for several milliseconds in these areas. In comparison, newly developed fluorescent phospholipid and cholesterol analogues with improved phase-partitioning properties show more homogenous diffusion, independent of the preference for liquid-ordered or disordered membrane environments. Our results do not support the presence of nanodomains based on lipid-phase separation in the basal membrane of our cultured nonstimulated cells, and show that alternative interactions are responsible for the strong local trapping of our sphingolipid analogue.


Analysis of metabolic networks using a pathway distance metric through linear programming.
The solution of the shortest path problem in biochemical systems constitutes an important step for studies of their evolution. In this paper, a linear programming (LP) algorithm for calculating minimal pathway distances in metabolic networks is studied. Minimal pathway distances are identified as the smallest number of metabolic steps separating two enzymes in metabolic pathways. The algorithm deals effectively with circularity and reaction directionality. The applicability of the algorithm is illustrated by calculating the minimal pathway distances for Escherichia coli small molecule metabolism enzymes, and then considering their correlations with genome distance (distance separating two genes on a chromosome) and enzyme function (as characterised by enzyme commission number). The results illustrate the effectiveness of the LP model. In addition, the data confirm that propinquity of genes on the genome implies similarity in function (as determined by co-involvement in the same region of the metabolic network), but suggest that no correlation exists between pathway distance and enzyme function. These findings offer insight into the probable mechanism of pathway evolution.


Biochemical and immunologic effects of rituximab in patients with primary biliary cirrhosis and an incomplete response to ursodeoxycholic acid.
The aim of this study was to determine the safety and potential efficacy of B-cell depletion with the anti-CD20 monoclonal antibody rituximab in patients with primary biliary cirrhosis (PBC) and an incomplete response to ursodeoxycholic acid (UDCA). This open-label study enrolled six patients with PBC and incomplete responses to UDCA to be treated with 2 doses of 1000 mg rituximab separated by 2 weeks and followed for 52 weeks. The primary endpoints were safety and changes in B-cell function. Two patients received only 1 dose of rituximab, one due to activation of latent varicella and the other due to a viral upper respiratory infection. Serum levels of total IgG, IgM, and IgA as well as anti-mitochondrial autoantibodies (AMAs) IgA and IgM decreased significantly from baseline by 16 weeks and returned to baseline levels by 36 weeks. Stimulation of B cells with CpG produced significantly less IgM at 52 weeks after treatment compared with B cells at baseline. In addition, transient decreases in memory B-cell and T-cell frequencies and an increase in CD25(high) CD4(+) T cells were observed after treatment. These changes were associated with significant increases in mRNA levels of FoxP3 and transforming growth factor-β (TGF-β) and a decrease in tumor necrosis factor-α (TNF-α) in CD4(+) T cells. Notably, serum alkaline phosphatase levels were significantly reduced up to 36 weeks following rituximab treatment. These data suggest that depletion of B cells influences the induction, maintenance, and activation of both B and T cells and provides a potential mechanism for treatment of patients with PBC with an incomplete response to UDCA.


Electron microscopic imaging revealed the flexible filamentous structure of the cell attachment protein P2 of Rice dwarf virus located around the icosahedral 5-fold axes.
The minor outer capsid protein P2 of Rice dwarf virus (RDV), a member of the genus Phytoreovirus in the family Reoviridae, is essential for viral cell entry. Here, we clarified the structure of P2 and the interactions to host insect cells. Negative stain electron microscopy (EM) showed that P2 proteins are monomeric and flexible L-shaped filamentous structures of ∼20 nm in length. Cryo-EM structure revealed the spatial arrangement of P2 in the capsid, which was prescribed by the characteristic virion structure. The P2 proteins were visualized as partial rod-shaped structures of ∼10 nm in length in the cryo-EM map and accommodated in crevasses on the viral surface around icosahedral 5-fold axes with hydrophobic interactions. The remaining disordered region of P2 assumed to be extended to the radial direction towards exterior. Electron tomography clearly showed that RDV particles were away from the cellular membrane at a uniform distance and several spike-like densities, probably corresponding to P2, connecting a viral particle to the host cellular membrane during cell entry. By combining the in vitro and in vivo structural information, we could gain new insights into the detailed mechanism of the cell entry of RDV.


Expression of CD99 in Multiple Myeloma: A Clinicopathologic and Immunohistochemical Study of 170 Cases.
Multiple myeloma (MM) is a heterogeneous and ultimately fatal disease. Risk stratification using prognostic biomarkers is crucial to individualize treatments. We sought to investigate the role of CD99, a transmembrane protein highly expressed in many hematopoietic cells including subpopulations of normal and neoplastic plasma cells, for MM risk stratification. CD99 expression was measured in paraffin samples of bone marrow and extramedullary biopsies of 170 patients with MM. Patients were divided into those with high score (moderately and strongly positive) and low score (negative and weakly positive), with all staining being cytoplasmic and/or membranous. High anti-CD99 immunostaining was observed in 72 of 136 (52.9%) bone marrow biopsies and 24 of 87 (27.6%) extramedullary biopsies in MM. High CD99 expression of extramedullary specimens was associated with significantly longer overall survival (OS; p=.016). High CD99 expression of extramedullary specimens was also associated with better prognosis in the nonautologous stem cell transplantation group of MM patients (p=.044). In multivariate analysis, International Staging System stage was an independent prognostic factor, whereas CD99 expression was no longer statistically significant. Expression of CD99 in extramedullary specimens was correlated with longer OS, suggesting that CD99 may be a helpful immunohistochemical marker for risk stratification.


