Dendritic cells enter lymph vessels by hyaluronan-mediated docking to the endothelial receptor LYVE-1.
Trafficking of tissue dendritic cells (DCs) via lymph is critical for the generation of cellular immune responses in draining lymph nodes (LNs). In the current study we found that DCs docked to the basolateral surface of lymphatic vessels and transited to the lumen through hyaluronan-mediated interactions with the lymph-specific endothelial receptor LYVE-1, in dynamic transmigratory-cup-like structures. Furthermore, we show that targeted deletion of the gene Lyve1, antibody blockade or depletion of the DC hyaluronan coat not only delayed lymphatic trafficking of dermal DCs but also blunted their capacity to prime CD8+ T cell responses in skin-draining LNs. Our findings uncovered a previously unknown function for LYVE-1 and show that transit through the lymphatic network is initiated by the recognition of leukocyte-derived hyaluronan.


A comprehensive functional characterization of BRCA2 variants associated with Fanconi anemia using mouse ES cell-based assay.
Biallelic mutations in the human breast cancer susceptibility gene, BRCA2, are associated with Fanconi anemia, implying that some persons who inherit 2 deleterious variants of BRCA2 are able to survive even though it is well established that BRCA2 is indispensable for viability in mice. One such variant, IVS7 + 2T > G, results in premature protein truncation because of skipping of exon 7. Surprisingly, the persons who are either IVS7 + 2T > G homozygous or compound heterozygous are born alive but die of malignancy associated with Fanconi anemia. Using a mouse embryonic stem cell-based functional assay, we found that the IVS7 + 2T > G allele produces an alternatively spliced transcript lacking exons 4-7, encoding an in-frame BRCA2 protein with an internal deletion of 105 amino acids (BRCA2(Δ105)). We demonstrate that BRCA2(Δ105) is proficient in homologous recombination-mediated DNA repair as measured by different functional assays. Evaluation of this transcript in normal and leukemia cells suggests that BRCA2(Δ105) may contribute to the viability of persons inheriting this mutation. In this study, we have also characterized 5 other BRCA2 variants and found 3 of these (p.L2510P, p.R2336H, and p.W2626C) to be deleterious and 2 (p.I2490T and p.K2729N) probably neutral. Such studies are important to understand the functional significance of unclassified BRCA2 variants.


Association of Gestational Age at Birth With Brain Morphometry.
Preterm and postterm births are associated with adverse neuropsychiatric outcomes. However, it remains unclear whether variation of gestational age within the 37- to 42-week range of term deliveries is associated with neurodevelopment. To investigate the association of gestational age at birth (GAB) with structural brain morphometry in children aged 10 years. This population-based cohort study included pregnant women living in Rotterdam, the Netherlands, with an expected delivery date between April 1, 2002, and January 31, 2006. The study evaluated 3079 singleton children with GAB ranging from 26.3 to 43.3 weeks and structural neuroimaging at 10 years of age from the Generation R Study, a longitudinal, population-based prospective birth cohort from early pregnancy onward in Rotterdam. Data analysis was performed from March 1, 2019, to February 28, 2020, and at the time of the revision based on reviewer suggestions. The GAB was calculated based on ultrasonographic assessment of crown-rump length (<12 weeks 5 days) or biparietal diameter (≥12 weeks 5 days) in dedicated research centers. Brain structure, including global and regional brain volumes and surface-based cortical measures (thickness, surface area, and gyrification), was quantified by magnetic resonance imaging. In the 3079 children (1546 [50.2%] female) evaluated at 10 years of age, GAB was linearly associated with global and regional brain volumes. Longer gestational duration was associated with larger brain volumes; for example, every 1-week-longer gestational duration corresponded to an additional 4.5 cm3/wk (95% CI, 2.7-6.3 cm3/wk) larger total brain volume. These associations persisted when the sample was restricted to children born at term (GAB of 37-42 weeks: 4.8 cm3/wk; 95% CI, 1.8-7.7 cm3/wk). No evidence of nonlinear associations between GA and brain morphometry was observed. In this cohort study, gestational duration was linearly associated with brain morphometry during childhood, including within the window of term delivery. These findings may have marked clinical importance, particularly given the prevalence of elective cesarean deliveries.


Structures of three diphtheria toxin repressor (DtxR) variants with decreased repressor activity.
The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae regulates the expression of the gene on corynebacteriophages that encodes diphtheria toxin (DT). Other genes regulated by DtxR include those that encode proteins involved in siderophore-mediated iron uptake. DtxR requires activation by divalent metals and holo-DtxR is a dimeric regulator with two distinct metal-binding sites per three-domain monomer. At site 1, three side chains and a sulfate or phosphate anion are involved in metal coordination. In the DtxR-DNA complex this anion is replaced by the side chain of Glu170 provided by the third domain of the repressor. At site 2 the metal ion is coordinated exclusively by constituents of the polypeptide chain. In this paper, five crystal structures of three DtxR variants focusing on residues Glu20, Arg80 and Cys102 are reported. The resolution of these structures ranges from 2.3 to 2.8 A. The side chain of Glu20 provided by the DNA-binding domain forms a salt bridge to Arg80, which in turn interacts with the anion. Replacing either of the salt-bridge partners with an alanine reduces repressor activity substantially and it has been inferred that the salt bridge could possibly control the wedge angle between the DNA-binding domain and the dimerization domain, thereby modulating repressor activity. Cys102 is a key residue of metal site 2 and its substitution into a serine abolishes repressor activity. The crystal structures of Zn-Glu20Ala-DtxR, Zn-Arg80Ala-DtxR, Cd-Cys102Ser-DtxR and apo-Cys102Ser-DtxR in two related space groups reveal that none of these substitutions leads to dramatic rearrangements of the DtxR fold. However, the five crystal structures presented here show significant local changes and a considerable degree of flexibility of the DNA-binding domain with respect to the dimerization domain. Furthermore, all five structures deviate significantly from the structure in the DtxR-DNA complex with respect to overall domain orientation. These results confirm the importance of the hinge motion for repressor activity. Since the third domain has often been invisible in previous crystal structures of DtxR, it is also noteworthy that the SH3-like domain could be traced in four of the five crystal structures.


Aldehyde Dehydrogenase 1B1 Is Associated with Altered Cell Morphology, Proliferation, Migration and Chemosensitivity in Human Colorectal Adenocarcinoma Cells.
Aldehyde dehydrogenases (ALDHs) are NAD(P)+-dependent enzymes that catalyze the oxidation of endogenous and exogenous aldehydes to their corresponding carboxylic acids. ALDHs participate in a variety of cellular mechanisms, such as metabolism, cell proliferation and apoptosis, as well as differentiation and stemness. Over the last few years, ALDHs have emerged as cancer stem cell markers in a wide spectrum of solid tumors and hematological malignancies. In this study, the pathophysiological role of ALDH1B1 in human colorectal adenocarcinoma was investigated. Human colon cancer HT29 cells were stably transfected either with human green fluorescent protein (GFP)-tagged ALDH1B1 or with an empty lentiviral expression vector. The overexpression of ALDH1B1 was correlated with altered cell morphology, decreased proliferation rate and reduced clonogenic efficiency. Additionally, ALDH1B1 triggered a G2/M arrest at 24 h post-cell synchronization, probably through p53 and p21 upregulation. Furthermore, ALDH1B1-overexpressing HT29 cells exhibited enhanced resistance against doxorubicin, fluorouracil (5-FU) and etoposide. Finally, ALDH1B1 induced increased migratory potential and displayed epithelial-mesenchymal transition (EMT) through the upregulation of ZEB1 and vimentin and the consequent downregulation of E-cadherin. Taken together, ALDH1B1 confers alterations in the cell morphology, cell cycle progression and gene expression, accompanied by significant changes in the chemosensitivity and migratory potential of HT29 cells, underlying its potential significance in cancer progression.


Early experience with continuous arteriovenous hemofiltration in critically ill pediatric patients.
The applicability of continuous arteriovenous hemofiltration (CAVH) for renal replacement therapy was evaluated in three infants and two young children with catastrophic medical and surgical illnesses. In the first four patients, CAVH was used in conjunction with either peritoneal or hemodialysis. In the fifth patient, CAVH was the sole renal replacement therapy employed; in this critically ill anuric infant, we were best able to evaluate the ability of CAVH to continuously control fluid, electrolyte, and acid-base balance, and allow the administration of adequate parenteral nutrition. The difficulties encountered were related to anticoagulation, establishment of adequate vascular access, and selection of an appropriate hemofilter for the performance of the technique. Despite the application of suction-assistance, we were unable to effectively employ a prototype pediatric hemofilter to attain a level of plasma ultrafiltration consistent with the objectives of therapy. However, we were able to effectively and safely employ an adult hemofilter for these purposes; modifications were made in the adult hemofilter system before its application in the smallest pediatric patients. Our experience suggests that, even in critically ill infants, CAVH can be successfully applied as an effective renal replacement therapy. However, further experience is required before its potential impact on patient survival can be assessed.


Dose verification in carcinoma of uterine cervix patients undergoing 3D conformal radiotherapy with Farmer type ion chamber.
External beam radiotherapy (EBRT) for carcinoma of uterine cervix is a basic line of treatment with three dimensional conformal radiotherapy (3DCRT) in large number of patients. There is need for an established method for verification dosimetry. We tried to document absorbed doses in a group of carcinoma cervix patients by inserting a 0.6 cc Farmer type ion chamber in the vaginal cavity. A special long perspex sleeve cap is designed to cover the chamber for using in the patient's body. Response of ionization chamber is checked earlier in water phantom with and without cap. Treatment planning was carried out with X-ray computed tomography (CT) scan and with the chamber along with cap in inserted position, and with the images Xio treatment planning system. Three measurements on 3 days at 5-6 fraction intervals were recorded in 12 patients. Electrometer measured charges are converted to absorbed dose at the chamber center, in vivo. Our results show good agreement with planned dose within 3% against prescribed dose. This study, is a refinement over our previous studies with transmission dosimetry and chemicals in ampules. This preliminary work shows promise that this can be followed as a routine dose check with special relevance to new protocols in the treatment of carcinoma cervix with EBRT.


