Sidestream smoke exposure increases the susceptibility of airway epithelia to adenoviral infection.
Although significant epidemiological evidence indicates that cigarette smoke exposure increases the incidence and severity of viral infection, the molecular mechanisms behind the increased susceptibility of the respiratory tract to viral pathogens are unclear. Adenoviruses are non-enveloped DNA viruses and important causative agents of acute respiratory disease. The Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for many adenoviruses. We hypothesized that cigarette smoke exposure increases epithelial susceptibility to adenovirus infection by increasing the abundance of apical CAR. Cultured human airway epithelial cells (CaLu-3) were used as a model to investigate the effect of sidestream cigarette smoke (SSS), mainstream cigarette smoke (MSS), or control air exposure on the susceptibility of polarized respiratory epithelia to adenoviral infection. Using a Cultex air-liquid interface exposure system, we have discovered novel differences in epithelial susceptibility between SSS and MSS exposures. SSS exposure upregulates an eight-exon isoform of CAR and increases adenoviral entry from the apical surface whilst MSS exposure is similar to control air exposure. Additionally, the level of cellular glycogen synthase kinase 3β (GSK3β) is downregulated by SSS exposure and treatment with a specific GSK3β inhibitor recapitulates the effects of SSS exposure on CAR expression and viral infection. This is the first time that SSS exposure has been shown to directly enhance the susceptibility of a polarized epithelium to infection by a common respiratory viral pathogen. This work provides a novel understanding of the impact of SSS on the burden of respiratory viral infections and may lead to new strategies to alter viral infections. Moreover, since GSK3β inhibitors are under intense clinical investigation as therapeutics for a diverse range of diseases, studies such as these might provide insight to extend the use of clinically relevant therapeutics and increase the understanding of potential side effects.


Prothrombin G20210A gene mutation, heparin cofactor II defects, primary (essential) thrombocythemia, and thrombohemorrhagic manifestations.
This article addresses the issue of thromboembolic disorders associated with the prothrombin G20210A gene mutation, with heparin cofactor II (HC-II) defects and with primary (essential) thrombocythemia. The prothrombin gene mutation is of recent discovery, is inherited as an autosomal dominant disorder, and seems to be highly prevalent in the general white population. The incidence is almost as high as that known for factor V Leiden. Both venous and arterial thromboses are noted, especially deep venous thrombosis, including cerebral venous events and myocardial infarction. As with other congenital thrombophilic states, additional risk factors or multiple defects seem to precipitate the events. Although initially elevated plasma prothrombin levels were described in these patients, this is no longer valid for all patients. At this time there is no easy screening test to detect this defect, but, because of the high prevalence, prothrombin G20210A gene mutation should routinely be assayed for in thrombophilic patients. The association between HC-II defects and thromboembolism is more controversial, and reports both confirming and denying this association have been described. The congenital form of HC-II defect is autosomal dominant. HC-II can be determined by its activity and immunologically. HC-II defects very likely play a role in conjunction with other congenital or acquired defects. Acquired HC-II defects are found in association with systemic disseminated intravascular coagulation (DIC) but not with local activation of the hemostasis system. HC-II levels are also decreased in preeclamptic women, and newborns have physiologically low levels. HC-II defects in thrombophilic patients should be considered after the more common disorders have been ruled out. Primary (essential) thrombocythemia can be associated with both thromboembolic events and bleeding. Typical thrombotic manifestations are erythromelalgia and microvascular thrombosis. Also, pregnant females suffer high rates of complications, such as spontaneous abortion. A number of treatment modalities are at present available to not only decrease platelet counts but also manage thromboembolic events.


Application of stable isotope analysis to study temporal changes in foraging ecology in a highly endangered amphibian.
Understanding dietary trends for endangered species may be essential to assessing the effects of ecological disturbances such as habitat modification, species introductions or global climate change. Documenting temporal variation in prey selection may also be crucial for understanding population dynamics. However, the rarity, secretive behaviours and obscure microhabitats of some endangered species can make direct foraging observations difficult or impossible. Furthermore, the lethality or invasiveness of some traditional methods of dietary analysis (e.g. gut contents analysis, gastric lavage) makes them inappropriate for such species. Stable isotope analysis facilitates non-lethal, indirect analysis of animal diet that has unrealized potential in the conservation of endangered organisms, particularly amphibians. I determined proportional contributions of aquatic macroinvertebrate prey to the diet of an endangered aquatic salamander Eurycea sosorum over a two-year period using stable isotope analysis of (13/12)C and (15/14)N and the Bayesian stable isotope mixing model SIAR. I calculated Strauss' dietary electivity indices by comparing these proportions with changing relative abundance of potential prey species through time. Stable isotope analyses revealed that a previously unknown prey item (soft-bodied planarian flatworms in the genus Dugesia) made up the majority of E. sosorum diet. Results also demonstrate that E. sosorum is an opportunistic forager capable of diet switching to include a greater proportion of alternative prey when Dugesia populations decline. There is also evidence of intra-population dietary variation. Effective application of stable isotope analysis can help circumvent two key limitations commonly experienced by researchers of endangered species: the inability to directly observe these species in nature and the invasiveness or lethality of traditional methods of dietary analysis. This study illustrates the feasibility of stable isotope analysis in identifying preferred prey species that can be used to guide conservation management of both wild and captive food sources for endangered species.


Early Development of Neural Speech Encoding Depends on Age but Not Native Language Status: Evidence From Lexical Tone.
We investigated the development of early-latency and long-latency brain responses to native and non-native speech to shed light on the neurophysiological underpinnings of perceptual narrowing and early language development. Specifically, we postulated a two-level process to explain the decrease in sensitivity to non-native phonemes toward the end of infancy. Neurons at the earlier stages of the ascending auditory pathway mature rapidly during infancy facilitating the encoding of both native and non-native sounds. This growth enables neurons at the later stages of the auditory pathway to assign phonological status to speech according to the infant's native language environment. To test this hypothesis, we collected early-latency and long-latency neural responses to native and non-native lexical tones from 85 Cantonese-learning children aged between 23 days and 24 months, 16 days. As expected, a broad range of presumably subcortical early-latency neural encoding measures grew rapidly and substantially during the first two years for both native and non-native tones. By contrast, long-latency cortical electrophysiological changes occurred on a much slower scale and showed sensitivity to nativeness at around six months. Our study provided a comprehensive understanding of early language development by revealing the complementary roles of earlier and later stages of speech processing in the developing brain.