Sodium-lithium countertransport, sodium-hydrogen exchange and membrane microviscosity in patients with hyperlipidaemia.
1. We measured sodium-lithium countertransport, sodium-hydrogen exchange and membrane micro-viscosity in 48 individuals with familial hypercholesterolaemia, 33 subjects with hypertriglyceridaemia and 54 normolipaemic controls. Full lipid profile, blood pressure, body mass index, fasting glucose and insulin levels were also measured. 2. Subjects with hypertriglyceridaemia had higher blood pressure, body mass index, fasting glucose and insulin levels than normal controls. 3. Vmax of the sodium-lithium countertransport was elevated in the hypertriglyceridaemic group compared with controls. Across the whole group log(e) triacylglycerols correlated with Vmax of the sodium-lithium countertransport. There was no difference in sodium-lithium countertransport K(m) between the groups. 4. Sodium-hydrogen maximal proton efflux rate (Vmax) and K(m) were not different between the three groups. There were no correlations between sodium-hydrogen exchange and sodium-lithium countertransport parameters. 5. Microviscosity as measured by diphenylhexatriene was reduced at the core of the membrane in subjects with hypertriglyceridaemia compared with those with familial hypercholesterolaemia or normolipaemic controls, suggesting an alteration in membrane structure. 6. Changes in sodium transport in hyperlipidaemia may be mediated by changes in membrane structure resulting in altered protein conformation or turnover.


Impact of angiotension I converting enzyme gene I/D polymorphism on running performance, lipid, and biochemical parameters in ultra-marathoners.
The insertion (I) or deletion (D) polymorphism in the angiotension I converting enzyme gene, (ACE I/D, rs1799752) is associated with human exercise endurance and performance. However, most of the aforementioned studies focus on marathons, swimming, and triathlons, while the ACE polymorphism in ultra-marathoners has not yet been reported. We studied the impact of ACE I/D polymorphism in ultra-marathoners and investigated its relationship with lipid profiles, interleukin-6 (IL-6), and high-sensitivity C-reactive protein (hs-CRP) levels in runners before and after ultra-marathon racing.This observational study used data from a 100-km ultra-marathon in Taipei, Taiwan. Twenty-four male participants were analyzed for their ACE insertion/deletion polymorphism, lipid profiles, hs-CRP, IL-6 in serum immediately before and after ultra-marathon running.In our 24 subjects analyzed, 7, 14, and 3 subjects were of I/I, I/D, and D/D genotypes, respectively. Runners with the D polymorphism (I/D and D/D) showed a trend of better performance in the 100-km ultra-marathon (measured by completion time in minutes, P = .036). In this group, the previous best marathon performance was also significantly better than the I/I group (P = .047). After adjusting for body mass index (BMI), the difference in performance was not significant. Ketone levels, IL-6, and hs-CRP levels were highly increased at immediately and 24-hour post-race. No correlation was found between different ACE polymorphisms and common biochemical parameters examined.We report the first study in the impact of the ACE I/D (rs1799752) on ultra-marathoners. Presence of the D polymorphism in ACE gene is associated with better performance, although the BMI of the runners contribute as a major factor. There was no difference in the biochemical or lipid parameters measured among different ACE polymorphisms.


How effective are 'age' tools at changing patient behaviour? A rapid review.
A common form of risk communication is to relay the relative risk (%) of an adverse outcome based on surrogate markers associated with the outcome. A novel way of communicating risk is through 'effective age' of a person or specific organ. These tools can be used to change patient behaviour. To determine the effect of 'effective age' tools on patient behaviour as compared with more traditional methods of risk communication. We performed a search of the PubMed database up to February 2019 for systematic reviews and randomised controlled trials (RCT) that answered our question. Interventions were 'effective age' tools, comparators were usual care or alternative risk communication tools. Primary outcomes were behavioural change measures. We included 1 overview of systematic reviews (level 1 evidence), 2 systematic reviews (level 1 evidence) and 13 RCTs (level 2 evidence). Both systematic reviews concluded the evidence base was not conclusive enough to make specific recommendations.Age tools assessed in the 13 RCTs were: 'lung age' (n=5), 'heart age' (n=3), 'health age' (n=2), 'cardiovascular age' (n=1), 'body age' (n=1) and 'net present value' (n=1). 7/13 (54%) RCTs demonstrated a clinical effect on behaviour change favouring the 'age' tool; 2/13 (15%) demonstrated a null effect; 4/13 (31%) favoured control. Our findings indicate that systematic review evidence needs updating. The evidence from RCTs on the effect of using age metrics on patient behaviour is poor. There is a need for high-quality trials to decrease uncertainty in the available evidence.


The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues.
The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 A of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical.


Supercoiling-dependent flexibility of adenosine-tract-containing DNA detected by a topological method.
Intrinsically bent DNA sequences have been implicated in the activation of transcription by promoting juxtaposition of DNA sequences near the terminal loop of a superhelical domain. We have developed a novel topological assay for DNA looping based on lambda integrative recombination to study the effects of intrinsically bent DNA sequences on the tertiary structure of negatively supercoiled DNA. Remarkably, the localization of adenosine-tract (A-tract) sequences in the terminal loop of a supercoiled plasmid is independent of the extent of intrinsic bending. The results suggest that A-tract-containing sequences have other properties that organize the structure of superhelical domains apart from intrinsic bending and may explain the lack of conservation in the degree of A-tract-dependent bending among DNA sequences located upstream of bacterial promoters.