Hypoglycaemic effects of myrtle oil in normal and alloxan-diabetic rabbits.
Myrtus communis L. (Myrtaceae) leaves as well as the volatile oil (Myrtii Oleum; MO) obtained from the leaves are used to lower the blood glucose level in type-2 diabetic patients in Turkish folk medicine. However, little attention has been paid to the therapeutic use of this plant. The present study was designed to investigate the oral hypoglycaemic activity of single and multiple doses of MO in normal and alloxan-diabetic rabbits. MO did not show any effect in normoglycaemic rabbits either in single or multiple dose administrations, but a good hypoglycaemic activity was observed 4 h after the administration to diabetic animals at 50 mg/kg. To investigate the effect of MO on repeated administration in both normal and diabetic rabbits, it was administered in 50 and 100 mg/kg doses once a day for one week. MO significantly lowered blood glucose by 51% in alloxan-diabetic rabbits on the fourth hour and the following days at a dose of 50 mg/kg (P < 0.001). The hypoglycaemic dose (50 mg/kg) was also determined by performing the oral glucose tolerance test (OGTT) in normal rabbits. The hypoglycaemic effect of the MO was 21% higher in rabbits, which received the glucose load orally, when compared with control group. However, MO did not affect serum insulin concentrations in normal and alloxan-diabetic rabbits but reduced the serum triglyceride concentrations by 14% in alloxan-diabetic rabbits. The above observations show that MO exerts hypoglycaemic as well as mild hypotriglyceridemic activity in diabetic animals. The reduction in blood glucose level may be due to the reversible inhibition of alpha-glucosidases present in the brush-border of the small intestinal mucosa, higher rate of glycolysis as envisaged by the higher activity of glucokinase, as one of the key enzymes of glycolysis, and enhanced rate of glycogenesis as evidenced by the higher amount of liver glycogen present after MO administration.


Impact of electronic reminders on venous thromboprophylaxis after admissions and transfers.
Clinical decision support has the potential to improve prevention of venous thromboembolism (VTE). The purpose of this prospective study was to analyze the effect of electronic reminders on thromboprophylaxis rates in wards to which patients were admitted and transferred. The latter was of particular interest since patient handoffs are considered to be critical safety issues. The trial involved two study periods in the six departments of a university hospital, three of which were randomly assigned to the intervention group displaying reminders during the second period. At 6 h after admission or transfer, the algorithm checked for prophylaxis orders within 0-30 h of the patient's arrival, increasing the specificity of the displayed reminders. The significant impact of the reminders could be seen by prophylaxis orders placed 6-24 h after admission (increasing from 8.6% (223/2579) to 12% (307/2555); p<0.0001) and transfer (increasing from 2.4% (39/1616) to 3.7% (63/1682); p=0.034). In admission wards, the rate of thromboprophylaxis increased from 62.4% to 67.7% (p<0.0001), and in transfer wards it increased from 80.2% to 84.3% (p=0.0022). Overall, the rate of prophylaxis significantly increased in the intervention group from 69.2% to 74.3% (p<0.0001). No significant changes were observed in the control group. Postponing prophylaxis checks to 6 h after admissions and transfers reduced the number of reminders by 62% and thereby minimized the risk of alert fatigue. The reminders improved awareness of VTE prevention in both admission and transfer wards. This approach may contribute to better quality of care and safer patient handoffs.


IL-15 deficient tax mice reveal a role for IL-1α in tumor immunity.
IL-15 is recognized as a promising candidate for tumor immunotherapy and has been described as both a promoter of cancer and a promoter of anti-cancer immunity. IL-15 was discovered in cells transformed by HTLV-1, the etiologic agent of adult T cell leukemia/lymphoma (ATL) and the human retrovirus that carries the Tax oncogene. We have developed the TAX-LUC mouse model of ATL in which Tax expression drives both malignant transformation and luciferase expression, enabling non-invasive imaging of tumorigenesis in real time. To identify the role of IL-15 in spontaneous development of lymphoma in vivo, an IL-15(-/-) TAX-LUC strain was developed and examined. The absence of IL-15 resulted in aggressive tumor growth and accelerated mortality and demonstrated that IL-15 was not required for Tax-mediated lymphoma but was essential for anti-tumor immunity. Further analysis revealed a unique transcriptional profile in tumor cells that arise in the absence of IL-15 that included a significant increase in the expression of IL-1α and IL-1α-regulated cytokines. Moreover, anti-IL-1α antibodies and an IL-1 receptor antagonist (Anakinra) were used to interrogate the potential of IL-1α targeted therapies in this model. Taken together, these findings identify IL-15 and IL-1α as therapeutic targets in lymphoma.


Quantum dynamics and entanglement of spins on a square lattice.
Bulk magnetism in solids is fundamentally quantum mechanical in nature. Yet in many situations, including our everyday encounters with magnetic materials, quantum effects are masked, and it often suffices to think of magnetism in terms of the interaction between classical dipole moments. Whereas this intuition generally holds for ferromagnets, even as the size of the magnetic moment is reduced to that of a single electron spin (the quantum limit), it breaks down spectacularly for antiferromagnets, particularly in low dimensions. Considerable theoretical and experimental progress has been made in understanding quantum effects in one-dimensional quantum antiferromagnets, but a complete experimental description of even simple two-dimensional antiferromagnets is lacking. Here we describe a comprehensive set of neutron scattering measurements that reveal a non-spin-wave continuum and strong quantum effects, suggesting entanglement of spins at short distances in the simplest of all two-dimensional quantum antiferromagnets, the square lattice Heisenberg system.


Prevalence of diabetes mellitus in rural Saudi Arabia.
Several studies have clearly shown the impact of modernization on the prevalence of diabetes mellitus in susceptible communities. Saudi Arabia has faced a rapid development program over the last two decades. In a recent study, we found a high prevalence of diabetes mellitus in urban Saudi Arabia. A total of 5222 rural subjects of both sexes were involved in a study of the prevalence of diabetes mellitus in the western region of Saudi Arabia. Random capillary blood glucose, body weight and height, and income were recorded. The results showed an overall prevalence of 4.3%. There was a rise of prevalence with age and higher-income groups. Prevalence also differed with sex. The overall prevalence in women (5.9%) was twice that for men (2.9%; P less than .001). Obesity occurred in 41.2% of our diabetic subjects compared to 29.3% in nondiabetic subjects (P less than .001). Multiple logistic regression analysis with body mass index (BMI) as the dependent variable showed that sex and income status were significant factors (P less than .0001 and P less than .04, respectively). When blood glucose was fixed as the dependent variable, the analysis showed that age, income, and BMI were significant factors (P less than .004, P less than .0001, and P less than .045, respectively).


The Utility of the Memorable Messages Framework as an Intermediary Evaluation Tool for Fruit and Vegetable Consumption in a Nutrition Education Program.
Additional strategies to evaluate the impact of community nutrition education programs on low-income individuals are needed. The objective of this qualitative study was to examine the use of the Memorable Messages Framework as an intermediary nutrition education program evaluation tool to determine what fruit and vegetable messages were reported as memorable and the characteristics of those memorable messages. A convenience sample of low-income, primarily African American adults (N = 58) who previously completed a series of community nutrition education lessons within an urban area of Indiana participated in a focus group (N = 8 focus groups). A lead moderator using a semistructured script conducted the focus groups to determine what information about fruits and vegetables was most memorable from the participants' nutrition lessons and why this information was memorable. All focus group audiotapes were transcribed verbatim and ATLAS.ti software was used to code and identify themes within the data. Participants cited quantity, variety, and the positive nutritional impact of eating fruits and vegetables as most memorable. Information given in the form of recipes was also cited as most memorable. For example, participants referred to the recipe demonstrations as not only fun but also key components of the program that helped with message retention and memorability. Key characteristics of memorable messages included personal relevance and message vividness. These findings indicated that the Memorable Messages Framework may serve as an intermediary program evaluation tool to identify what information and messages are most influential to participants in community nutrition education programs.


Donor gametes and embryos: who wants to know what about whom, and why?
Many treatments for infertility require the use of donated gametes or embryos. Arguments have been made that all parties involved (donors, recipients, and children) should have open access to information about one another. The present article reports a survey of attitudes of 77 donors and 327 recipients in the state of Western Australia. Donors and recipients endorsed a register of nonidentifying information, but were less keen on a register of identifying information. They believed that medical personnel should have access to such registers, and that donors and recipients (but not children) should have access to nonidentifying, but not identifying, information. Typically, the sort of information respondents wanted to access pertained to health status and physical characteristics. Overwhelmingly, both donors and recipients saw gamete and embryo donation as more like blood donation than like adoption.


Hydroxysafflor Yellow A Alleviates Ovalbumin-Induced Asthma in a Guinea Pig Model by Attenuateing the Expression of Inflammatory Cytokines and Signal Transduction.
Hydroxysafflor yellow A (HSYA) is an effective ingredient of the Chinese herb Carthamus tinctorius L. In this study, we aimed to evaluate the effects of HSYA on ovalbumin (OVA)-induced asthma in guinea pigs, and to elucidate the underlying mechanisms. We established a guinea pig asthma model by intraperitoneal injection and atomized administration OVA. Guinea pigs were injected intraperitoneally with HSYA (50, 75, 112.5 mg/kg) once daily from days 2 to 22 before OVA administration. We examined biomarkers including lung function, pulmonary histopathology, immunoglobulin E (IgE), Th1/Th2 relative inflammatory mediators, and related pathways. Pathological changes in lung tissues were detected by hematoxylin and eosin and periodic acid-Schiff staining. Phosphorylation levels of JNK mitogen-activated protein kinase (MAPK), p38 MAPK, ERK MAPK, and inhibitor of nuclear factor κBα (IκBα) were detected by western blot. plasma levels of total IgE, platelet-activating factor (PAF), and interleukin (IL)-3 were detected by enzyme-linked immunosorbent assay (ELISA). Expression levels of tumor necrosis factor (TNF)-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-13, and interferon (IFN)-γ were detected by ELISA and real-time quantitative polymerase chain reaction. HSYA significantly reduced airway resistance, improved dynamic lung compliance, and attenuated the pathologic changes. HSYA also inhibited the phosphorylation of JNK MAPK, p38 MAPK, ERK MAPK, and IκBα, and inhibited the OVA-induced elevations of IgE, PAF, IL-1β, IL-6, IL-4, IL-5, and IL-13 and the decreases in TNF-α, IFN-γ, IL-2, and IL-3. These findings suggest that HSYA has a protective effect on OVA-induced asthma through inhibiting the Th1/Th2 cell imbalance and inhibiting activation of the MAPK signaling pathway.