FLI-1 distinguishes Ewing sarcoma from small cell osteosarcoma and mesenchymal chondrosarcoma.
Small cell osteosarcoma and mesenchymal chondrosarcoma are 2 primary bone tumors with a small round blue cell component, which can mimic the appearance of Ewing sarcoma. Distinguishing these tumors from each other on biopsy material is important clinically, as optimal therapy differs according to the tumor type. However, separating these entities on morphology alone can be challenging. FLI-1 has been described to be a useful marker for Ewing sarcoma, particularly when hematolymphoid markers are negative. In small cell osteosarcoma and mesenchymal chondrosarcoma, the FLI-1 staining pattern has not been adequately characterized. Using a monoclonal FLI-1 antibody, nuclear immunoreactivity in tumor cells was evaluated in 10 small cell osteosarcomas, 10 mesenchymal chondrosarcomas, and 8 Ewing sarcomas, together with a number of other small, round, blue cell tumors. None of the small cell osteosarcomas or mesenchymal chondrosarcomas exhibited FLI-1 staining in the tumor cells, in contrast to the positive nuclear FLI-1 staining in the stromal endothelial cells. In comparison, 6 of the 8 Ewing sarcomas showed moderate-to-strong nuclear FLI-1 staining of the tumor cells in addition to strong staining of the stromal endothelial cell nuclei. With the exception of lymphoblastic lymphomas, FLI-1 positivity was not seen in the other small round blue cell tumors examined. These findings show that, in contrast to Ewing sarcoma, small cell osteosarcoma and mesenchymal chondrosarcoma lack FLI-1 immunoreactivity. FLI-1 is therefore useful in the differential diagnosis of small round blue cell tumors of the bone.


Extracting the mean size across the visual field in patients with mild, chronic unilateral neglect.
Previous studies suggest that normal vision pools information from groups of objects in a display to extract statistical summaries (e.g., mean size). Here we explored whether patients with mild, chronic left neglect were able to extract statistical summaries on the right and left sides of space in a typical manner. We tested four patients using a visual search task and varied the mean size of a group of circles within the display. On each trial, a single circle first appeared in the center of the screen (the target). This circle varied in size from trial to trial. Then a multi-item display appeared with circles of various sizes grouped together either on the left or right side of the display. The instructions were to search the circles and determine whether the target was present or not. The circles were always accompanied by a group of task-irrelevant triangles that appeared on the opposite side of the display. On half the trials, the mean size of the circles was the size of the target. On the other half the mean size was different from the target. The patients were not told that this was the case, and no explicit report of the statistics was required. The results showed that when the targets were absent patients produced more false alarms to the mean than non-mean size when the circles were on the left (neglected) side of the display. This finding demonstrates that statistical information was implicitly extracted from the left group of circles. However, summary statistics on the right side were not limited to the circles. Rather it appears that participants pooled the distractors with the target circles, yielding a skewed statistical summary on the right side. These findings are discussed as they relate to statistical summary processing, visual search and segregation of right and left items in patients with mild, chronic unilateral neglect.


Efficacy of triple therapies including ionising radiation, agonistic TRAIL antibodies and cisplatin.
The detection of molecular targeted agents is a milestone in cancer treatment. Despite the achievements, the efficacy of single targeted agents in combination with radiotherapy is limited by putative treatment resistance. We therefore tested a rationally designed triple therapy consisting of an agonistic antibody against either TRAIL-R1 (mapatumumab/HGS-ETR1) or TRAIL-R2 (lexatumumab/HGS-ETR2) in combination with the established chemotherapeutic drug cisplatin in a panel of solid tumour cell lines derived from head and neck as well as colorectal carcinomas. Induction of apoptosis after monotherapy, double or triple treatment was determined in FaDu (squamous cancer cell line of the head and neck), Colo205 and HCT116 cells (colorectal adenocarcinoma cell lines) by Hoechst 33342 stain. Double and triple therapies were compared using analysis of variance followed by post hoc tests. The degree of interaction was determined by 3D-isobologram analysis. A knockout variant of HCT116 was used to examine Bax-dependence of the triple therapy to gain insight into the underlying molecular signaling pathways possibly responsible for the observed effects. Dose-response relationships revealed different baseline activities of the modalities dependent on cell type. Triple therapy was more effective than double therapy in most cases according to the induction of apoptosis. Furthermore, a synergistic efficacy of the triple therapy was demonstrated in a subset of tumour cell lines. The efficacy of this multimodal approach was highly dependent on the presence of Bax. Our data suggest that targeted agents can be effectively added to existing multimodal therapy approaches which might open new perspectives in radiation oncology.


Early Experiences With COVID-19 Testing in Transplantation.
The early effects of coronavirus disease 2019 (COVID-19) on transplantation are dramatic: >75% of kidney and liver programs are either suspended or operating under major restrictions. To resume transplantation, it is important to understand the prevalence of COVID-19 among transplant recipients, donors, and healthcare workers (HCWs) and its associated mortality. To investigate this, we studied severe acute respiratory syndrome coronavirus 2 diagnostic test results among patients with end-stage renal disease or kidney transplants from the Johns Hopkins Health System (n = 235), and screening test results from deceased donors from the Southwest Transplant Alliance Organ Procurement Organization (n = 27), and donors, candidates, and HCWs from the National Kidney Registry and Viracor-Eurofins (n = 253) between February 23 and April 15, 2020. We found low rates of COVID-19 among donors and HCWs (0%-1%) who were screened, higher rates of diagnostic tests among patients with end-stage renal disease or kidney transplant (17%-20%), and considerable mortality (7%-13%) among those who tested positive. These findings suggest the threat of COVID-19 for the transplant population is significant and ongoing data collection and reporting is critical to inform transplant practices during and after the pandemic.


High-Intensity Interval Training Is Equivalent to Moderate-Intensity Continuous Training for Short- and Medium-Term Outcomes of Glucose Control, Cardiometabolic Risk, and Microvascular Complication Markers in Men With Type 2 Diabetes.
We sought to determine the efficacy of 12 weeks high-intensity interval training (HIIT), compared to moderate-intensity continuous training (MICT) on glucose control, cardiometabolic risk and microvascular complication markers in men living with type 2 diabetes (T2D). Both modalities were combined with resistance training (RT). Additionally, the study aimed to determine the medium-term durability of effects. After a 12-week, thrice weekly, training intervention incorporating either MICT+RT (n = 11) or HIIT+RT (n = 12), the study concluded with a 6-month follow-up analysis. The middle-aged study participants were obese, had moderate duration T2D and were taking multiple medications including insulin, statins and beta-blockers. Participants, randomized via the method of minimization, performed MICT (progressing to 26-min at 55% maximum estimated workload [eWLmax]) or HIIT (progressing to two variations in which twelve 1-min bouts at 95% eWLmax interspersed with 1-min recovery bouts, alternated with eight 30-s bouts at 120% eWLmax interspersed with 2:15 min recovery bouts) under supervision at an exercise physiology facility. To account for fixed and random effects within the study sample, mixed-effect models were used to determine the significance of change following the intervention and follow-up phases and to evaluate group*time interactions. Beyond improvements in aerobic capacity (P < 0.001) for both groups, both training modalities elicited similar group*time interactions (P > 0.05) while experiencing benefits for glycated hemoglobin (HbA1c; P = 0.01), subcutaneous adiposity (P < 0.001), and heart rate variability (P = 0.02) during the 12-week intervention. Adiposity (P < 0.001) and aerobic capacity (P < 0.001) were significantly maintained in both groups at the 6-month follow-up. In addition, during the intervention, participants in both MICT+RT and HIIT+RT experienced favorable reductions in their medication usage. The study reported the inter-individual variability of change within both groups, the exaggerated acute physiological responses (using exercise termination indicators) that occurred during the interventions as well as the incidence of precautionary respite afforded in such a study sample. To reduce hyperglycaemia, and prevent further deterioration of cardiometabolic risk and microvascular complication markers (in both the short- and medium-term), future strategies that integrate the adoption and maintenance of physical activity as a cornerstone in the treatment of T2M for men should (cognisant of appropriate supervision) include either structured MICT+RT, or HIIT+RT. Clinical Trials Registration Number: ACTRN12617000582358 http://www.anzctr.org.au/default.aspx.