Decision Making for Treatment After ACL Injury From an Orthopaedic Surgeon and Patient Perspective: Results From the NACOX Study.
In the treatment of anterior cruciate ligament (ACL) injuries, there is little evidence of when and why a decision for ACL reconstruction (ACLR) or nonoperative treatment (non-ACLR) is made. To (1) describe the key characteristics of ACL injury treatment decisions and (2) compare patient-reported knee instability, function, and preinjury activity level between patients with non-ACLR and ACLR treatment decisions. Cohort study; Level of evidence, 2. A total of 216 patients with acute ACL injury were evaluated during the first year after injury. The treatment decision was non-ACLR in 73 patients and ACLR in 143. Reasons guiding treatment decision were obtained from medical charts and questionnaires to patients and orthopaedic surgeons. Patient-reported instability and function were obtained via questionnaires and compared between patients with non-ACLR and ACLR treatment decisions. The ACLR treatment group was classified retrospectively by decision phase: acute phase (decision made between injury day and 31 days after injury), subacute phase (decision made between 32 days and up to 5 months after injury), and late phase (decision made 5-12 months after injury). Data were evaluated using descriptive statistics, and group comparisons were made using parametric or nonparametric tests as appropriate. The main reasons for a non-ACLR treatment decision were no knee instability and no problems with knee function. The main reasons for an ACLR treatment decision were high activity demands and knee instability. Patients in the non-ACLR group were significantly older (P = .031) and had a lower preinjury activity level than did those in the acute-phase (P < .01) and subacute-phase (P = .006) ACLR decision groups. There were no differences in patient-reported instability and function between treatment decision groups at baseline, 4 weeks after injury, or 3 months after injury. Activity demands, not patient-reported knee instability, may be the most important factor in the decision-making process for treatment after ACL injury. We suggest a decision-making algorithm for patients with ACL injuries and no high activity demands; waiting for >3 months can help distinguish those who need surgical intervention from those who can undergo nonoperative management. NCT02931084 (ClinicalTrials.gov identifier).


Structure, mechanism, and substrate profile for Sco3058: the closest bacterial homologue to human renal dipeptidase .
Human renal dipeptidase, an enzyme associated with glutathione metabolism and the hydrolysis of beta-lactams, is similar in sequence to a cluster of approximately 400 microbial proteins currently annotated as nonspecific dipeptidases within the amidohydrolase superfamily. The closest homologue to the human renal dipeptidase from a fully sequenced microbe is Sco3058 from Streptomyces coelicolor. Dipeptide substrates of Sco3058 were identified by screening a comprehensive series of l-Xaa-l-Xaa, l-Xaa-d-Xaa, and d-Xaa-l-Xaa dipeptide libraries. The substrate specificity profile shows that Sco3058 hydrolyzes a broad range of dipeptides with a marked preference for an l-amino acid at the N-terminus and a d-amino acid at the C-terminus. The best substrate identified was l-Arg-d-Asp (k(cat)/K(m) = 7.6 x 10(5) M(-1) s(-1)). The three-dimensional structure of Sco3058 was determined in the absence and presence of the inhibitors citrate and a phosphinate mimic of l-Ala-d-Asp. The enzyme folds as a (beta/alpha)(8) barrel, and two zinc ions are bound in the active site. Site-directed mutagenesis was used to probe the importance of specific residues that have direct interactions with the substrate analogues in the active site (Asp-22, His-150, Arg-223, and Asp-320). The solvent viscosity and kinetic effects of D(2)O indicate that substrate binding is relatively sticky and that proton transfers do not occurr during the rate-limiting step. A bell-shaped pH-rate profile for k(cat) and k(cat)/K(m) indicated that one group needs to be deprotonated and a second group must be protonated for optimal turnover. Computational docking of high-energy intermediate forms of l/d-Ala-l/d-Ala to the three-dimensional structure of Sco3058 identified the structural determinants for the stereochemical preferences for substrate binding and turnover.


Evidence of Early Household Transmission of SARS-CoV-2 Involving a School-aged Child.
Little is known about the role of school-aged children and household transmission at the start of the SARS-CoV-2 pandemic. To evaluate for SARS-CoV-2 in school-aged children and assess household transmission, we performed reverse transcription polymerase chain reaction on 670 archived specimens that were collected between September 1, 2019 and June 30, 2020 as part of a community-based study. A single SARS-CoV-2 case was detected in an 11-year-old girl on March 18, 2020, resulting in very low prevalence (0.15% [95% CI, 0.03-0.84]) in this population. This case was associated with SARS-CoV-2 detection in all other household members. Symptoms were reported as mild to moderate. Whole genome sequencing supported household transmission of near-identical viruses within the 19B clade. This case represents the earliest known household cluster of SARS-CoV2 in Wisconsin. This case suggests that household transmission associated with school-aged children may have contributed to wide seeding across populations.