Human tolerance to heat strain during exercise: influence of hydration.
This study determined whether 1) exhaustion from heat strain occurs at the same body temperatures during exercise in the heat when subjects are euhydrated as when they are hypohydrated, 2) aerobic fitness influences the body temperature at which exhaustion from heat strain occurs, and 3) curves could be developed to estimate exhaustion rates at a given level of physiological strain. Seventeen heat-acclimated men [maximal oxygen uptake (VO2max) from 45 to 65 ml.kg-1.min-1] attempted two heat stress tests (HSTs): one when euhydrated and one when hypohydrated by 8% of total body water. The HSTs consisted of 180 min of rest and treadmill walking (45% VO2max) in a hot-dry (ambient temperature 49 degrees C, relative humidity 20%) environment. The required evaporative cooling (Ereq) exceeded the maximal evaporative cooling capacity of the environment (Emax); thus thermal equilibrium could not be achieved and 27 of 34 HSTs ended by exhaustion from heat strain. Our findings concerning exhaustion from heat strain are 1) hypohydration reduced the core temperature that could be tolerated; 2) aerobic fitness, per se, did not influence the magnitude of heat strain that could be tolerated; 3) curves can be developed to estimate exhaustion rates for a given level of physiological strain; and 4) exhaustion was rarely associated with a core temperature up to 38 degrees C, and it always occurred before a temperature of 40 degrees C was achieved. These findings are applicable to heat-acclimated individuals performing moderate-intensity exercise under conditions where Ereq approximates or exceeds Emax and who have high skin temperatures.


Evaluation and Comparison of Occurrence of Tooth Discoloration after the Application of Various Calcium Silicate-based Cements: An Ex Vivo Study.
Biodentine (Septodont, Saint Maur des Fossés, France), OrthoMTA (BioMTA, Seoul, Korea), and EndoSequence Root Repair Material (ERRM; Brasseler, Savannah, GA) have been developed to overcome the shortcomings of mineral trioxide aggregate (MTA). The purpose of this study was to compare tooth discoloration after the application of ProRoot MTA (Dentsply Tulsa Dental Products, Tulsa, OK) and 3 recently introduced calcium silicate-based cements in the presence and absence of blood. In total, 104 human anterior teeth were prepared; 96 were randomly divided into 2 groups (blood and saline contamination). Each group was subdivided into 4 experimental subgroups (n = 12) of ProRoot MTA, Biodentine, OrthoMTA, and ERRM that were used to fill the pulp chambers. The remaining 8 teeth served as the saline and blood groups. Color analysis of tooth crowns was performed using a spectroradiometer before the application of materials and at 24 hours, 1 month, and 6 months after application. Repeated measures analysis of variance was used to evaluate the effects of blood, material, and time on color change (ΔE*). Tooth color change in all experimental groups increased over time (P < .05). Blood contamination significantly increased ΔE* (P < .05), but no significant difference occurred between the 4 groups in this respect in the presence of blood. However, in the absence of blood, the ΔE* of Biodentine and ERRM was significantly less than that of OrthoMTA (P < .05). There was no significant difference between tooth discolorations with materials in the presence of blood. However, in the absence of blood, Biodentine and ERRM exhibited less tooth discoloration than OrthoMTA.


Total allowable concentrations of monomeric inorganic aluminum and hydrated aluminum silicates in drinking water.
Maximum contaminant levels are used to control potential health hazards posed by chemicals in drinking water, but no primary national or international limits for aluminum (Al) have been adopted. Given the differences in toxicological profiles, the present evaluation derives total allowable concentrations for certain water-soluble inorganic Al compounds (including chloride, hydroxide, oxide, phosphate and sulfate) and for the hydrated Al silicates (including attapulgite, bentonite/montmorillonite, illite, kaolinite) in drinking water. The chemistry, toxicology and clinical experience with Al materials are extensive and depend upon the particular physical and chemical form. In general, the water solubility of the monomeric Al materials depends on pH and their water solubility and gastrointestinal bioavailability are much greater than that of the hydrated Al silicates. Other than Al-containing antacids and buffered aspirin, food is the primary source of Al exposure for most healthy people. Systemic uptake of Al after ingestion of the monomeric salts is somewhat greater from drinking water (0.28%) than from food (0.1%). Once absorbed, Al accumulates in bone, brain, liver and kidney, with bone as the major site for Al deposition in humans. Oral Al hydroxide is used routinely to bind phosphate salts in the gut to control hyperphosphatemia in people with compromised renal function. Signs of chronic Al toxicity in the musculoskeletal system include a vitamin D-resistant osteomalacia (deranged membranous bone formation characterized by accumulation of the osteoid matrix and reduced mineralization, reduced numbers of osteoblasts and osteoclasts, decreased lamellar and osteoid bands with elevated Al concentrations) presenting as bone pain and proximal myopathy. Aluminum-induced bone disease can progress to stress fractures of the ribs, femur, vertebrae, humerus and metatarsals. Serum Al ≥100 µg/L has a 75-88% positive predictive value for Al bone disease. Chronic Al toxicity is also manifest in the hematopoietic system as an erythropoietin-resistant microcytic hypochromic anemia. Signs of Al toxicity in the central nervous system (speech difficulty to total mutism to facial grimacing to multifacial seizures and dyspraxia) are related to Al accumulation in the brain and these symptoms can progress to frank encephalopathy. There are four groups of people at elevated risk of systemic Al intoxication after repeated ingestion of monomeric Al salts: the preterm infant, the infant with congenital uremia and children and adults with kidney disease. There is a dose-dependent increase in serum and urinary Al in people with compromised renal function, and restoration of renal function permits normal handling of systemically absorbed Al and resolution of Al bone disease. Clinical experience with 960 mg/day of Al(OH)(3) (~5 mg Al/kg-day) given by mouth over 3 months to men and women with compromised renal function found subclinical reductions in hemoglobin, hematocrit and serum ferritin. Following adult males and females with reduced kidney function found that ingestion of Al(OH)(3) at 2.85 g/day (~40 mg/kg-day Al) over 7 years increased bone Al, but failed to elicit significant bone toxicity. There was one report of DNA damage in cultured lymphocytes after high AlCl(3) exposure, but there is no evidence that ingestion of common inorganic Al compounds presents an increased carcinogenic risk or increases the risk for adverse reproductive or developmental outcomes. A number of studies of Al exposure in relation to memory in rodents have been published, but the results are inconsistent. At present, there is no evidence to substantiate the hypothesis that the pathogenesis of Alzheimer's Disease is caused by Al found in food and drinking water at the levels consumed by people living in North America and Western Europe. Attapulgite (palygorskite) has been used for decades at oral doses (recommended not to exceed two consecutive days) of 2,100 mg/day in children of 3-6 years, 4,200 mg/day in children of 6-12 years, and 9,000 mg/day in adults. Chronic ingestion of insoluble hydrated Al silicates (in kg) can result in disturbances in iron and potassium status, primarily as a result of clay binding to intestinal contents and enhanced fecal iron and zinc elimination. Sufficiently high doses of ingested Al silicates (≥50 g/day) over prolonged periods of time can elicit a deficiency anemia that can be corrected with oral Fe supplements. There is essentially no systemic Al uptake after ingestion of the hydrated Al silicates. Rats fed up to 20,000 ppm Ca montmorillonite (equivalent to 1,860 ppm total Al as the hydrated Al silicate) for 28 weeks failed to develop any adverse signs. The results of dietary Phase I and II clinical trials conducted in healthy adult volunteers over 14 days and 90 days with montmorillonite found no adverse effects after feeding up to 40 mg/kg-day as Al. Since the Al associated with ingestion of hydrated Al silicates is not absorbed into the systemic circulation, the hydrated Al silicates seldom cause medical problems unless the daily doses consumed are substantially greater than those used clinically or as dietary supplements. A no-observable-adverse-effect-level (NOAEL) of 13 mg/kg-day as total Al can be identified based on histologic osteomalacia seen in adult hemodialysis patients given Al hydroxide for up to 7 years as a phosphate binder. Following U.S. EPA methods for calculation of an oral reference dose (RfD), an intraspecies uncertainty factor of 10x was applied to that value results in a chronic oral reference dose (RfD) of 1.3 mg Al/kg-day; assuming a 70-kg adult consumes 2 L of drinking water per day and adjusting for a default 20% relative source contribution that value corresponds to a drinking water maximum concentration of 9 mg/L measured as total Al. A chronic NOAEL for montmorillonite as representative of the hydrated Al silicates was identified from the highest dietary concentration (20,000 ppm) fed in a 28-week bioassay with male and female Sprague-Dawley rats. Since young rats consume standard laboratory chow at ~23 g/day, this concentration corresponds to 56 mg Al/kg-day. Application of 3x interspecies uncertainty factor and a 3x factor to account for study duration results in a chronic oral RfD of 6 mg Al/kg-day. Of note, this RfD is 5-10 fold less than oral doses of Al silicates consumed by people who practice clay geophagy and it corresponds to a maximum drinking water concentration of 40 mg Al/L. To utilize the values derived here, the risk manager must recognize the particular product (e.g., alum) or source (e.g., groundwater, river water, clay or cement pipe) of the Al found in tap water, apply the appropriate analytical methods (atomic absorption, energy dispersive X-ray diffraction, infrared spectral analysis and/or scanning transmission electron microscopy) and compare the results to the most relevant standard. The drinking water concentrations derived here are greater than the U.S. EPA secondary maximum contaminant level (MCL) for total Al of 0.05-0.2 mg/L [40 CFR 143.3]. As such, domestic use of water with these concentrations is likely self-limiting given that its cloudy appearance will be greater than the maximum permitted (0.5-5.0 nephalometric turbidity units; 40 CFR Parts 141 and 142). Therefore, the organoleptic properties of Al materials in water determine public acceptance of potable water as contrast to any potential health hazard at the concentrations ordinarily present in municipal drinking water.