Computational sensing of herpes simplex virus using a cost-effective on-chip microscope.
Caused by the herpes simplex virus (HSV), herpes is a viral infection that is one of the most widespread diseases worldwide. Here we present a computational sensing technique for specific detection of HSV using both viral immuno-specificity and the physical size range of the viruses. This label-free approach involves a compact and cost-effective holographic on-chip microscope and a surface-functionalized glass substrate prepared to specifically capture the target viruses. To enhance the optical signatures of individual viruses and increase their signal-to-noise ratio, self-assembled polyethylene glycol based nanolenses are rapidly formed around each virus particle captured on the substrate using a portable interface. Holographic shadows of specifically captured viruses that are surrounded by these self-assembled nanolenses are then reconstructed, and the phase image is used for automated quantification of the size of each particle within our large field-of-view, ~30 mm2. The combination of viral immuno-specificity due to surface functionalization and the physical size measurements enabled by holographic imaging is used to sensitively detect and enumerate HSV particles using our compact and cost-effective platform. This computational sensing technique can find numerous uses in global health related applications in resource-limited environments.


Child Passenger Safety.
Child passenger safety has dramatically evolved over the past decade; however, motor vehicle crashes continue to be the leading cause of death for children 4 years and older. This policy statement provides 4 evidence-based recommendations for best practices in the choice of a child restraint system to optimize safety in passenger vehicles for children from birth through adolescence: (1) rear-facing car safety seats as long as possible; (2) forward-facing car safety seats from the time they outgrow rear-facing seats for most children through at least 4 years of age; (3) belt-positioning booster seats from the time they outgrow forward-facing seats for most children through at least 8 years of age; and (4) lap and shoulder seat belts for all who have outgrown booster seats. In addition, a fifth evidence-based recommendation is for all children younger than 13 years to ride in the rear seats of vehicles. It is important to note that every transition is associated with some decrease in protection; therefore, parents should be encouraged to delay these transitions for as long as possible. These recommendations are presented in the form of an algorithm that is intended to facilitate implementation of the recommendations by pediatricians to their patients and families and should cover most situations that pediatricians will encounter in practice. The American Academy of Pediatrics urges all pediatricians to know and promote these recommendations as part of child passenger safety anticipatory guidance at every health supervision visit.


Methods for evaluating the benefits and harms of antenatal and newborn screening programmes adopted by health economic assessments: protocol for a systematic review.
Complex organisational arrangements are required to deliver antenatal and newborn screening programmes. Decision-makers consider the benefits and harms of screening when reviewing the evidence about these programmes. Economic evaluations contribute one important part of this assessment process. However, it is not fully understood what approaches health economic assessments have adopted to measure and value benefits and harms. This study aims to systematically review and critique the published and grey literature on methods for identifying, measuring and valuing the benefits and harms of antenatal and newborn screening adopted by health economic assessments. Nine bibliographic databases will be searched from 2000 onwards. These search strategies will be supplemented by manual reference searching of bibliographies, forward citation searching, contacts with experts, author searching and web searching for grey literature. Studies will be selected for review if they report health economic assessments of an antenatal or newborn screening programme. Assessments of title and abstracts and full reports will be undertaken independently with disagreements resolved through discussion. Data extraction will include fields to assess the reporting quality of the studies using the Consolidated Health Economic Evaluation Reporting Standards statement and a bespoke ancillary form to assess how benefits and harms have been accounted for. This is an evidence synthesis review from already published materials and hence ethics committee approval or written informed consent will not be required. Our results will be disseminated by publishing in high-impact peer-review journals and presenting at relevant conferences. CRD42020165236.


Molecular biology of high-grade gliomas: what should the clinician know?
The current World Health Organization classification system of primary brain tumors is solely based on morphologic criteria. However, there is accumulating evidence that tumors with similar histology have distinct molecular signatures that significantly impact treatment response and survival. Recent practice-changing clinical trials have defined a role for routine assessment of O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation in glioblastoma patients, especially in the elderly, and 1p and 19q codeletions in patients with anaplastic glial tumors. Recently discovered molecular alterations including mutations in IDH-1/2, epidermal growth factor receptor (EGFR), and BRAF also have the potential to become targets for future drug development. This article aims to summarize current knowledge on the molecular biology of high-grade gliomas relevant to daily practice.


DNA Barcode Reveals the Bycatch of Endangered Batoids Species in the Southwest Atlantic: Implications for Sustainable Fisheries Management and Conservation Efforts.
Today, elasmobranchs are one the most threatened vertebrate groups worldwide. In fact, at least 90% of elasmobranch species are listed in the International Union for Conservation of Nature (IUCN) Red List, while more than 40% are data-deficient. Although these vertebrates are mainly affected by unsustainable fishery activities, bycatch is also one of the major threats to sharks and batoids worldwide, and represents a challenge for both sustainable fishery management and for biodiversity and conservational efforts. Thus, in this study, DNA barcode methodology was used to identify the bycatch composition of batoid species from small-scale industrial fisheries in the southwest Atlantic and artisanal fisheries from southeast Brazil. A total of 228 individuals belonging to four Chondrichthyes orders, seven families, and at least 17 distinct batoid species were sequenced; among these individuals, 131 belonged to species protected in Brazil, 101 to globally threatened species, and some to species with trade restrictions provided by Appendix II of the Convention on International Trade in Endangered Species (CITES). These results highlight the impacts on marine biodiversity of bycatch by small-scale industrial and unmanaged artisanal fisheries from the southwest Atlantic, and support the implementation of DNA-based methodologies for species-specific identification in data-poor fisheries as a powerful tool for improving the quality of fisheries' catch statistics and for keeping precise bycatch records.