Variation in mycorrhizal development and growth stimulation by 20 Pisolithus isolates inoculated on to Eucalyptus grandis W. Hill ex Maiden.
Twenty isolates of Pisolithus, covering u range of hosts, basidiocarp types and geographic locations were compared on their ability to form mycorrhiza in vitro with Eucalyptus grandis and to stimulate seedling growth in vivo. There was a large variation between isolates in the rate of mycorrhizal development and this was used to categorize isolates into six types. Mycorrhiza of seedlings inoculated with type I isolates were the most advanced with a dual-layered mantle, a fully-developed Harris net and radially elongated epidermal cells. Mycorrhiza of seedlings inoculated with types II V isolates were progressively less advanced so that mycorrhiza of type V isolates had a thin single-layered mantle, the Hartig net was only partially developed and the epidermal cells were not elongated. Root lips of seedlings inoculated with type- VI isolates were non-mycorrhizal. Inoculated seedlings were transplanted directly into undrained pots containing a yellow sand deficient in phosphorus. Growth stimulation of E. grandis seedlings in the glasshouse varied greatly between Pisolithus isolates, with dry weight increases ranging from two to 45 times that of the non-inoculated control seedlings. Extent of mycorrhizal development was positively correlated to growth stimulation in the glasshouse and it is proposed that this could be used as an indicator of isolate aggressiveness and consequently the potential of an isolate to promote tree growth.


Learning Curve in Anatomo-Electrophysiological Correlations in Subthalamic Nucleus Stimulation.
Advances in neuroradiological planning techniques in deep brain stimulation have put the need for intraoperative electrophysiological monitoring into doubt. Moreover intraoperative monitoring prolongs surgical time and there is potential association between the use of microelectrodes and increased incidence of hemorrhagic complications. The aim of this study was to analyze the correlation between the anatomically planned trajectory and the final subthalamic electrode placement after electrophysiological monitoring in patients with Parkinson"s disease and its change with the increasing experience of the surgical team. The trajectories of right (first implanted) and left electrodes were compared in the first 50 patients operated on (Group 1) and the next 50 patients (Group 2). In Group 1, 52% of central trajectories were on the right and 38% on the left; in Group 2, the percentage of central trajectories was 76% on the right and 78% on the left; the difference was statistically significant (p=0.021 and 0.001). The difference in the percentage of posterior trajectories reflecting brain shift between the right and left sides was statistically insignificant in Groups 1 (26% and 28%, p=0.999) and 2 (18% and 12%, p=0.549). The percentage of bilateral central electrodes was 14% and 62% in Groups 1 and 2, respectively. The correlation between anatomically planned trajectory and final electrode placement markedly improves with the number of patients. However the significant percentage of patients with final electrode trajectory differing from anatomically planned target supports the use of intraoperative monitoring.


Tumor Necrosis Factor and Schistosoma mansoni egg antigen omega-1 shape distinct aspects of the early egg-induced granulomatous response.
Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of the Schistosoma mansoni egg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, the S. mansoni secreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response to Schistosoma mansoni eggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg.


Using Single Colors and Color Pairs to Communicate Basic Tastes.
Recently, it has been demonstrated that people associate each of the basic tastes (e.g., sweet, sour, bitter, and salty) with specific colors (e.g., red, green, black, and white). In the present study, we investigated whether pairs of colors (both associated with a particular taste or taste word) would give rise to stronger associations relative to pairs of colors that were associated with different tastes. We replicate the findings of previous studies highlighting the existence of a robust crossmodal correspondence between individual colors and basic tastes. However, while there was evidence that pairs of colors could indeed communicate taste information more consistently than single colors, our participants took more than twice as long to match the color pairs with tastes than the single colors. Possible reasons for these results are discussed.


Prevalence and Impact of Dietary Avoidance among Individuals with Hidradenitis Suppurativa.
People with hidradenitis suppurativa (HS) are interested in dietary alterations to manage the condition. However, there are few data on the prevalence of this or the impact on HS activity. The objective of this study was to investigate the prevalence and impact of dietary alterations made by people with HS. A cross-sectional survey was sent to people with HS through multiple sources. Participants reported food alterations in the prior 6 months. -Results: Overall, 242 complete surveys were included in this analysis; the mean age was 35.8 years, and most (87.5%) were women. The majority (75.8%, n = 182) altered at least one food from their diet. Within this group, 154 (84.6%) made changes to multiple food groups. The top 5 food groups that were altered were gluten (48.8%), dairy (44.2%), refined sugars (40.0%), tomatoes (36.7%), and alcohol (37.1%). Smoking was eliminated in 27.5% of participants. Some participants (30.9%) reported the change made the HS "much better." Dietary alteration to manage HS was common among participants in this study. Some people reported improvement in HS activity, but some noted worsening. Additional research is needed to evaluate the efficacy of dietary alteration to manage HS and to better understand the underlying pathomechanisms.


Access to pediatric rheumatology subspecialty care in British Columbia, Canada.
Early recognition and treatment of pediatric rheumatic diseases is associated with improved outcome. We documented access to pediatric rheumatology subspecialty care for children in British Columbia (BC), Canada, referred to the pediatric rheumatology clinic at BC Children's Hospital, Vancouver. An audit of new patients attending the outpatient clinic from May 2006 to February 2007 was conducted. Parents completed a questionnaire through a guided interview at the initial clinic assessment. Referral dates were obtained from the referral letters. Patients were classified as having rheumatic disease, nonrheumatic disease, or a pain syndrome based on final diagnosis by a pediatric rheumatologist. Data were collected from 124 of 203 eligible new patients. Before pediatric rheumatology assessment, a median of 3 healthcare providers were seen (range 1-11) for a median of 5 visits (range 1-39). Overall, the median time interval from symptom onset to pediatric rheumatology assessment was 268 days (range 13-4989), and the median time interval from symptom onset to referral to pediatric rheumatology was 179 days (range 3-4970). Among patients ultimately diagnosed with rheumatic diseases (n = 53), there was a median of 119 days (range 3-4970) from symptom onset to referral, and 169 days (range 31-4989) from onset to pediatric rheumatology assessment. Children and adolescents with rheumatic complaints see multiple care providers for multiple visits before referral to pediatric rheumatology, and there is often a long interval between symptom onset and this referral.