Financial hardship and health risk behavior during COVID-19 in a large US national sample of women.
COVID-19 has caused over 300,000 US deaths thus far, but its long-term health consequences are not clear. Policies to contain the pandemic have led to widespread economic problems, which likely increase stress and resulting health risk behaviors, particularly among women, who have been hardest hit both by job loss and caregiving responsibilities. Further, women with pre-existing disadvantage (e.g., those without health insurance) may be most at risk for stress and consequent health risk behavior. Our objective was to estimate the associations between financial stressors from COVID-19 and health risk behavior changes since COVID-19, with potential effect modification by insurance status. We used multilevel logistic regression to assess the relationships between COVID-19-related financial stressors (job loss, decreases in pay, trouble paying bills) and changes in health risk behavior (less exercise, sleep, and healthy eating; more smoking/vaping and drinking alcohol), controlling for both individual-level and zip code-level confounders, among 90,971 US women who completed an online survey in March-April 2020. Almost 40% of women reported one or more COVID-19-related financial stressors. Each financial stressor was significantly associated with higher odds of each type of health risk behavior change. Overall, reporting one or more financial stressors was associated with 56% higher odds (OR = 1.56; 95% CI: 1.51, 1.60) of reporting two or more health risk behavior changes. This association was even stronger among women with no health insurance (OR = 2.46; 95% CI: 1.97, 3.07). COVID-19-related economic stress is thus linked to shifts in health risk behaviors among women, which may have physical health consequences for years to come. Further, the relationship between financial hardship and health risk behavior among women may be modified by health insurance status, as a marker for broader socioeconomic context and resources. The most socioeconomically vulnerable women are likely at highest risk for long-term health effects of COVID-19 financial consequences.


MED16 and MED23 of Mediator are coactivators of lipopolysaccharide- and heat-shock-induced transcriptional activators.
Transcriptional activators interact with diverse proteins and recruit transcriptional machinery to the activated promoter. Recruitment of the Mediator complex by transcriptional activators is usually the key step in transcriptional activation. However, it is unclear how Mediator recognizes different types of activator proteins. To systematically identify the subunits responsible for the signal- and activator-specific functions of Mediator in Drosophila melanogaster, each Mediator subunit was depleted by RNA interference, and its effect on transcriptional activation of endogenous as well as synthetic promoters was examined. The depletion of some Mediator gene products caused general transcriptional defects, whereas depletion of others caused defects specifically related to activation. In particular, MED16 and MED23 were required for lipopolysaccharide- and heat-shock-specific gene expression, respectively, and their activator-specific functions appeared to result from interaction with specific activators. The corequirement of MED16 for other forms of differentiation-inducing factor-induced transcription was confirmed by microarray analysis of differentiation-inducing factor (DIF)- and MED16-depleted cells individually. These results suggest that distinct Mediator subunits interact with specific activators to coordinate and transfer activator-specific signals to the transcriptional machinery.


Identification of eczema clusters and their association with filaggrin and atopic comorbidities: analysis of five birth cohorts.
Longitudinal modelling of the presence/absence of current eczema through childhood has identified similar phenotypes, but their characteristics often differ between studies. To demonstrate that a more comprehensive description of longitudinal pattern of symptoms may better describe trajectories than binary information on eczema presence. We derived six multidimensional variables of eczema spells from birth to 18 years of age (including duration, temporal sequencing and the extent of persistence/recurrence). Spells were defined as consecutive observations of eczema separated by no eczema across 5 epochs in five birth cohorts: infancy (first year); early childhood (age 2-3 years); preschool/early school age (4-5 years); middle childhood (8-10 years); adolescence (14-18 years). We applied Partitioning Around Medoids clustering on these variables to derive clusters of the temporal patterns of eczema. We then investigated the stability of the clusters, within-cluster homogeneity and associated risk factors, including FLG mutations. Analysis of 7464 participants with complete data identified five clusters: (i) no eczema (51.0%); (ii) early transient eczema (21.6%); (iii) late-onset eczema (LOE; 8.1%); (iv) intermittent eczema (INT; 7.5%); and (v) persistent eczema (PE; 11.8%). There was very-high agreement between the assignment of individual children into clusters when using complete or imputed (n = 15 848) data (adjusted Rand index = 0.99; i.e. the clusters were very stable). Within-individual symptom patterns across clusters confirmed within-cluster homogeneity, with consistent patterns of symptoms among participants within each cluster and no overlap between the clusters. Clusters were characterized by differences in associations with risk factors (e.g. parental eczema was associated with all clusters apart from LOE; sensitization to inhalant allergens was associated with all clusters, with the highest risk in the PE cluster). All clusters apart from LOE were associated with FLG mutations. Of note, the strongest association was for PE [relative risk ratio (RRR) 2.70, 95% confidence interval (CI) 2.24-3.26; P < 0.001] followed by INT (RRR 2.29, 95% CI 1.82-2.88; P < 0.001). Clustering of multidimensional variables identified stable clusters with different genetic architectures. Using multidimensional variables may capture eczema development and derive stable and internally homogeneous clusters. However, deriving homogeneous symptom clusters does not necessarily mean that these are underpinned by completely unique mechanisms.


Electrical activity in the heart during hyperkalemic hypothermic cardioplegic arrest: site of origin and relationship to specialized conduction tissue.
Previous studies from this laboratory have shown that low-amplitude electrical activity (LEA) may occur during standard hyperkalemic hypothermic cardioplegic arrest and be undetected by routine monitoring techniques. The present study was designed to elucidate the electrophysiological nature of LEA. Ten dogs were monitored continuously during standard cardioplegic arrest using a 32-channel data acquisition system. In 7 animals (Part I), electrophysiological mapping of the lower right atrial septum during arrest was performed. The initial site of activation of LEA was consistently recorded from the region of the lower atrial septum prior to atrial or ventricular electrical activation. The site of origin of LEA was thus localized to the anatomical region of the atrial septum containing the atrioventricular nodal conduction tissue. In Part II, the electrophysiological mechanism of LEA was investigated in the remaining 3 animals utilizing an intrinsic property of specialized conduction tissue. Inclusion of a calcium channel-blocking agent in standard cardioplegic solution completely prevented the development of LEA in all 3 animals, which is in contrast to findings in previous studies using standard cardioplegic solution alone. These data suggest that LEA may be related to calcium-mediated activation of specialized conduction tissue. This mechanism of activation may explain why LEA cannot be detected by the intraoperative monitoring techniques routinely employed.


Potent trypanocidal curcumin analogs bearing a monoenone linker motif act on trypanosoma brucei by forming an adduct with trypanothione.
We have previously reported that curcumin analogs with a C7 linker bearing a C4-C5 olefinic linker with a single keto group at C3 (enone linker) display midnanomolar activity against the bloodstream form of Trypanosoma brucei. However, no clear indication of their mechanism of action or superior antiparasitic activity relative to analogs with the original di-ketone curcumin linker was apparent. To further investigate their utility as antiparasitic agents, we compare the cellular effects of curcumin and the enone linker lead compound 1,7-bis(4-hydroxy-3-methoxyphenyl)hept-4-en-3-one (AS-HK014) here. An AS-HK014-resitant line, trypanosomes adapted to AS-HK014 (TA014), was developed by in vitro exposure to the drug. Metabolomic analysis revealed that exposure to AS-HK014, but not curcumin, rapidly depleted glutathione and trypanothione in the wild-type line, although almost all other metabolites were unchanged relative to control. In TA014 cells, thiol levels were similar to untreated wild-type cells and not significantly depleted by AS-HK014. Adducts of AS-HK014 with both glutathione and trypanothione were identified in AS-HK014-exposed wild-type cells and reproduced by chemical reaction. However, adduct accumulation in sensitive cells was much lower than in resistant cells. TA014 cells did not exhibit any changes in sequence or protein levels of glutathione synthetase and γ-glutamylcysteine synthetase relative to wild-type cells. We conclude that monoenone curcuminoids have a different mode of action than curcumin, rapidly and specifically depleting thiol levels in trypanosomes by forming an adduct. This adduct may ultimately be responsible for the highly potent trypanocidal and antiparasitic activity of the monoenone curcuminoids.


Expectations regarding action sequences modulate electrophysiological correlates of the gaze-cueing effect.
Predictive mechanisms of the brain are important for social cognition, as they enable inferences about others' goals and intentions, thereby allowing for generation of expectations regarding what will happen next in the social environment. Therefore, attentional selection is modulated by expectations regarding behavior of others (Perez-Osorio, Müller, Wiese, & Wykowska, 2015). In this article, we examined-using the ERPs of the EEG signal-which stages of processing are influenced by expectations about others' action steps. We used a paradigm in which a gaze-cueing procedure was embedded in successively presented naturalistic photographs composing an action sequence. Our results showed (a) behavioral gaze-cueing effects modulated by whether the observed agent gazed at an object that was expected to be gazed at, according to the action sequence; (b) the N1 component locked to the onset of a target was modulated both by spatial gaze validity and participants' expectations about where the agent would gaze to perform an action; (c) a more positive amplitude, locked to the shift of gaze direction for action-congruent gaze, relative to incongruent and neutral conditions-over parieto-occipital areas in the time window between 280 and 380 ms. Taken together, these findings revealed that confirmation or violation of expectations concerning others' goal-oriented actions modulate attentional selection processes, as indexed by early ERP components.


Recurrence of esophageal intestinal metaplasia after endoscopic mucosal resection and radiofrequency ablation of Barrett's esophagus: results from a US Multicenter Consortium.
Radiofrequency ablation (RFA) is an established treatment for dysplastic Barrett's esophagus (BE). Although short-term end points of ablation have been ascertained, there have been concerns about recurrence of intestinal metaplasia (IM) after ablation. We aimed to estimate the incidence and identify factors that predicted the recurrence of IM after successful RFA. We analyzed data from 592 patients with BE treated with RFA from 2003 through 2011 at 3 tertiary referral centers. Complete remission of intestinal metaplasia (CRIM) was defined as eradication of IM (in esophageal and gastroesophageal junction biopsy specimens), documented by 2 consecutive endoscopies. Recurrence was defined as the presence of IM or dysplasia after CRIM in surveillance biopsies. Two experienced gastrointestinal pathologists confirmed pathology findings. Based on histology analysis, before RFA, 71% of patients had high-grade dysplasia or esophageal adenocarcinoma, 15% had low-grade dysplasia, and 14% had nondysplastic BE. Of patients treated, 448 (76%) were assessed after RFA. Fifty-five percent of patients underwent endoscopic mucosal resection before RFA. The median time to CRIM was 22 months, with 56% of patients in CRIM by 24 months. Increasing age and length of BE segment were associated with longer times to CRIM. Twenty-four months after CRIM, the incidence of recurrence was 33%; 22% of all recurrences observed were dysplastic BE. There were no demographic or endoscopic factors associated with recurrence. Complications developed in 6.5% of subjects treated with RFA; strictures were the most common complication. Of patients with BE treated by RFA, 56% were in complete remission after 24 months. However, 33% of these patients had disease recurrence within the next 2 years. Most recurrences were nondysplastic and endoscopically manageable, but continued surveillance after RFA is essential.