Cloning of the rat progesterone receptor gene 5'-region and identification of two functionally distinct promoters.
To examine some of the molecular mechanisms controlling transcription of the rat progesterone receptor (PR) gene, we have cloned and sequenced the 5'-region of the gene. Northern blot analyses with a series of probes identified two regions where distinct subsets of the multiple PR gene transcripts initiated, suggesting the presence of two promoters in the gene. Promoter activities for two gene fragments encompassing these regions, -131/+65 (P; distal) and +461/+675 (P'; proximal), were demonstrated in transient transfection experiments using reporter constructs containing the gene fragments linked individually upstream of the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of P-CAT or P'-CAT constructs containing two upstream GAL4 binding sites into primary cultures of rat uterine cells with a vector expressing a GAL4 DNA binding domain-VP16 activating region fusion protein resulted in a 10-fold increase in CAT activity relative to cells transfected with either reporter and a vector expressing only the GAL4 DNA binding domain. The estrogen inducibility of the promoter-CAT constructs was assessed by transfection into MCF-7 breast cancer cells, which contain high levels of estrogen receptor (ER). P'-CAT, but not P-CAT, was induced by estradiol (E2; 8-fold). In primary rat uterine cells, which contain lower levels of ER, P'-CAT required the addition of one upstream consensus estrogen response element (ERE) to be estrogen inducible, whereas P-CAT required the addition of two EREs. Point and deletion mutants of the proximal promoter region in the P'-CAT reporter, screened in MCF-7 cells, were used to identify a 20-base pair fragment (+617/+636) that retained the promoter activity and 50% of the estrogen inducibility of P'. This fragment contained an ERE-like sequence conserved in 8 of 10 positions relative to the consensus ERE. Two copies of this sequence conferred estrogen inducibility (4-fold) when placed upstream of the distal promoter in P-CAT. To examine ER-dependent stimulation of the two PR gene promoters by cAMP, P-CAT and P'-CAT reporter constructs containing two upstream consensus EREs were cotransfected into ER-negative 3T3 cells with an ER expression vector. Induction by E2 was greater than 50-fold for both constructs. Treatment of the cells with agents that increase intracellular cAMP levels, namely cholera toxin plus isobutyl methylxanthine, resulted in CAT activity that was 8% and 51% of the E2-stimulated activity for the P and P' constructs, respectively.


SARS-CoV-2-Host Chimeric RNA-Sequencing Reads Do Not Necessarily Arise From Virus Integration Into the Host DNA.
The human genome bears evidence of extensive invasion by retroviruses and other retroelements, as well as by diverse RNA and DNA viruses. High frequency of somatic integration of the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the DNA of infected cells was recently suggested, based on a number of observations. One key observation was the presence of chimeric RNA-sequencing (RNA-seq) reads between SARS-CoV-2 RNA and RNA transcribed from human host DNA. Here, we examined the possible origin specifically of human-SARS-CoV-2 chimeric reads in RNA-seq libraries and provide alternative explanations for their origin. Chimeric reads were frequently detected also between SARS-CoV-2 RNA and RNA transcribed from mitochondrial DNA or episomal adenoviral DNA present in transfected cell lines, which was unlikely the result of SARS-CoV-2 integration. Furthermore, chimeric reads between SARS-CoV-2 RNA and RNA transcribed from nuclear DNA were highly enriched for host exonic, rather than intronic or intergenic sequences and often involved the same, highly expressed host genes. Although these findings do not rule out SARS-CoV-2 somatic integration, they nevertheless suggest that human-SARS-CoV-2 chimeric reads found in RNA-seq data may arise during library preparation and do not necessarily signify SARS-CoV-2 reverse transcription, integration in to host DNA and further transcription.


Alteration of χ recognition by RecBCD reveals a regulated molecular latch and suggests a channel-bypass mechanism for biological control.
The RecBCD enzyme is a complex heterotrimeric helicase/nuclease that initiates recombination at double-stranded DNA breaks. In Escherichia coli, its activities are regulated by the octameric recombination hotspot, χ (5'-GCTGGTGG), which is read as a single-stranded DNA sequence while the enzyme is unwinding DNA at over ∼1,000 bp/s. Previous studies implicated the RecC subunit as the "χ-scanning element" in this process. Site-directed mutagenesis and phenotypic analyses identified residues in RecC responsible for χ recognition [Handa N, et al., (2012) Proc Natl Acad Sci USA, 10.1073/pnas.1206076109]. The genetic analyses revealed two classes of mutants. Here we use ensemble and single-molecule criteria to biochemically establish that one class of mutants (type 1) has lost the capacity to recognize χ (lost-recognition), whereas the second class (type 2) has a lowered specificity for recognition (relaxed-specificity). The relaxed-specificity mutants still recognize canonical χ, but they have gained the capacity to precociously recognize single-nucleotide variants of χ. Based on the RecBCD structure, these mutant classes define an α-helix responsible for χ recognition that is allosterically coupled to a structural latch. When opened, we propose that the latch permits access to an alternative exit channel for the single-stranded DNA downstream of χ, thereby avoiding degradation by the nuclease domain. These findings provide a unique perspective into the mechanism by which recognition of a single-stranded DNA sequence switches the translocating RecBCD from a destructive nuclease to a constructive component of recombinational DNA repair.


[Body acupuncture combined with auricular acupressure for menstrual headache: a randomized controlled clinical trial].
To observe the therapeutic effect of body acupuncture combined with auricular acupressure for treatment of menstrual headache of hyperactivity of "liver fire". A total of 85 menstrual headache patients with hyperactivity of "liver-fire" were randomly divided into control group (n = 42) and treatment group (n = 43). Patients of the control group were treated by oral administration of Flunarizine Hydrochloride Capsules (Sibelium, 5 mg/time, twice daily) for 5 days beginning at the headache attack in the first menstrual cycle, and for two weeks (except weekends) in the second and third menstrual cycles. Patients of the treatment group were treated by manual acupuncture stimulation of body acupoints Baihui (GV 20), Fengchi (GB 20), Sanyinjiao (SP 6), Taichong (LR 3), Xiaxi (GB 43), Taiyang (EX-HN 5), Hanyan (GB 4), Xuanlu (GB 5) and Shuaigu (GB 8) beginning from the headache attack, and otopoint-pellet pressure of otopoints unilateral Endocrine, Ovaries, Shenmen, Cortex, Liver, Spleen and Kidney (beginning 5 days before menstrual onset) and bilateral Shenmen, Nie, Liver, Gallbladder, Subcortex and Jiaogan during headache attack. Clinical symptom scores were assessed according to the "Guiding Principles for Clinical Research on New Chinese Herbal Drugs" issued by the Chinese Ministry of Health. After the treatment, of the 42 and 43 cases in the control and treatment groups, 9 (21.43%) and 20 (46.51%) were cured, 12 (28.57%) and 14 (32.56%) had a marked improvement, 13 (30.95%) and 7 (16.28%) were effective, 8 (19.05%) and 2 (4.65%) invalid, with the effective rates being 80.95% and 95.35% , respectively. The curative effect of the treatment group was significantly better than that of the control group (P<0.05). The symptom score and VAS score after the treatment and 3 months post-treatment in the treatment group were significantly lower than those of the control group (P<0.01). The headache scores and VAS scores were significantly reduced following the treatment and 3 months after the treatment in both control and treatment groups (P<0.01). After the treatment for 2-3 therapeutic courses, the headache attack duration during menstruation was markedly and gradually shortened in both groups (P<0.01), and therapeutic effect of the treatment group was significantly better than that of the control group (P<0.01). Body acupuncture combined with auricular acupressure treatment can effectively relieve menstrual headache in menstrual women with hyperactivity of "liver fire".