Treatment with tumor necrosis factor inhibitors in axial spondyloarthritis: comparison between private rheumatology practices and academic centers in a large observational cohort.
To evaluate the initiation of and response to tumor necrosis factor (TNF) inhibitors for axial spondyloarthritis (axSpA) in private rheumatology practices versus academic centers. We compared newly initiated TNF inhibition for axSpA in 363 patients enrolled in private practices with 100 patients recruited in 6 university hospitals within the Swiss Clinical Quality Management (SCQM) cohort. All patients had been treated with ≥ 1 nonsteroidal antiinflammatory drug and > 70% of patients had a baseline Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) ≥ 4 before anti-TNF agent initiation. The proportion of patients with nonradiographic axSpA (nr-axSpA) treated with TNF inhibitors was higher in hospitals versus private practices (30.4% vs 18.7%, p = 0.02). The burden of disease as assessed by patient-reported outcomes at baseline was slightly higher in the hospital setting. Mean levels (± SD) of the Ankylosing Spondylitis Disease Activity Score were, however, virtually identical in private practices and academic centers (3.4 ± 1.0 vs 3.4 ± 0.9, p = 0.68). An Assessment of SpondyloArthritis international Society (ASAS40) response at 1 year was reached for ankylosing spondylitis in 51.7% in private practices and 52.9% in university hospitals (p = 1.0) and for nr-axSpA in 27.5% versus 25.0%, respectively (p = 1.0). With the exception of a lower proportion of patients with nr-axSpA newly treated with anti-TNF agents in private practices in comparison to academic centers, adherence to ASAS treatment recommendations for TNF inhibition was equally high, and similar response rates to TNF blockers were achieved in both clinical settings.


Epigenetics: molecular mechanisms and implications for disease.
Epigenetics is rising to prominence in biology as a mechanism by which environmental factors have intermediate-term effects on gene expression without changing the underlying genetic sequence. This can occur through the selective methylation of DNA bases and modification of histones. There are wide-ranging implications for the gene-environment debate and epigenetic mechanisms are causing a reevaluation of many traditional concepts such as heritability. The reversible nature of epigenetics also provides plausible treatment or prevention prospects for diseases previously thought hard-coded into the genome. Here, we consider how growing knowledge of epigenetics is altering our understanding of biology and medicine, and its implications for future research.


Vitamin A status of healthy children in Manisa, Turkey.
Vitamin A deficiency is a major public health nutrition problem in the developing world. Even subclinical Vitamin A deficiency is associated with increased childhood mortality. Severe maternal vitamin A deficiency may cause increased mortality in the first months of life. There have been a limited number of studies regarding vitamin A status in Turkey. The aim of this study was to assess vitamin A status of healthy children in Manisa, Turkey. Vitamin A status of 100 healthy children aged 36-48 months is evaluated. The children were seen during routine examination. Serum retinol concentrations were measured by high-performance liquid chromatography. Duration of breast feeding, age solid foods introduced, use of supplementary vitamins, weight and height, and intake of specific groups of nutrients on a daily, weekly and monthly basis were collected from a questionnaire completed by the mothers. Height and weight z-scores were calculated according to national standards. Mothers of 20 of the 100 children were known to have normal serum and breast milk retinol concentrations. Children with normal serum retinol concentration were compared with the children with VAD. Student's t-test and Mann-Whitney test were used to compare independent variables. The Pearson correlation analysis test was used to test relation between numeric variables. Mean retinol concentration was 0.98 ± 0.32 μmol/L in the whole study group. Serum retinol concentrations were normal (>0.70 μmol/L) in 89% of the children. When children with normal serum retinol concentrations were compared with those with retinol concentrations lower than 0.70 μmol/L, there was no difference in terms of age, gender, weight and height at the time of study, z-scores, birth weight, birth length, duration of breast feeding, time to begin solid food, rate of supplementary vitamin use, and rate of infections (P > 0.05). There was not any relation between vitamin A concentrations and weight and height at the time of study, z-scores, birth weight, birth length, duration of breast feeding, time to begin solid food, vitamin use, and frequency of intake of specific groups of nutrients (P > 0.05). This study showed that VAD is a moderate health problem in Manisa.


Regulatory elements required for the activation and repression of the protocadherin-alpha gene cluster.
The mouse protocadherin (Pcdh) -α, -β, and -γ gene clusters encode more than 50 protein isoforms, the combinatorial expression of which generates vast single-cell diversity in the brain. At present, the mechanisms by which this diversity is expressed are not understood. Here we show that two transcriptional enhancer elements, HS5-1 and HS7, play a critical role in Pcdhα gene expression in mice. We show that the HS5-1 element functions as an enhancer in neurons and a silencer in nonneuronal cells. The enhancer activity correlates with the binding of zinc finger DNA binding protein CTCF to the target promoters, and the silencer activity requires the binding of the REST/NRSF repressor complex in nonneuronal cells. Thus, the HS5-1 element functions as a neuron-specific enhancer and nonneuronal cell repressor. In contrast, the HS7 element functions as a Pcdhα cluster-wide transcription enhancer element. These studies reveal a complex organization of regulatory elements required for generating single cell Pcdh diversity.