Mental Health Functioning in the Human Rights Field: Findings from an International Internet-Based Survey.
Human rights advocates play a critical role in promoting respect for human rights world-wide, and engage in a broad range of strategies, including documentation of rights violations, monitoring, press work and report-writing, advocacy, and litigation. However, little is known about the impact of human rights work on the mental health of human rights advocates. This study examined the mental health profile of human rights advocates and risk factors associated with their psychological functioning. 346 individuals currently or previously working in the field of human rights completed an internet-based survey regarding trauma exposure, depression, posttraumatic stress disorder (PTSD), resilience and occupational burnout. PTSD was measured with the Posttraumatic Stress Disorder Checklist-Civilian Version (PCL-C) and depression was measured with the Patient History Questionnaire-9 (PHQ-9). These findings revealed that among human rights advocates that completed the survey, 19.4% met criteria for PTSD, 18.8% met criteria for subthreshold PTSD, and 14.7% met criteria for depression. Multiple linear regressions revealed that after controlling for symptoms of depression, PTSD symptom severity was predicted by human rights-related trauma exposure, perfectionism and negative self-appraisals about human rights work. In addition, after controlling for symptoms of PTSD, depressive symptoms were predicted by perfectionism and lower levels of self-efficacy. Survey responses also suggested high levels of resilience: 43% of responders reported minimal symptoms of PTSD. Although survey responses suggest that many human rights workers are resilient, they also suggest that human rights work is associated with elevated rates of PTSD and depression. The field of human rights would benefit from further empirical research, as well as additional education and training programs in the workplace about enhancing resilience in the context of human rights work.


The frequency and severity of extinction after stroke affecting different vascular territories.
We examined the frequency and severity of visual versus tactile extinction based on data from a large group of sub-acute patients (n=454) with strokes affecting different vascular territories. After right hemisphere damage visual and tactile extinction were equally common. However, after left hemisphere damage tactile extinction was more common than visual. The frequency of extinction was significantly higher in patients with right compared to left hemisphere damage in both visual and tactile modalities but this held only for strokes affecting the MCA and PCA territories and not for strokes affecting other vascular territories. Furthermore, the severity of extinction did not differ as a function of either the stimulus modality (visual versus tactile), the affected hemisphere (left versus right) or the stroke territory (MCA, PCA or other vascular territories). We conclude that the frequency but not severity of extinction in both modalities relates to the side of damage (i.e. left versus right hemisphere) and the vascular territories affected by the stroke, and that left hemisphere dominance for motor control may link to the greater incidence of tactile than visual extinction after left hemisphere stroke. We discuss the implications of our findings for understanding hemispheric lateralization within visuospatial attention networks.


Cortex-sparing infarctions in patients with occlusion of the middle cerebral artery.
In patients with a middle cerebral artery (MCA) occlusion, the involvement of the cortex may be affected by the presence of leptomeningeal anastomoses between the cerebral arteries. The authors enrolled consecutive patients with acute infarctions in the MCA territory and MCA occlusion on angiographic studies. Infarct patterns were classified into three categories based on the extent of cortical surface involvement: total cortex (TC), partial cortex (PC) and no cortex (NC). The authors analysed the infarction patterns by stroke subtype, and investigated factors that resulted in cortex sparing. Out of 73 total patients, cortex-sparing infarctions were seen in 53 patients (72.6%, NC in 39 (53.5%) and PC in 14 (19.1%)). The extent of cortical involvement differed according to stroke subtype (p=0.036). TC was more frequent (42.9% vs 22.2%), and PC was less frequent (10.7% vs 27.9%, p=0.037) in cardioembolism than large-artery atherosclerosis. However, the proportion of patients with complete cortical sparing (NC) was similar between cardioembolism and large-artery atherosclerosis (46.4% vs 49.9%). In the upstream of leptomeningeal collateral arteries, the extent of cortical involvement was associated with significant stenosis of the ipsilateral anterior or posterior cerebral artery (p=0.011). This study suggests that pre-existing arteriolar connections, which may cover almost entire cortical surfaces of the MCA territory, exist in many patients. The findings also suggest that the extent of cortical involvement is different between stroke subtypes, and is critically affected by the status of upstream collateral arteries.


Increased mitochondrial activity in a novel IDH1-R132H mutant human oligodendroglioma xenograft model: in situ detection of 2-HG and α-KG.
Point mutations in genes encoding NADP+-dependent isocitrate dehydrogenases (especially IDH1) are common in lower grade diffuse gliomas and secondary glioblastomas and occur early during tumor development. The contribution of these mutations to gliomagenesis is not completely understood and research is hampered by the lack of relevant tumor models. We previously described the development of the patient-derived high-grade oligodendroglioma xenograft model E478 that carries the commonly occurring IDH1-R132H mutation. We here report on the analyses of E478 xenografts at the genetic, histologic and metabolic level. LC-MS and in situ mass spectrometric imaging by LESA-nano ESI-FTICR revealed high levels of the proposed oncometabolite D-2-hydroxyglutarate (D-2HG), the product of enzymatic conversion of α-ketoglutarate (α-KG) by IDH1-R132H, in the tumor but not in surrounding brain parenchyma. α-KG levels and total NADP+-dependent IDH activity were similar in IDH1-mutant and -wildtype xenografts, demonstrating that IDH1-mutated cancer cells maintain α-KG levels. Interestingly, IDH1-mutant tumor cells in vivo present with high densities of mitochondria and increased levels of mitochondrial activity as compared to IDH1-wildtype xenografts. It is not yet clear whether this altered mitochondrial activity is a driver or a consequence of tumorigenesis. The oligodendroglioma model presented here is a valuable model for further functional elucidation of the effects of IDH1 mutations on tumor metabolism and may aid in the rational development of novel therapeutic strategies for the large subgroup of gliomas carrying IDH1 mutations.


Functional interaction of hormone-sensitive lipase and perilipin in lipolysis.
Adipocyte lipolysis is controlled by complex interactions of lipases, cofactors, and structural proteins associated with lipid droplets. Perilipin (Plin) A is a major droplet-associated protein that functions as a scaffold, both suppressing basal and facilitating cAMP-dependent protein kinase (PKA)-stimulated lipolysis. Plin is required for the translocation of hormone-sensitive lipase (HSL) from the cytosol to lipid droplets upon stimulation. In these studies, we provide direct evidence for a physical interaction of HSL with Plin. By coexpressing HSL with truncation mutations of Plin, we demonstrate using coimmunoprecipitation that HSL can interact with an N-terminal region located between amino acids 141 and 200 of Plin A as well as with a C-terminal region located between amino acids 406 and 480. The N-terminal construct, Plin 1-200, which does not associate with lipid droplets but interacts with HSL, can function as a dominant negative for PKA-stimulated lipolysis. Using confocal microscopy of Plin truncations, we demonstrate that sequences between amino acids 463 and 517 may be important for or participate in lipid targeting. The results suggest the translocation of HSL to the lipid droplet occurs by virtue of Plin localization to the surface of lipid droplets and a physical interaction of HSL occurring with sequences within the N-terminal region of Plin.


Characterization of Bacillus Subtilis Viruses vB_BsuM-Goe2 and vB_BsuM-Goe3.
The Spounavirinae viruses are ubiquitous in nature and have an obligatory virulent lifestyle. They infect Firmicutes, a bacterial phylum containing an array of environmental non-pathogenic and pathogenic organisms. To expand the knowledge of this viral subfamily, new strains were isolated and investigated in this study. Here we present two new viruses, vB_BsuM-Goe2 and vB_BsuM-Goe3, isolated from raw sewage and infecting Bacillus species. Both were morphologically classified via transmission electron microscopy (TEM) as members of the Spounavirinae subfamily belonging to the Myoviridae family. Genomic sequencing and analyses allowed further affiliation of vB_BsuM-Goe2 to the SPO1-like virus group and vB_BsuM-Goe3 to the Bastille-like virus group. Experimentally determined adsorption constant, latency period, burst size and host range for both viruses revealed different survival strategies. Thus vB_BsuM-Goe2 seemed to rely on fewer host species compared to vB_BsuM-Goe3, but efficiently recruits those. Stability tests pointed out that both viruses are best preserved in LB-medium or TMK-buffer at 4 or 21 °C, whereas cryopreservation strongly reduced viability.


Development of a universal approach to increase physical activity among adolescents: the GoActive intervention.
To develop a physical activity (PA) promotion intervention for adolescents using a process addressing gaps in the literature while considering participant engagement. We describe the initial development stages; (1) existing evidence, (2) large scale opinion gathering and (3) developmental qualitative work, aiming (A) to gain insight into how to increase PA among the whole of year 9 (13-14 years-old) by identifying elements for intervention inclusion (B) to improve participant engagement and (C) to develop and refine programme design. Relevant systematic reviews and longitudinal analyses of change were examined. An intervention was developed iteratively with older adolescents (17.3 ± 0.5 years) and teachers, using the following process: (1) focus groups with (A) adolescents (n=26) and (B) teachers (n=4); (2) individual interviews (n=5) with inactive and shy adolescents focusing on engagement and programme acceptability. Qualitative data were analysed thematically. Limitations of the existing literature include lack of evidence on whole population approaches, limited adolescent involvement in intervention development, and poor participant engagement. Qualitative work suggested six themes which may encourage adolescents to do more PA; choice, novelty, mentorship, competition, rewards and flexibility. Teachers discussed time pressures as a barrier to encouraging adolescent PA and suggested between-class competition as a strategy. GoActive aims to increase PA through increased peer support, self-efficacy, group cohesion, self-esteem and friendship quality, and is implemented in tutor groups using a student-led tiered-leadership system. We have followed an evidence-based iterative approach to translate existing evidence into an adolescent PA promotion intervention. Qualitative work with adolescents and teachers supported intervention design and addressed lack of engagement with health promotion programmes within this age group. Future work will examine the feasibility and effectiveness of GoActive to increase PA among adolescents while monitoring potential negative effects. The approach developed is applicable to other population groups and health behaviours. ISRCTN31583496.