Recovery from viral encephalomyelitis: immune-mediated noncytolytic virus clearance from neurons.
Viral encephalomyelitis is caused by virus infections of neurons in the brain and spinal cord. Recovery is dependent on immune-mediated control and clearance of virus from these terminally differentiated essential cells. Preservation of neuronal function is essential for prevention of neurologic sequelae such as paralysis, seizures and cognitive deficits. Using the model system of Sindbis virus-induced encephalomyelitis in mice, we have shown that immune-mediated clearance of infectious virus from neurons is a noncytolytic process. The major effectors are antibody to the E2 surface glycoprotein produced by B cells, and interferon-gamma produced by T cells. These effectors work in synergy, but neuronal populations differ in their responses to each. Virus is least likely to be cleared from brain neurons and most likely to be cleared from motor neurons in the cervical and thoracic regions of the spinal cord. Because the infected neurons are not eliminated, viral RNA persists and long-term control is needed to prevent virus reactivation. Virus-specific antibody-secreting cells residing in the nervous system after recovery from infection are likely to be important for long-term control.


A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)-Cell Interaction and the Resultant Bioeffects at the Single-cell Level.
In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.


A common variant mapping to CACNA1A is associated with susceptibility to exfoliation syndrome.
Exfoliation syndrome (XFS) is the most common recognizable cause of open-angle glaucoma worldwide. To better understand the etiology of XFS, we conducted a genome-wide association study (GWAS) of 1,484 cases and 1,188 controls from Japan and followed up the most significant findings in a further 6,901 cases and 20,727 controls from 17 countries across 6 continents. We discovered a genome-wide significant association between a new locus (CACNA1A rs4926244) and increased susceptibility to XFS (odds ratio (OR) = 1.16, P = 3.36 × 10(-11)). Although we also confirmed overwhelming association at the LOXL1 locus, the key SNP marker (LOXL1 rs4886776) demonstrated allelic reversal depending on the ancestry group (Japanese: OR(A allele) = 9.87, P = 2.13 × 10(-217); non-Japanese: OR(A allele) = 0.49, P = 2.35 × 10(-31)). Our findings represent the first genetic locus outside of LOXL1 surpassing genome-wide significance for XFS and provide insight into the biology and pathogenesis of the disease.


The need for permanent pacemaker after restoration of conduction following atrioventricular block: a retrospective cohort study.
A permanent pacemaker (PPM) is necessary for patients with a symptomatic third-degree or advanced second-degree atrioventricular (AV) block. An AV block due to medication use can often be reversed; however, subsequent relapse can occur and necessitate subsequent PPM implantation. The aim of this study was to explore the course and prognosis of patients with an AV block. This historical cohort study was conducted between January 2013 and June 2018. A total of 1900 patient records were analyzed and 1123 subjects with an AV block on admission were enrolled. The patients were categorized into 2 groups: Group 1 comprised patients with an AV block due to medication use (n=316, 28%) and Group 2 included patients with an AV block caused by other etiologies (n=807, 72%). Data of the cause of AV block, recurrence, and PPM implantation were analyzed. Patients in both groups who did not require a PPM during the index admission were followed up regarding subsequent implantation of a PPM. AV conduction was recovered in 38 (12%) patients in Group 1 and 48 (6%) patients in Group 2 during the index hospitalization. However, recurrence of the AV block was observed in 18% of Group 1 patients and 40% of Group 2 patients. Only 25 patients in each group (4.5% of the whole study population) remained PPM-free during a median 3-year follow-up period. The study findings suggest that drug-induced AV blocks may not be as benign as previously thought. The high relapse rate indicates that watchful follow-up may be required despite discontinuation of the responsible medication and that consideration of earlier PPM implantation in cases of early recurrence may be warranted.


Covalent labeling of cell-surface proteins for in-vivo FRET studies.
Fluorescence resonance energy transfer (FRET) is a powerful technique to reveal interactions between membrane proteins in live cells. Fluorescence labeling for FRET is typically performed by fusion with fluorescent proteins (FP) with the drawbacks of a limited choice of fluorophores, an arduous control of donor-acceptor ratio and high background fluorescence arising from intracellular FPs. Here we show that these shortcomings can be overcome by using the acyl carrier protein labeling technique. FRET revealed interactions between cell-surface neurokinin-1 receptors simultaneously labeled with a controlled ratio of donors and acceptors. Moreover, using FRET the specific binding of fluorescent agonists could be monitored.


Risk factors for development of microalbuminuria in insulin dependent diabetic patients: a cohort study. Microalbuminuria Collaborative Study Group, United Kingdom.
To investigate the risk factors for the development of persistent microalbuminuria in insulin dependent diabetic patients. Four year follow up of a cohort of diabetic patients. Outpatient departments of teaching and district general hospitals in England. 148 non-microalbuminuric, non-hypertensive insulin dependent diabetic patients. Urinary albumin excretion rate and arterial blood pressure. 137 patients completed four year follow up, of whom 11 developed persistent microalbumin-uria (albumin excretion rate > or = 30 micrograms/min on at least two consecutive occasions), giving a cumulative frequency of 8%. 103 remained normoalbuminuric and 23 exhibited intermittent microalbuminuria. At baseline the persistent microalbuminuric group had had significantly raised blood pressure (mean 137.9 (SD 14.9)/82.3 (7.6) v 123.5 (13.2)/72.8 (9.1) mm Hg), glycated haemoglobin concentration 10.4% (2.0%) v 8-9% (2.0%), and albumin excretion rate (median (interquartile range) 17.5 (13.0-22.3) v 4.8 (3.7-6.7) micrograms/min) (p < 0.05 for all) compared with the normo-albuminuric group. Blood pressure and glycated haemoglobin remained significantly higher in the persistent microalbuminuric group throughout the study (p < 0.05). Multiple regression analysis showed initial albumin excretion rate, blood pressure, and smoking to be significant determinants of persistent microalbuminuria (p < 0.02). In insulin dependent diabetic patients with poor glucose control, which may initially increase albumin excretion rate, an early rise of arterial pressure and smoking are implicated in the development of persistent microalbuminuria.