The relation between parietal GABA concentration and numerical skills.
Several scientific, engineering, and medical advancements are based on breakthroughs made by people who excel in mathematics. Our current understanding of the underlying brain networks stems primarily from anatomical and functional investigations, but our knowledge of how neurotransmitters subserve numerical skills, the building block of mathematics, is scarce. Using 1H magnetic resonance spectroscopy (N = 54, 3T, semi-LASER sequence, TE = 32 ms, TR = 3.5 s), the study examined the relation between numerical skills and the brain's major inhibitory (GABA) and excitatory (glutamate) neurotransmitters. A negative association was found between the performance in a number sequences task and the resting concentration of GABA within the left intraparietal sulcus (IPS), a key region supporting numeracy. The relation between GABA in the IPS and number sequences was specific to (1) parietal but not frontal regions and to (2) GABA but not glutamate. It was additionally found that the resting functional connectivity of the left IPS and the left superior frontal gyrus was positively associated with number sequences performance. However, resting GABA concentration within the IPS explained number sequences performance above and beyond the resting frontoparietal connectivity measure. Our findings further motivate the study of inhibition mechanisms in the human brain and significantly contribute to our current understanding of numerical cognition's biological bases.


Psychological status in childhood cancer survivors: a report from the Childhood Cancer Survivor Study.
Psychological quality of life (QOL), health-related QOL (HRQOL), and life satisfaction outcomes and their associated risk factors are reviewed for the large cohort of survivors and siblings in the Childhood Cancer Survivor Study (CCSS). This review includes previously published manuscripts that used CCSS data focused on psychological outcome measures, including the Brief Symptom Inventory (BSI-18), the Medical Outcomes Survey Short Form-36 (SF-36), the Cantril Ladder of Life, and other self-report questionnaires. Comparisons and contrasts are made between siblings and survivors, and to normative data when available, in light of demographic/health information and abstracted data from the medical record. These studies demonstrate that a significant proportion of survivors report more symptoms of global distress and poorer physical, but not emotional, domains of HRQOL. Other than brain tumor survivors, most survivors report both good present and expected future life satisfaction. Risk factors for psychological distress and poor HRQOL are female sex, lower educational attainment, unmarried status, annual household income less than $20,000, unemployment, lack of health insurance, presence of a major medical condition, and treatment with cranial radiation and/or surgery. Cranial irradiation impacted neurocognitive outcomes, especially in brain tumor survivors. Psychological distress also predicted poor health behaviors, including smoking, alcohol use, fatigue, and altered sleep. Psychological distress and pain predicted use of complementary and alternative medicine. Overall, most survivors are psychologically healthy and report satisfaction with their lives. However, certain groups of childhood cancer survivors are at high risk for psychological distress, neurocognitive dysfunction, and poor HRQOL, especially in physical domains. These findings suggest targeting interventions for groups at highest risk for adverse outcomes and examining the positive growth that remains despite the trauma of childhood cancer.


Molecular cloning and functional analysis of nine cinnamyl alcohol dehydrogenase family members in Populus tomentosa.
Nine CAD/CAD-like genes in P. tomentosa were classified into four classes based on expression patterns, phylogenetic analysis and biochemical properties with modification for the previous claim of SAD. Cinnamyl alcohol dehydrogenase (CAD) functions in monolignol biosynthesis and plays a critical role in wood development and defense. In this study, we isolated and cloned nine CAD/CAD-like genes in the Populus tomentosa genome. We investigated differential expression using microarray chips and found that PtoCAD1 was highly expressed in bud, root and vascular tissues (xylem and phloem) with the greatest expression in the root. Differential expression in tissues was demonstrated for PtoCAD3, PtoCAD6 and PtoCAD9. Biochemical analysis of purified PtoCADs in vitro indicated PtoCAD1, PtoCAD2 and PtoCAD8 had detectable activity against both coniferaldehyde and sinapaldehyde. PtoCAD1 used both substrates with high efficiency. PtoCAD2 showed no specific requirement for sinapaldehyde in spite of its high identity with so-called PtrSAD (sinapyl alcohol dehydrogenase). In addition, the enzymatic activity of PtoCAD1 and PtoCAD2 was affected by temperature. We classified these nine CAD/CAD-like genes into four classes: class I included PtoCAD1, which was a bone fide CAD with the highest activity; class II included PtoCAD2, -5, -7, -8, which might function in monolignol biosynthesis and defense; class III genes included PtoCAD3, -6, -9, which have a distinct expression pattern; class IV included PtoCAD12, which has a distinct structure. These data suggest divergence of the PtoCADs and its homologs, related to their functions. We propose genes in class II are a subset of CAD genes that evolved before angiosperms appeared. These results suggest CAD/CAD-like genes in classes I and II play a role in monolignol biosynthesis and contribute to our knowledge of lignin biosynthesis in P. tomentosa.


Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect.
Cross-hybridization of repetitive sequences in genomic and expression arrays is reported to be suppressed with repeat-blocking nucleic acids (C(o)t-1 DNA). Contrary to expectation, we demonstrated that C(o)t-1 also enhanced non-specific hybridization between probes and genomic targets. When added to target DNA, C(o)t-1 enhanced hybridization (2.2- to 3-fold) to genomic probes containing conserved repetitive elements. In addition to repetitive sequences, C(o)t-1 was found to be enriched for linked single copy (sc) sequences. Adventitious association between these sequences and probes distort quantitative measurements of the probes hybridized to desired genomic targets. Quantitative microarray hybridization studies using C(o)t-1 DNA are also susceptible to these effects, especially for probes that map to genomic regions containing conserved repetitive sequences. Hybridization measurements with such probes are less reproducible in the presence of C(o)t-1 than for probes derived from sc regions or regions containing divergent repeat elements, a finding with significant ramifications for genomic and expression microarray studies. We mitigated the requirement for C(o)t-1 either by hybridizing with computationally defined sc probes lacking repeats or by substituting synthetic repetitive elements complementary to sequences in genomic probes.


Increase in the reduction potential of uranyl upon interaction with graphene oxide surfaces.
Coordination of uranyl (U(vi)) with carboxylate groups on functionalized graphene oxide (GO) surfaces has been shown to alter the reduction potential of the sorbed uranium ion. A quantitative measure of the reduction potential and qualitative estimation of sorption/desorption processes were conducted using cyclic voltammetry, and the proposed coordination environment was determined using the surface sensitive attenuated total reflection mode of infrared spectroscopy (ATR-FTIR). GO is a nanostructured material possessing a large amount of oxygen-containing functional groups both on basal planes and at the edges, which can form strong surface complexes with radionuclides. The presence of these functional groups on the surface of GO allows efficient immobilization of uranium due to sorption of uranyl (UO22+) to carboxylate, hydroxide, or sulfonate functional groups and the potential for enhanced reduction of U(vi) to more strongly sorbing and insoluble U(iv). Herein, binding of U(vi) to carboxylate groups on the GO surface is proposed as the primary sorption mechanism based on the FTIR study. Furthermore, the coordination of uranium with the surface increases the reduction potential of the U(vi)/U(iv) redox couple as compared to the case of the aqueous U(vi)/U(iv) species. This is consistent with the alteration of the electronic structure of the sorbed ion, which can be determined in our case due to the use of a GO-coated working electrode. Thus, GO-coated glassy carbon electrodes and other semi-conducting electrodes with high ion sorption capacities may provide a means of examining the oxidation/reduction potentials of sorbed ions.


Protein-tyrosine phosphatase H1 controls growth hormone receptor signaling and systemic growth.
Several protein-tyrosine phosphatases (PTPs) have been implicated in the control of growth hormone receptor (GHR) signaling, but none have been shown to affect growth in vivo. We have applied a battery of molecular and cellular approaches to test a family-wide panel of PTPs for interference with GHR signaling. Among the subset of PTPs that showed activity in multiple readouts, we selected PTP-H1/PTPN3 for further in vivo studies and found that mice lacking the PTP-H1 catalytic domain show significantly enhanced growth over their wild type littermates. In addition, PTP-H1 mutant animals had enhanced plasma and liver mRNA expression of insulin-like growth factor 1, as well as increased bone density and mineral content. These observations point to a controlling role for PTP-H1 in modulating GHR signaling and systemic growth through insulin-like growth factor 1 secretion.


Decreased serum zinc is an effect of ageing and not Alzheimer's disease.
We examined the distribution of zinc in the periphery (erythrocytes and serum) in a large, well-characterised cohort, the Australian Imaging, Biomarkers and Lifestyle (AIBL) study, in order to determine if there is systemic perturbation in zinc homeostasis in Alzheimer's disease (AD). We observed an age dependent decrease in serum zinc of approximately 0.4% per year. When correcting for the age dependent decline in serum zinc no significant difference between healthy controls (HC), mildly cognitively impaired (MCI) or AD subjects was observed.


Immunohistochemical localization and functional characterization of somatostatin receptor subtypes in a corticotropin releasing hormone-secreting adrenal phaeochromocytoma: review of the literature and report of a case.
Somastostatin receptors are frequently expressed in phaeochromocytoma but data on somatostatin receptor subtyping are scanty and the functional response to the somatostatin analogue octretide is still debated.We report an unusual case of pheochromocytoma, causing ectopic Cushing's syndrome due to CRH production by the tumour cells, in a 50-yr-old woman. Abdominal computed tomography revealed an inhomogeneous, 9-cm mass in the right adrenal gland, and [111In-DTPA0] octreotide scintigraphy showed an abnormal uptake of the radiotracer in the right perirenal region, corresponding to the adrenal mass. The patient underwent laparoscopic surgery and formalin-fixed and paraffin-embedded samples were studied. The tumour was extensively characterized by immunohistochemistry and somatostatin receptor (SSTRs) subtypes expression was analyzed. Histological and immunohistochemical examination of the surgical specimens displayed a typical pheochromocytoma, which was found to be immunoreative to S-100, chromogranin A and neurofilaments. Immunostaining for SSTR subtypes showed a positive reaction for SSTR1, SSTR2A, SSTR2B, antisera on tumour cells. The intense and diffuse immunostaining for corticotropin releasing hormone (CRH) antiserum indicated that Cushing's disease was dependent on CRH overproduction by the pheochromocytoma, in which no immunostaining for adrenocorticotropic hormone was found. Our report confirms the heterogeneity of the pattern of SSTR expression in pheochromocytomas, and provide further evidence for functional SSTR subtype SSTR2a in a subgroup of pheochromocytomas, suggesting that these tumours may represent potential target for octreotide treatment.