Tools from viruses: bacteriophage successes and beyond.
Viruses are ubiquitous and can infect any of the three existing cellular lineages (Archaea, Bacteria and Eukarya). Despite the persisting negative public perception of these entities, scientists learnt how to domesticate some of them. The study of molecular mechanisms essential to the completion of viral cycles has greatly contributed to deciphering fundamental processes in biology. Nowadays, viruses have entered the biotechnological era and numerous applications have already been developed. Viral-derived tools are used to manipulate genetic information, detect, diagnose, control and cure infectious diseases, or even design new structural assemblies. With the recent advances in the field of metagenomics, an overwhelming amount of information on novel viruses has become available. As current tools have been historically developed from a limited number of viruses, the potential of discoveries from new archaeal, bacterial and eukaryotic viruses may be limited only by our understanding of the multiple facets of viral cycles.


Favorable outcomes with machine perfusion and longer pump times in kidney transplantation: a single-center, observational study.
Hypothermic machine perfusion (MP) preservation is used for all deceased donor kidney transplants at our center. Kidneys are placed in cold storage at retrieval, then transferred to MP on arrival. Because a lack of consensus regarding optimal use of MP still exists, we evaluated the overall impact of using MP at our center and the prognostic value of MP (Pump) time. We retrospectively analyzed 339 adult, primary deceased donor kidney transplant recipients who were pooled across three prospective, randomized immunosuppression trials (since 2000) at our center. In addition to providing overall results for delayed graft function (DGF) (requirement for dialysis in the first week), slow graft function (SGF), first biopsy-proven acute rejection (BPAR), and graft failure, stepwise logistic and Cox regression analyses were used to determine the prognostic value of pump time, particularly after controlling for other significant prognosticators. Mean cold storage and pump times were 6.6 and 26.7 hr, consistent across immunosuppression protocols. Overall DGF and SGF rates were 4.4% (15/339) and 12.1% (41/339). DGF was equally low for pump time less than 24 vs. more than or equal to 24 hr, 5.2% (6/116) vs. 4.0% (9/223) (P=0.63), with similar results after adjusting for known DGF predictors. A significantly lower first BPAR rate was observed for longer pump time (as a continuous variable) among more immunologically active recipients (those having more risk factors: DGF, age <50 yr, and non-white) (univariable P=0.005; multivariable P=0.009), with an estimated hazard ratio of 0.43 (P=0.006) favoring pump time more than or equal to 24 hr among those with more than or equal to two risk factors. CONCLUSIONS.: In this single-center, observational study, MP with prolonged pump times was associated with low DGF/SGF and first BPAR rates, supporting continued use of MP.


Oxidative DNA damage during sleep periods among nightshift workers.
Oxidative DNA damage may be increased among nightshift workers because of suppression of melatonin, a cellular antioxidant, and/or inflammation related to sleep disruption. However, oxidative DNA damage has received limited attention in previous studies of nightshift work. From two previous cross-sectional studies, urine samples collected during a night sleep period for 217 dayshift workers and during day and night sleep (on their first day off) periods for 223 nightshift workers were assayed for 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, using high-performance liquid chromatography with electrochemical detection. Urinary measures of 6-sulfatoxymelatonin (aMT6s), a marker of circulating melatonin levels, and actigraphy-based sleep quality data were also available. Nightshift workers during their day sleep periods excreted 83% (p=0.2) and 77% (p=0.03) of the 8-OH-dG that dayshift workers and they themselves, respectively, excreted during their night sleep periods. Among nightshift workers, higher aMT6s levels were associated with higher urinary 8-OH-dG levels, and an inverse U-shaped trend was observed between 8-OH-dG levels and sleep efficiency and sleep duration. Reduced excretion of 8-OH-dG among nightshift workers during day sleep may reflect reduced functioning of DNA repair machinery, which could potentially lead to increased cellular levels of oxidative DNA damage. Melatonin disruption among nightshift workers may be responsible for the observed effect, as melatonin is known to enhance repair of oxidative DNA damage. Quality of sleep may similarly impact DNA repair. Cellular levels of DNA damage will need to be evaluated in future studies to help interpret these findings.


Comparative Proteomics Analysis of Four Commonly Used Methods for Identification of Novel Plasma Membrane Proteins.
Plasma membrane proteins perform a variety of important tasks in the cells. These tasks can be diverse as carrying nutrients across the plasma membrane, receiving chemical signals from outside the cell, translating them into intracellular action, and anchoring the cell in a particular location. When these crucial roles of plasma membrane proteins are considered, the need for their characterization becomes inevitable. Certain characteristics of plasma membrane proteins such as hydrophobicity, low solubility, and low abundance limit their detection by proteomic analyses. Here, we presented a comparative proteomics study in which the most commonly used plasma membrane protein enrichment methods were evaluated. The methods that were utilized include biotinylation, selective CyDye labeling, temperature-dependent phase partition, and density-gradient ultracentrifugation. Western blot analysis was performed to assess the level of plasma membrane protein enrichment using plasma membrane and cytoplasmic protein markers. Quantitative evaluation of the level of enrichment was performed by two-dimensional electrophoresis (2-DE) and benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE) from which the protein spots were cut and identified. Results from this study demonstrated that density-gradient ultracentrifugation method was superior when coupled with 16-BAC/SDS-PAGE. This work presents a valuable contribution and provides a future direction to the membrane sub-proteome research by evaluating commonly used methods for plasma membrane protein enrichment.


Hydrogels from phospholipid vesicles.
It is shown that phospholipid dispersions with a few percent of diacylphosphocholine PC in water can be swollen to single-phase lyotropic liquid crystalline Lα-phases by the addition of co-solvents like glycerol, 1,3-butyleneglycol BG or 1,2-propyleneglycol PG. The birefringent Lα-phases contain small unilamellar and multilamellar vesicles if the temperature of the samples is above the Krafft-Temperature Tm of the phospholipid. When such transparent birefringent viscous samples are cooled down below Tm the samples are transformed into birefringent gels. Cryo-TEM and FF-TEM measurements show that the bilayers of the vesicles are transformed from the liquid to the crystalline state during the transformation while the vesicle structure remains. The bilayers of the crystalline vesicles form adhesive contacts in the gel. Pulsed-field gradient NMR measurements show that two different kinds of water or co-solvent can be distinguished in the gels. One type of solvent molecules can diffuse like normal solvent in a continuous bulk phase. A second type of water diffuses much more slowly. This type of solvent is obviously trapped in the vesicles. The permeability of the crystalline vesicles for water and solvent molecules is much lower in the crystalline state than in the fluid state. Maximum swelling of the diacylphosphocholin dispersions occurs when the refractive index of the solvent is matched to the refractive index of the bilayers. The attraction between the bilayers is at a minimum in this state and the liquid crystalline Lα-phase's undulation forces between the bilayers push the bilayers apart. On transformation to the gel state the crystalline bilayers assume a high elastic bending rigidity. Undulations of the bilayers are now suppressed, and the bilayers can form adhesive contacts. Oscillating rheological measurements show that the gels with only 1% of phospholipids can have a storage modulus of 1000Pa. The gels are very brittle. They break when they are deformed by a few percent.


The protective effect of blueberry anthocyanins against perfluorooctanoic acid-induced disturbance in planarian (Dugesia japonica).
The influence of blueberry anthocyanins on perfluorooctanoic acid (PFOA)-induced stress response in planarian mitochondria was investigated. PFOA at 15mg/L and anthocyanins at 10 or 20mg/L were individually and simultaneously administered to planarians for up to 10d. The results showed PFOA treatment induced an increase in mitochondrial permeability transition pore opening and a decrease antioxidant capacity and enzyme activities. In anthocyanin treated animals, the activity of succinate dehydrogenase, cytochrome oxidase and monoamine oxidase increased, but mitochondrial permeability transition pore opening decreased and total antioxidant capacity increased. An improvement in above-mentioned physiological and biochemical parameters was found in the combined PFOA and anthocyanin treated animals, in a dose-dependent manner. Anthocyanins attenuated the PFOA induced toxicity; antioxidant capacity and enzyme activities are involved in the protective mechanism of anthocyanins.


Emergence and Spread of Basal Lineages of Yersinia pestis during the Neolithic Decline.
Between 5,000 and 6,000 years ago, many Neolithic societies declined throughout western Eurasia due to a combination of factors that are still largely debated. Here, we report the discovery and genome reconstruction of Yersinia pestis, the etiological agent of plague, in Neolithic farmers in Sweden, pre-dating and basal to all modern and ancient known strains of this pathogen. We investigated the history of this strain by combining phylogenetic and molecular clock analyses of the bacterial genome, detailed archaeological information, and genomic analyses from infected individuals and hundreds of ancient human samples across Eurasia. These analyses revealed that multiple and independent lineages of Y. pestis branched and expanded across Eurasia during the Neolithic decline, spreading most likely through early trade networks rather than massive human migrations. Our results are consistent with the existence of a prehistoric plague pandemic that likely contributed to the decay of Neolithic populations in Europe.