The challenge of studying parallel behaviors in humans and animal models.
The use of animal models is essential in carrying out research into clinical phenomena such as addiction. However, the complexity of the clinical condition inevitably means that even the best animal models are inadequate representations of the condition they seek to mimic. Such mismatches may account for apparent inconsistencies between discoveries in animal models, including the identification of potential novel therapies, and the translation of such discoveries to the clinic. We argue that it is overambitious to attempt to model human disorders such as addiction in animals, and especially in rodents, where "validity" of such models is often limited to superficial similarities, referred to as "face validity" that reflect quite different underlying phenomena and biological processes from the clinical situation. Instead, we suggest a more profitable approach may be to identify (a) well-defined intermediate human behavioral phenotypes that reflect defined, limited aspects of the human clinical disorder, and (b) to develop animal models that are homologous with those discrete human behavioral phenotypes in terms of psychological processes, and underlying neurobiological mechanisms. Examples of current weaknesses and suggestions for more limited approaches that may allow better homology between the test animal and human condition are made.


Going down to the local: incorporating social organisation and political culture into assessments of decentralised health care.
The social organisation and political culture of the society in which an organisation is embedded can have major effects on the way in which organisational policy is implemented and on how that organisation functions. Research on health sector reforms has paid scant attention to this aspect. If the claims made for decentralised management in the health sector are to be evaluated seriously, it is critical to develop concepts and methods to evaluate not only the formal organisation and the outputs of the health system, but also the aspects of local social organisation and political culture within which that local health system is embedded that may mediate their relationship. The paper explores three cases of district health systems in Northeast Brazil in order to identify aspects of local social organisation and political culture that appear to influence the implementation of the reforms and thereby potentially impact upon the quality of the care provided. The results of the study indicate the importance that aspects of local social organisation and political culture may exert on the operations of a decentralised health system. Key aspects identified are: the space for autonomy; the space for local voice in political life; personalized and institutionalised influences on autonomy and local voice; differences of involvement of health staff with the district; different spaces of acceptable practice and accountability. These factors are seen to moderate the intent of the health reforms at all stages in their implementation. Three possibilities are discussed for the nature of the interaction in terms of cause and effect between the formal organisation of the health system and its local context. Seeing this relationship as one of a dialogue offers some cautious optimism for the potential of the reform agenda. The paper closes with suggestions on how to take this line of research forward.


Time-elapsed synchrotron-light microstructural imaging of femoral neck fracture.
Time-elapsed micro-computed-tomography (μCT) imaging allows studying bone micromechanics. However, no study has yet performed time-elapsed μCT imaging of human femoral neck fractures. We developed a protocol for time-elapsed synchrotron μCT imaging of the microstructure in the entire proximal femur, while inducing clinically-relevant femoral neck fractures. Three human cadaver femora (females, age: 75-80 years) were used. The specimen-specific force to be applied at each load step was based on the specimens' strength estimated a priori using finite-element analysis of clinical CT images. A radio-transparent compressive stage was designed for loading the specimens while recording the applied load during synchrotron μCT scanning. The total μCT scanning field of view was 146 mm wide and 131 mm high, at 29.81 µm isotropic pixel size. Specimens were first scanned unloaded, then under incremental load steps, each equal to 25% of the estimated specimens' strength, and ultimately after fracture. Fracture occurred after 4-5 time-elapsed load steps, displaying sub-capital fracturing of the femoral neck, in agreement with finite-element predictions. Time-elapsed μCT images, co-registered to those of the intact specimen, displayed the proximal femur microstructure under progressive deformation up to fracture. The images showed (1) a spatially heterogeneous deformation localized in the proximal femoral head; (2) a predominantly elastic recovery, after load removal, of the diaphyseal and trochanteric regions and; (3) post-fracture residual displacements, mainly localized in the fractured region. The time-elapsed μCT imaging protocol developed and the high resolution images generated, made publicly available, may spur further research into human femur micromechanics and fracture.


Shank3 mutation in a mouse model of autism leads to changes in the S-nitroso-proteome and affects key proteins involved in vesicle release and synaptic function.
Mutation in the SHANK3 human gene leads to different neuropsychiatric diseases including Autism Spectrum Disorder (ASD), intellectual disabilities and Phelan-McDermid syndrome. Shank3 disruption in mice leads to dysfunction of synaptic transmission, behavior, and development. Protein S-nitrosylation, the nitric oxide (NO•)-mediated posttranslational modification (PTM) of cysteine thiols (SNO), modulates the activity of proteins that regulate key signaling pathways. We tested the hypothesis that Shank3 mutation would generate downstream effects on PTM of critical proteins that lead to modification of synaptic functions. SNO-proteins in two ASD-related brain regions, cortex and striatum of young and adult InsG3680(+/+) mice (a human mutation-based Shank3 mouse model), were identified by an innovative mass spectrometric method, SNOTRAP. We found changes of the SNO-proteome in the mutant compared to WT in both ages. Pathway analysis showed enrichment of processes affected in ASD. SNO-Calcineurin in mutant led to a significant increase of phosphorylated Synapsin1 and CREB, which affect synaptic vesicle mobilization and gene transcription, respectively. A significant increase of 3-nitrotyrosine was found in the cortical regions of the adult mutant, signaling both oxidative and nitrosative stress. Neuronal NO• Synthase (nNOS) was examined for levels and localization in neurons and no significant difference was found in WT vs. mutant. S-nitrosoglutathione concentrations were higher in mutant mice compared to WT. This is the first study on NO•-related molecular changes and SNO-signaling in the brain of an ASD mouse model that allows the characterization and identification of key proteins, cellular pathways, and neurobiological mechanisms that might be affected in ASD.


Creation and preclinical evaluation of genetically attenuated malaria parasites arresting growth late in the liver.
Whole-sporozoite (WSp) malaria vaccines induce protective immune responses in animal malaria models and in humans. A recent clinical trial with a WSp vaccine comprising genetically attenuated parasites (GAP) which arrest growth early in the liver (PfSPZ-GA1), showed that GAPs can be safely administered to humans and immunogenicity is comparable to radiation-attenuated PfSPZ Vaccine. GAPs that arrest late in the liver stage (LA-GAP) have potential for increased potency as shown in rodent malaria models. Here we describe the generation of four putative P. falciparum LA-GAPs, generated by CRISPR/Cas9-mediated gene deletion. One out of four gene-deletion mutants produced sporozoites in sufficient numbers for further preclinical evaluation. This mutant, PfΔmei2, lacking the mei2-like RNA gene, showed late liver growth arrest in human liver-chimeric mice with human erythrocytes, absence of unwanted genetic alterations and sensitivity to antimalarial drugs. These features of PfΔmei2 make it a promising vaccine candidate, supporting further clinical evaluation. PfΔmei2 (GA2) has passed regulatory approval for safety and efficacy testing in humans based on the findings reported in this study.