Tumor-targeted, systemic delivery of therapeutic viral vectors using hitchhiking on antigen-specific T cells.
Antigen-specific T cells circulate freely and accumulate specifically at sites of antigen expression. To enhance the survival and targeting of systemically delivered viral vectors, we exploited the observation that retroviral particles adhere nonspecifically, or 'hitchhike,' to the surface of T cells. Adoptive transfer of antigen-specific T cells, loaded with viruses encoding interleukin (IL)-12 or Herpes Simplex Virus thymidine kinase (HSVtk), cured established metastatic disease where adoptive T-cell transfer alone was not effective. Productive hand off correlated with local heparanase expression either from malignant tumor cells and/or as a result of T-cell activation by antigen, providing high levels of selectivity for viral transfer to metastatic tumors in vivo. Protection, concentration and targeting of viruses by adsorption to cell carriers represent a new technique for systemic delivery of vectors, in fully immunocompetent hosts, for a variety of diseases in which delivery of genes may be therapeutically beneficial.


Porencephaly in a fetus and HANAC in her father: variable expression of COL4A1 mutation.
COL4A1-associated disorders encompass a wide range of hereditary vasculopathy, including porencephaly and HANAC (adult-onset hemorrhagic stroke with cerebral aneurysm and retinal arterial tortuosity, renal cysts, and thenar muscle cramp). It remains elusive whether or not porencephaly and HANAC are molecularly distinctive disorders due to different classes of mutations. We report on a girl with porencephaly and an episode of microangiopathic hemolysis in infancy and her father with HANAC, both of whom had a heterozygous missense mutation of COL4A1 (c.3715G>A, p.G1239R). The current observation implies phenotypic diversities of COL4A1 mutations.


A potent and selective peptide blocker of the Kv1.3 channel: prediction from free-energy simulations and experimental confirmation.
The voltage-gated potassium channel Kv1.3 is a well-established target for treatment of autoimmune diseases. ShK peptide from a sea anemone is one of the most potent blockers of Kv1.3 but its application as a therapeutic agent for autoimmune diseases is limited by its lack of selectivity against other Kv channels, in particular Kv1.1. Accurate models of Kv1.x-ShK complexes suggest that specific charge mutations on ShK could considerably enhance its specificity for Kv1.3. Here we evaluate the K18A mutation on ShK, and calculate the change in binding free energy associated with this mutation using the path-independent free energy perturbation and thermodynamic integration methods, with a novel implementation that avoids convergence problems. To check the accuracy of the results, the binding free energy differences were also determined from path-dependent potential of mean force calculations. The two methods yield consistent results for the K18A mutation in ShK and predict a 2 kcal/mol gain in Kv1.3/Kv1.1 selectivity free energy relative to wild-type peptide. Functional assays confirm the predicted selectivity gain for ShK[K18A] and suggest that it will be a valuable lead in the development of therapeutics for autoimmune diseases.


BRAF mutation and RASSF1A expression in thyroid carcinoma of southern Italy.
Aim of this work is to provide a detailed comparison of clinical-pathologic features between well-differentiated and poorly differentiated tumors according to their BRAF and RASSF1A status. We analyzed RASSF1A methylation by MSP and BRAF mutation by LCRT-PCR with LightMix® kit BRAF V600E in neoplastic thyroid tissues. Immunohistochemical evaluation of RASSF1A expression was also performed by standard automated LSAB-HRP technique. An overall higher degree of RASSF1A over-expression than normal thyroid parenchyma surrounding tumors (P < 0.05) has been found in all malignant well-differentiated lesions. Moreover, statistically significant higher levels of RASSF1A expression were observed in differentiated cancers associated to an inflammatory autoimmune background (P = 0.01). Amplifiable DNA for LC PCR with LightMix® kit BRAF V600E was obtained in nine PTCs, four FVPTCs, five ATCs, and one control. The V600E mutation was found in 13 of 18 (72%) tumors. BRAF was mutated in 6 of 9 (66%) classical PTC, in 2 of 4 (50%) follicular variant PTC and in all ACs (100%). The overall frequency of RASSF1A promoter methylation observed was 20.5% (9 cases out 44). Hypermethylation of RASSF1A in primary tumors was variable according to histotypes ranging from100% (5/5) in ACs to only 12.5% (4/32) in PTCs. We show a correlation between RASSF1A methylation status and RASSF1A protein expression. Finally, we conclude that BRAF V600E mutation and RASSF1A methylation were pathogenetic event restricted to a subgroup of PTC/FVPTCs in early stage and to clinically aggressive ATCs.


β₁-Adrenoceptor stimulation suppresses endothelial IK(Ca)-channel hyperpolarization and associated dilatation in resistance arteries.
In small arteries, small conductance Ca²⁺-activated K⁺ channels (SK(Ca)) and intermediate conductance Ca²⁺-activated K⁺ channels (IK(Ca)) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI₂-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK(Ca) channels can be negatively regulated by PKA, we investigated whether β-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation. Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²⁺]i , mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording. Intraluminal perfusion of β-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM-10 μM) without modifying the associated changes in endothelial cell [Ca²⁺]i . The inhibitory effect of β-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the β-adrenoceptor antagonists propranolol (non-selective), atenolol (β₁) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (β₂) or glibenclamide (ATP-sensitive K⁺ channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by β-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK(Ca) {with 1 μM 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK(Ca) (50 nM apamin) channels prevented β-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh. In resistance arteries, endothelial cell β₁-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK(Ca) channels and may be one consequence of raised circulating catecholamines.