School difficulties at adolescence in a regional cohort of children who were extremely low birth weight.
To compare measures of psychometric assessment and school difficulties in a cohort of extremely low birth weight (ELBW) teenagers and term controls, and to determine whether there is stability in psychometric measures between age 8 and the teen years. Longitudinal follow-up; geographically defined region. 150 of 169 (89%) ELBW survivors born between 1977 and 1982 and 124 of 145 (86%) sociodemographically matched term controls between 12 and 16 years of age. Psychometric measures: Wechsler Intelligence Scale for Children-Revised, Wide Range Achievement Test-Revised, and a validated parent questionnaire. Neurosensory impairments were present in 28% of ELBW and 1% of controls. The mean Wechsler Intelligence Scale for Children-Revised scores were ELBW: 89 +/- 19 and controls: 102 +/- 13. ELBW children did less well on Wide Range Achievement Test-Revised Reading, Spelling, and Arithmetic measures with mean scores in the range from 75 to 85. ELBW children <750 g were more disadvantaged, compared with those >/=750 g. A significantly higher proportion of ELBW children were receiving special educational assistance and/or had repeated a grade (ELBW: 58%; controls: 13%; odds ratio: 9.0). Paired analysis of within-cohort data at age 8 and teen years showed that for both cohorts Arithmetic scores declined, but there were small improvements in other measures, predominantly in the term children. Differences of 13 to 18 points in psychometric measures in ELBW teens compared with controls are both statistically significant and clinically relevant. Decreasing birth weight was associated with increased risk on all measures. The high utilization of special educational resources has economic implications, and the incremental cost attributable to being extremely premature needs to be determined.


Cannabinoids: the lows and the highs of chemotherapy-induced nausea and vomiting.
Despite remaining one of the most widely abused drugs worldwide, Cannabis sativa exhibits remarkable medicinal properties. The phytocannabinoids, cannabidiol and Δ-9-tetrahydrocannabinol, reduce nausea and vomiting, particularly during chemotherapy. This is attributed to their ability to reduce the release of serotonin from enterochromaffin cells in the small intestine, which would otherwise orchestrate the vomiting reflex. Although there are many preclinical and clinical studies on the effects of Δ-9-tetrahydrocannabinol during nausea and vomiting, little is known about the role that cannabidiol plays in this scenario. Since cannabidiol does not induce psychotropic effects, in contrast to other cannabinoids, its use as an anti-emetic is of great interest. This review aims to summarize the available literature on cannabinoid use, with a specific focus on the nonpsychotropic drug cannabidiol, as well as the roles that cannabinoids play in preventing several other adverse side effects of chemotherapy including organ toxicity, pain and loss of appetite.


Systematic literature review on the effects of occupational safety and health (OSH) interventions at the workplace.
Objectives The aim of this review was to assess the evidence that occupational safety and health (OSH) legislative and regulatory policy could improve the working environment in terms of reduced levels of industrial injuries and fatalities, musculoskeletal disorders, worker complaints, sick leave and adverse occupational exposures. Methods A systematic literature review covering the years 1966‒2017 (February) was undertaken to capture both published and gray literature studies of OSH work environment interventions with quantitative measures of intervention effects. Studies that met specified in- and exclusion criteria went through an assessment of methodological quality. Included studies were grouped into five thematic domains: (i) introduction of OHS legislation, (ii) inspection/enforcement activity, (iii) training, such as improving knowledge, (iv), campaigns, and (v) introduction of technical device, such as mechanical lifting aids. The evidence synthesis was based on meta-analysis and a modified Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. Results The search for peer-reviewed literature identified 14 743 journal articles of which 45 fulfilled the inclusion criteria and were eligible for meta-analysis. We identified 5181 articles and reports in the gray literature, of which 16 were evaluated qualitatively. There was moderately strong evidence for improvement by OHS legislation and inspections with respect to injuries and compliance. Conclusions This review indicates that legislative and regulatory policy may reduce injuries and fatalities and improve compliance with OHS regulation. A major research gap was identified with respect to the effects of OSH regulation targeting psychological and musculoskeletal disorders.


Association of breast cancer risk loci with breast cancer survival.
The survival of breast cancer patients is largely influenced by tumor characteristics, such as TNM stage, tumor grade and hormone receptor status. However, there is growing evidence that inherited genetic variation might affect the disease prognosis and response to treatment. Several lines of evidence suggest that alleles influencing breast cancer risk might also be associated with breast cancer survival. We examined the associations between 35 breast cancer susceptibility loci and the disease over-all survival (OS) in 10,255 breast cancer patients from the National Cancer Institute Breast and Prostate Cancer Cohort Consortium (BPC3) of which 1,379 died, including 754 of breast cancer. We also conducted a meta-analysis of almost 35,000 patients and 5,000 deaths, combining results from BPC3 and the Breast Cancer Association Consortium (BCAC) and performed in silico analyses of SNPs with significant associations. In BPC3, the C allele of LSP1-rs3817198 was significantly associated with improved OS (HRper-allele =0.70; 95% CI: 0.58-0.85; ptrend = 2.84 × 10(-4) ; HRheterozygotes = 0.71; 95% CI: 0.55-0.92; HRhomozygotes = 0.48; 95% CI: 0.31-0.76; p2DF = 1.45 × 10(-3) ). In silico, the C allele of LSP1-rs3817198 was predicted to increase expression of the tumor suppressor cyclin-dependent kinase inhibitor 1C (CDKN1C). In the meta-analysis, TNRC9-rs3803662 was significantly associated with increased death hazard (HRMETA =1.09; 95% CI: 1.04-1.15; ptrend = 6.6 × 10(-4) ; HRheterozygotes = 0.96 95% CI: 0.90-1.03; HRhomozygotes = 1.21; 95% CI: 1.09-1.35; p2DF =1.25 × 10(-4) ). In conclusion, we show that there is little overlap between the breast cancer risk single nucleotide polymorphisms (SNPs) identified so far and the SNPs associated with breast cancer prognosis, with the possible exceptions of LSP1-rs3817198 and TNRC9-rs3803662.


[A case of pancreatic metastasis from renal cell carcinoma 27 years after nephrectomy].
A 78-year-old man was admitted to Ogikubo Hospital for pancreatic tumors detected by computed tomography (CT). The patient had undergone right nephrectomy for renal cell carcinoma (RCC) 27 years previously. Dynamic CT revealed a hypervascular mass in the pancreatic head and a cystic mass in the pancreatic body that were approximately 35 mm and 20 mm in size, respectively. Total pancreatectomy and splenectomy were performed. Histological examination of the resected specimens revealed metastatic tumors from RCC and they were diagnosed as clear cell type. Metastatic carcinoma of the pancreas is uncommon. Pancreatic metastasis from RCC is rare; however, it could occur many years after the initial diagnosis and treatment of the primary tumor. A long and careful follow-up that includes examination of the pancreas is mandatory after nephrectomy for RCC. In this paper, we discuss a case of RCC metastasis to the pancreas and report it in the literature.


Risk factors for requirement of filtration surgery after vitrectomy in patients with proliferative diabetic retinopathy.
We retrospectively reviewed patients with postoperative neovascular glaucoma (NVG) after vitrectomy for proliferative diabetic retinopathy to investigate how variables assessed before, during, and after vitrectomy are associated with the requirement for filtration surgery. The subjects in this retrospective, observational, comparative study were 55 consecutive patients (61 eyes) who underwent vitrectomy for proliferative diabetic retinopathy at Toho University Sakura Medical Center between December 2011 and November 2016, were followed up for at least 6 months after surgery, and developed NVG within 2 years after surgery. They comprised 44 men and 11 women of mean age 52.4±9.1 years, who were followed up for a mean 7.1±6.1 months. We collected data on the following 16 variables: sex, age, history of panretinal photocoagulation completed within 3 months before vitrectomy, presence/absence of a lens, obvious iris/angle neovascularization, tractional retinal detachment, diabetic macular edema, vitreous hemorrhage, visual acuity and intraocular pressure before vitrectomy and at the onset of NVG, glycated hemoglobin, fasting blood glucose, estimated glomerular filtration rate, and use of intraoperative gas tamponade. Logistic regression analysis with the backward elimination method identified preoperative fasting hyperglycemia (P=0.08), high intraocular pressure at the onset of NVG (P=0.04), and use of gas tamponade during vitrectomy (P=0.008) to be significant risk factors for requirement of filtration surgery. Preoperative fasting hyperglycemia, high intraocular pressure at the onset of NVG, and use of gas tamponade during vitrectomy predispose patients to require filtration surgery in the event of postoperative NVG.


Biochemical and biophysical approaches to probe CFTR structure.
The cystic fibrosis transmembrane regulator (CFTR) is a multi-domain integral membrane protein central to epithelial fluid secretion (see Chapter 21). Its activity is defective in the recessive genetic disease cystic fibrosis (CF). The most common CF-causing mutation is F508del in the first nucleotide binding domain (NBD1) of CFTR. This mutation is found on at least one allele of more than 90% of all CF patients. It is known to interfere with the trafficking/maturation of CFTR through the secretory pathway, leading to a loss-of-function at the plasma membrane. Notably, correction of the trafficking defect by addition of intragenic second-site suppressor mutations, or the alteration of bulk solvent conditions, such as by reducing the temperature or adding osmolytes, leads to appearance of functional channels at the membrane--thus, the rescued F508del-CFTR retains measurable function. High-resolution structural models of NBD1 from X-ray crystallographic data indicate that F508 is exposed on the surface of the domain in a position predicted by homologous ABC transporter structures to lie at the interface with the intracellular loops (ICLs) connecting the transmembrane spans. Determining the relative impact of the F508del mutation directly on NBD1 folding or on steps of domain assembly or both domain folding and assembly requires methods for evaluating the structure and stability of the isolated domain.


Oral bioavailability of glyphosate: studies using two intestinal cell lines.
Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.


Single-agent lapatinib for HER2-overexpressing advanced or metastatic breast cancer that progressed on first- or second-line trastuzumab-containing regimens.
This phase II study evaluated the efficacy and safety of lapatinib in patients with human epidermal growth factor receptor 2 (HER2)-positive advanced or metastatic breast cancer that progressed during prior trastuzumab therapy. Women with stage IIIB/IV HER2-overexpressing breast cancer were treated with single-agent lapatinib 1250 or 1500 mg once daily after protocol amendment. Tumor response according to RECIST was assessed every 8 weeks. HER2 expression was assessed in tumor tissue by immunohistochemistry and FISH. Seventy-eight patients were enrolled in the study. Investigator and independent review response rates [complete response (CR) or partial response (PR)] were 7.7% and 5.1%, and clinical benefit rates (CR, PR, or stable disease for >or=24 weeks) were 14.1% and 9.0%, respectively. Median time to progression was 15.3 weeks by independent review, and median overall survival was 79 weeks. The most common treatment-related adverse events were rash (47%), diarrhea (46%), nausea (31%), and fatigue (18%). Single-agent lapatinib has clinical activity with manageable toxic effects in HER2-overexpressing breast cancer that progressed on trastuzumab-containing therapy. Studies of lapatinib-based combination regimens with chemotherapy and other targeted therapies in metastatic and earlier stages of breast cancer are warranted.