Effects of long workhours on life-style, stress and quality of life among intermediate Japanese managers.
In accordance with international coordination of labor conditions a Japanese campaign advocating less work finally got under way recently in the form of work-reducing policies of the government to prevent occupational and stress-related diseases. However, long workhours among intermediate managers, who are key persons in most organizations in Japanese industry, are still considered to be prevalent. This study was conducted to examine the workhours of intermediate managers and clarify the effects of long workhours on the life-style, subjective stress, and subjective quality of life among them. Questionnaires were administered concerning workhours, life-styles, subjective stress, and subjective quality of life to 3870 heads of a division or a section and 2666 foremen in 110 firms in Japan. The prevalence of > or = 10 workhours was 69.7% for the divisional or sectional heads and 53.2% for the foremen. Long workhours had significant effects on the managers' life-style, such as sleeping pattern and regularity of daily life and meals. The divisional or sectional managers with long workhours perceived higher stress [odds ratio (OR) 2.51, 95% confidence interval (95% CI) 2.17-2.90] and lower quality of life (OR 1.17, 95% CI 1.02-1.36) than those who worked relatively short hours. The foremen with long workhours perceived higher stress (OR 2.35, 95% CI 2.01-2.75) and lower quality of life (OR 1.26, 95% CI 1.08-1.46) than those who worked relatively short hours. Long workhours may be associated with poorer life-style, higher stress, and lower quality of life among managers at the intermediate level.


Olympic boxing is associated with elevated levels of the neuronal protein tau in plasma.
The aim of this study was to investigate if olympic (amateur) boxing is associated with elevation of brain injury biomarkers in peripheral blood compared to controls. Thirty olympic boxers competing in at least 47 bouts were compared to 25 controls. Blood was collected from the controls at one occasion and from the boxers within 1-6 days after a bout and after a rest period of at least 14 days. Tau concentration in plasma was determined using a novel single molecule ELISA assay and S-100B, glial fibrillary acidic protein, brain-derived neurotrophic factor and amyloid β 1-42 were determined using standard immunoassays. None of the boxers had been knocked-out during the bout. Plasma-tau was significantly increased in the boxers after a bout compared to controls (mean ± SD, 2.46 ± 5.10 vs. 0.79 ± 0.961 ng L(-1), p = 0.038). The other brain injury markers did not differ between the groups. Plasma-tau decreased significantly in the boxers after a resting period compared to after a bout (p = 0.030). Olympic boxing is associated with elevation of tau in plasma. The repetitive minimal head injury in boxing may lead to axonal injuries that can be diagnosed with a blood test.


Relationship Between Temperament and Character Traits, Mood, and Medications in Bipolar I Disorder.
Bipolar I disorder is an illness causing mood shifts that can result in personality and character trait alterations. The relationship between mood and personality and character traits in bipolar I disorder is unclear at this time. We conducted a study from February 2009 to March 2010 that included 42 subjects with bipolar I disorder, which was confirmed using the Structured Clinical Interview for DSM-IV Axis I Disorders. Mood was assessed via the Young Mania Rating Scale (YMRS) and the 30-item Clinician-rated Inventory of Depressive Symptomatology (IDS-C). Temperament and character traits were assessed via the Temperament and Character Inventory (TCI). Multivariate analysis was used to test relationships between mood and temperament and character traits with the effects of possible cofactors taken into account (eg, age, gender, medications). We noted a positive correlation between YMRS scores and persistence (P = .046) and a trend toward positive correlation with novelty seeking (P = .054). There was a positive correlation between higher IDS-C scores and harm avoidance (P < .001) and a negative correlation with self-directedness scores (P < .001). Antipsychotic use was positively correlated with the character trait self-directedness (P = .008), with a trend toward a positive correlation with reward dependence (P = .056). Lithium was negatively correlated with reward dependence (P = .047) and self-transcendence (P = .028), with a trend toward a negative correlation with novelty seeking (P = .053). The findings of our study suggest that some personality and character traits may vary according to mood state and medications in patients with bipolar I disorder. Prospective and longitudinal studies are required to fully characterize the relationships between personality and character traits and mood state in bipolar I disorder.


Depth of the patient-doctor relationship and content of general practice consultations: cross-sectional study.
Patient-doctor continuity is valued by both parties, yet the effect of the depth of the patient-doctor relationship on the content of consultations in general practice is unknown. To assess whether differences in the depth of relationship between a patient and their GP affects the length of consultations, and the number and type of problems and issues raised during a consultation. Cross-sectional study in 22 GP practices in the UK. GP consultations (n = 229) were videotaped and the number of problems and aspects of those problems and issues identified. Patients completed the Patient-Doctor Depth of Relationship (PDDR) and General Practice Assessment Questionnaire-communication (GPAQc) scales. Associations were explored using multivariable linear and logistic regression. Complete data were available on 190 participants consulting 30 GPs. In unadjusted analysis, patients with a deep relationship with their GP discussed more problems (mean 2.8) and issues (mean 4.7) compared with those with a moderate (2.4 problems; 4.0 issues) or shallow (2.3 problems; 3.8 issues) relationship. Patients with deep relationships had consultations that were on average 118 seconds (95% CI = 1 to 236) longer than those with shallow relationships. Adjustment for participant and GP factors attenuated these relationships, although the main trends persisted. A greater number of problems and issues may be raised in a consultation when patients have a deeper relationship with their GP. Over several clinical encounters each year, this may be associated with significant benefits to patients and efficiencies in GP consultations and warrants further investigation.


Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants.
Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed.


Sodium and potassium competition in potassium-selective and non-selective channels.
Potassium channels selectively conduct K(+), primarily to the exclusion of Na(+), despite the fact that both ions can bind within the selectivity filter. Here we perform crystallographic titration and single-channel electrophysiology to examine the competition of Na(+) and K(+) binding within the filter of two NaK channel mutants; one is the potassium-selective NaK2K mutant and the other is the non-selective NaK2CNG, a CNG channel pore mimic. With high-resolution structures of these engineered NaK channel constructs, we explicitly describe the changes in K(+) occupancy within the filter upon Na(+) competition by anomalous diffraction. Our results demonstrate that the non-selective NaK2CNG still retains a K(+)-selective site at equilibrium, whereas the NaK2K channel filter maintains two high-affinity K(+) sites. A double-barrier mechanism is proposed to explain K(+) channel selectivity at low K(+) concentrations.


Phylogenetic uncertainty revisited: Implications for ecological analyses.
Ecologists and biogeographers usually rely on a single phylogenetic tree to study evolutionary processes that affect macroecological patterns. This approach ignores the fact that each phylogenetic tree is a hypothesis about the evolutionary history of a clade, and cannot be directly observed in nature. Also, trees often leave out many extant species, or include missing species as polytomies because of a lack of information on the relationship among taxa. Still, researchers usually do not quantify the effects of phylogenetic uncertainty in ecological analyses. We propose here a novel analytical strategy to maximize the use of incomplete phylogenetic information, while simultaneously accounting for several sources of phylogenetic uncertainty that may distort statistical inferences about evolutionary processes. We illustrate the approach using a clade-wide analysis of the hummingbirds, evaluating how different sources of uncertainty affect several phylogenetic comparative analyses of trait evolution and biogeographic patterns. Although no statistical approximation can fully substitute for a complete and robust phylogeny, the method we describe and illustrate enables researchers to broaden the number of clades for which studies informed by evolutionary relationships are possible, while allowing the estimation and control of statistical error that arises from phylogenetic uncertainty. Software tools to carry out the necessary computations are offered.