Nasolacrimal obstruction after inferior meatus nasal antrostomy.
Transient epiphora following rhinoplasty or intranasal procedures is a common occurrence. Permanent nasolacrimal duct obstruction, however, is rare. This article documents four cases of nasolacrimal duct obstruction following intranasal antrostomy. Three patients were cured by dacryocystorhinostomy and a fourth refused surgery. The anatomy of the nasolacrimal duct in the inferior meatus has considerable variation. Although the duct typically opens in the inferior meatus immediately under the insertion of the inferior turbinate, the orifice can be a single hole, a slit, multiple holes, or a trough, and can be located anywhere from 30 to 40 mm dorsal to the anterior nares. We review the embryology and anatomy of the nasolacrimal orifice in the nose and make recommendations for safe surgery in the inferior meatus.


The Impact of Physical Activity on Mitigation of Health Care Costs Related to Diabetes Mellitus: Findings from Developed and Developing Settings.
Over the last decades, the prevalence of diabetes mellitus (DM) has been increasing globally such that nowadays the disease constitutes an important outcome related to early mortality among adults. In parallel with the high prevalence, healthcare costs related to DM treatment have increased significantly, exacerbating its burden on modern society. The scientific literature points out that obesity and physical inactivity have a central role in the development of most DM cases. In fact, either physical exercise practice or an increase in the level of physical activity, constitute relevant tools in the guidelines for treatment of the disease. On the other hand, the effect of physical activity on the economic consequences of DM is not completely clear. The identification of the actual burden of lifestyle changes on the reduction of healthcare costs related to DM is relevant, primarily for developing nations, where it could represent a cheaper strategy for treating the disease and its complications than paying for drug treatment, which is commonly related to collateral effects. That being said, the prevention of DM and other diseases and consequently the mitigation of the costs related to these outcomes seem to depend essentially on the promotion of healthy habits. The aim of the present review was therefore to discuss recent evidence on the effects of physical activity/exercise on mitigation of health care cost related to DM.


Analysis of allele-specific gene expression.
The analysis of allele-specific gene expression has been of long-standing interest in the study of genomic imprinting, but there is growing awareness that differences in allelic expression are widespread among autosomal nonimprinted genes. Recent research into cis-acting regulatory polymorphisms has utilized the analysis of allele-specific gene expression to identify functionally important regulatory haplotypes and specific genetic polymorphisms. Allele-specific effects are typically of modest magnitude, requiring techniques for analysis of high sensitivity and specificity. Here, strategic approaches to the analysis of allele-specific gene expression are reviewed with protocols for in vivo analysis. These include analysis of the relative allelic abundance of transcribed RNA and of transcription factor recruitment and Pol II loading by chromatin immunoprecipitation.


Multiple modes of proepicardial cell migration require heartbeat.
The outermost layer of the vertebrate heart, the epicardium, forms from a cluster of progenitor cells termed the proepicardium (PE). PE cells migrate onto the myocardium to give rise to the epicardium. Impaired epicardial development has been associated with defects in valve development, cardiomyocyte proliferation and alignment, cardiac conduction system maturation and adult heart regeneration. Zebrafish are an excellent model for studying cardiac development and regeneration; however, little is known about how the zebrafish epicardium forms. We report that PE migration occurs through multiple mechanisms and that the zebrafish epicardium is composed of a heterogeneous population of cells. Heterogeneity is first observed within the PE and persists through epicardium formation. Using in vivo imaging, histology and confocal microscopy, we show that PE cells migrate through a cellular bridge that forms between the pericardial mesothelium and the heart. We also observed the formation of PE aggregates on the pericardial surface, which were released into the pericardial cavity. It was previously reported that heartbeat-induced pericardiac fluid advections are necessary for PE cluster formation and subsequent epicardium development. We manipulated heartbeat genetically and pharmacologically and found that PE clusters clearly form in the absence of heartbeat. However, when heartbeat was inhibited the PE failed to migrate to the myocardium and the epicardium did not form. We isolated and cultured hearts with only a few epicardial progenitor cells and found a complete epicardial layer formed. However, pharmacologically inhibiting contraction in culture prevented epicardium formation. Furthermore, we isolated control and silent heart (sih) morpholino (MO) injected hearts prior to epicardium formation (60 hpf) and co-cultured these hearts with "donor" hearts that had an epicardium forming (108 hpf). Epicardial cells from donor hearts migrated on to control but not sih MO injected hearts. Epicardial cells stem from a heterogeneous population of progenitors, suggesting that the progenitors in the PE have distinct identities. PE cells attach to the heart via a cellular bridge and free-floating cell clusters. Pericardiac fluid advections are not necessary for the development of the PE cluster, however heartbeat is required for epicardium formation. Epicardium formation can occur in culture without normal hydrodynamic and hemodynamic forces, but not without contraction.


Identification of Nitrogen-Fixing Bradyrhizobium Associated With Roots of Field-Grown Sorghum by Metagenome and Proteome Analyses.
Sorghum (Sorghum bicolor) is cultivated worldwide for food, bioethanol, and fodder production. Although nitrogen fixation in sorghum has been studied since the 1970s, N2-fixing bacteria have not been widely examined in field-grown sorghum plants because the identification of functional diazotrophs depends on the culture method used. The aim of this study was to identify functional N2-fixing bacteria associated with field-grown sorghum by using "omics" approaches. Four lines of sorghum (KM1, KM2, KM4, and KM5) were grown in a field in Fukushima, Japan. The nitrogen-fixing activities of the roots, leaves, and stems were evaluated by acetylene reduction and 15N2-feeding assays. The highest nitrogen-fixing activities were detected in the roots of lines KM1 and KM2 at the late growth stage. Bacterial cells extracted from KM1 and KM2 roots were analyzed by metagenome, proteome, and isolation approaches and their DNA was isolated and sequenced. Nitrogenase structural gene sequences in the metagenome sequences were retrieved using two nitrogenase databases. Most sequences were assigned to nifHDK of Bradyrhizobium species, including non-nodulating Bradyrhizobium sp. S23321 and photosynthetic B. oligotrophicum S58T. Amplicon sequence and metagenome analysis revealed a relatively higher abundance (2.9-3.6%) of Bradyrhizobium in the roots. Proteome analysis indicated that three NifHDK proteins of Bradyrhizobium species were consistently detected across sample replicates. By using oligotrophic media, we purified eight bradyrhizobial isolates. Among them, two bradyrhizobial isolates possessed 16S rRNA and nif genes similar to those in S23321 and S58T which were predicted as functional diazotrophs by omics approaches. Both free-living cells of the isolates expressed N2-fixing activity in a semi-solid medium according to an acetylene reduction assay. These results suggest that major functional N2-fixing bacteria in sorghum roots are unique bradyrhizobia that resemble photosynthetic B. oligotrophicum S58T and non-nodulating Bradyrhizobium sp. S23321. Based on our findings, we discuss the N2-fixing activity level of sorghum plants, phylogenetic and genomic comparison with diazotrophic bacteria in other crops, and Bradyrhizobium diversity in N2 fixation and nodulation.


Role of 5-HT7 receptors in the immune system in health and disease.
In mammalians, serotonin (5-HT) has critical roles in the central nervous system (CNS), including mood stability, pain tolerance, or sleep patterns. However, the vast majority of serotonin is produced by intestinal enterochromaffin cells of the gastrointestinal tract and circulating blood platelets, also acting outside of the CNS. Serotonin effects are mediated through its interaction with 5-HT receptors (5-HTRs), a superfamily with a repertoire of at least fourteen well-characterized members. 5-HT7 receptors are the last 5-HTR member to be identified, with well-defined functions in the nervous, gastrointestinal, and vascular systems. The effects of serotonin on the immune response are less well understood. Mast cells are known to produce serotonin, while T cells, dendritic cells, monocytes, macrophages and microglia express 5-HT7 receptor. Here, we review the known roles of 5-HT7 receptors in the immune system, as well as their potential therapeutic implication in inflammatory and immune-mediated disorders.


A dynamic interaction between CD19 and the tetraspanin CD81 controls B cell co-receptor trafficking.
CD81 and its binding partner CD19 are core subunits of the B cell co-receptor complex. While CD19 belongs to the extensively studied Ig superfamily, CD81 belongs to a poorly understood family of four-pass transmembrane proteins called tetraspanins. Tetraspanins play important physiological roles by controlling protein trafficking and other processes. Here, we show that CD81 relies on its ectodomain to traffic CD19 to the cell surface. Moreover, the anti-CD81 antibody 5A6, which binds selectively to activated B cells, recognizes a conformational epitope on CD81 that is masked when CD81 is bound to CD19. Mutations of CD81 in this interface suppress its CD19 export activity. These data indicate that the CD81 - CD19 interaction is dynamically regulated upon B cell activation and this dynamism can be exploited to regulate B cell function. These results are not only valuable for understanding B cell biology, but also have important implications for understanding tetraspanin function generally.


NUMB does not impair growth and differentiation status of experimental gliomas.
The cell fate determinant NUMB orchestrates asymmetric cell division in flies and mammals and has lately been suggested to have a tumor suppressor function in breast and lung cancer. Here, we studied NUMB in the context of malignant gliomas. We used ectopic expression of NUMB in order to inhibit proliferation and induce differentiation in glioma cells by alteration of Notch, Hedgehog and p53 signaling. We found that NUMB is consistently expressed in glioma biopsies with predominance of NUMB2/4 isoforms as determined by isoform-specific real-time PCR and Western blotting. Upon lentiviral overexpression, in vitro proliferation rate and the grade of differentiation as assessed by morphology and expression of neural and glial markers remained unchanged. Orthotopic xenografts of NUMB-transduced human U87 glioma cells could be established in nude rats without impairing engraftment or causing significant changes in morphology based on magnetic resonance imaging (MRI). The previously reported alteration of Hedgehog and p53 signaling by NUMB could not be recapitulated in glioma cells. We thus show that in experimental gliomas, NUMB overexpression most likely does not exert a tumor suppressor function such as seen in epithelial cancers.