In vitro biocompatibility tests of glass ionomer cements impregnated with collagen or bioactive glass to fibroblasts.
To evaluate the biocompatibility of glass ionomer cement (GIC) impregnated with collagen or bioactive glass to BHK-21 fibroblasts in vitro. Mineral Trioxide Aggregate was used as the standard for comparison. Human maxillary central incisors (n = 70) were instrumented with a rotary NiTi system and filled. Following resection of the apical 3mm, root end cavities were prepared and restored with conventional GIC (group 1) or GIC with 0.01%, 0.1% or 1% collagen (groups 2, 3, 4 respectively) or, 10%, 30% or 50% bioactive glass (groups 5, 6, 7 respectively), or Mineral Trioxide Aggregate (group 8). The root slices were incubated in tissue culture plates with BHK-21 fibroblast cell line. Phase contrast and scanning electron microscopes were used to score cell quantity, morphology and cell attachment. The data were statistically analyzed by one way ANOVA with Post Hoc Tukey HSD test (p = 0.05). Group 5 showed the highest scores which was significantly higher than all other groups (p < 0.05) except group 8, with which there was no significant difference (p > 0.05). Glass ionomer cement with 10% bioactive glass showed better adhesion and spreading of cells than glass ionomer cement with 0.01% collagen. The biocompatibility of collagen and bioactive glass was concentration dependent. The addition of bioactive glass improved the biocompatibility of glass ionomer cement to fibroblasts better than addition of collagen.


Position of the Academy of Nutrition and Dietetics: Vegetarian Diets.
It is the position of the Academy of Nutrition and Dietetics that appropriately planned vegetarian, including vegan, diets are healthful, nutritionally adequate, and may provide health benefits for the prevention and treatment of certain diseases. These diets are appropriate for all stages of the life cycle, including pregnancy, lactation, infancy, childhood, adolescence, older adulthood, and for athletes. Plant-based diets are more environmentally sustainable than diets rich in animal products because they use fewer natural resources and are associated with much less environmental damage. Vegetarians and vegans are at reduced risk of certain health conditions, including ischemic heart disease, type 2 diabetes, hypertension, certain types of cancer, and obesity. Low intake of saturated fat and high intakes of vegetables, fruits, whole grains, legumes, soy products, nuts, and seeds (all rich in fiber and phytochemicals) are characteristics of vegetarian and vegan diets that produce lower total and low-density lipoprotein cholesterol levels and better serum glucose control. These factors contribute to reduction of chronic disease. Vegans need reliable sources of vitamin B-12, such as fortified foods or supplements.


Benzodiazepines and risk of dementia: true association or reverse causation?
According to a recently published population study conducted in France, exposure to benzodiazepines may be associated with an approximately 50% increase in the risk of dementia in the elderly. However, the clinical interpretation of this finding raised some concerns. A causal link between benzodiazepine use and diagnosis of dementia may be real, but it is nevertheless possible that the increased risk might be due to other confounding factors. In this article, the main strengths and weaknesses of this study are briefly analysed, including the possibility of reverse causation. Implications for research and current practice are discussed.


The Effects of the Driver's Mental State and Passenger Compartment Conditions on Driving Performance and Driving Stress.
Globalization has increased the number of road trips and vehicles. The result has been an intensification of traffic accidents, which are becoming one of the most important causes of death worldwide. Traffic accidents are often due to human error, the probability of which increases when the cognitive ability of the driver decreases. Cognitive capacity is closely related to the driver's mental state, as well as other external factors such as the CO2 concentration inside the vehicle. The objective of this work is to analyze how these elements affect driving. We have conducted an experiment with 50 drivers who have driven for 25 min using a driving simulator. These drivers completed a survey at the start and end of the experiment to obtain information about their mental state. In addition, during the test, their stress level was monitored using biometric sensors and the state of the environment (temperature, humidity and CO2 level) was recorded. The results of the experiment show that the initial level of stress and tiredness of the driver can have a strong impact on stress, driving behavior and fatigue produced by the driving test. Other elements such as sadness and the conditions of the interior of the vehicle also cause impaired driving and affect compliance with traffic regulations.


Copper Complex in Poly(vinyl chloride) as a Nitric Oxide-Generating Catalyst for the Control of Nitrifying Bacterial Biofilms.
In this study, catalytic generation of nitric oxide by a copper(II) complex embedded within a poly(vinyl chloride) matrix in the presence of nitrite (source of nitric oxide) and ascorbic acid (reducing agent) was shown to effectively control the formation and dispersion of nitrifying bacteria biofilms. Amperometric measurements indicated increased and prolonged generation of nitric oxide with the addition of the copper complex when compared to that with nitrite and ascorbic acid alone. The effectiveness of the copper complex-nitrite-ascorbic acid system for biofilm control was quantified using protein analysis, which showed enhanced biofilm suppression when the copper complex was used in comparison to that with nitrite and ascorbic acid treatment alone. Confocal laser scanning microscopy (CLSM) and LIVE/DEAD staining revealed a reduction in cell surface coverage without a loss of viability with the copper complex and up to 5 mM of nitrite and ascorbic acid, suggesting that the nitric oxide generated from the system inhibits proliferation of the cells on surfaces. Induction of nitric oxide production by the copper complex system also triggered the dispersal of pre-established biofilms. However, the addition of a high concentration of nitrite and ascorbic acid to a pre-established biofilm induced bacterial membrane damage and strongly decreased the metabolic activity of planktonic and biofilm cells, as revealed by CLSM with LIVE/DEAD staining and intracellular adenosine triphosphate measurements, respectively. This study highlights the utility of the catalytic generation of nitric oxide for the long-term suppression and removal of nitrifying bacterial biofilms.


Robot-Assisted Rehabilitation of Ankle Plantar Flexors Spasticity: A 3-Month Study with Proprioceptive Neuromuscular Facilitation.
In this paper, we aim to investigate the effect of proprioceptive neuromuscular facilitation (PNF)-based rehabilitation for ankle plantar flexors spasticity by using a Robotic Ankle-foot Rehabilitation System (RARS). A modified robot-assisted system was proposed, and seven poststroke patients with hemiplegic spastic ankles participated in a 3-month robotic PNF training. Their impaired sides were used as the experimental group, while their unimpaired sides as the control group. A robotic intervention for the experimental group started from a 2-min passive stretching to warming-up or relaxing the soleus and gastrocnemius muscles and also ended with the same one. Then a PNF training session including 30 trials was activated between them. The rehabilitation trainings were carried out three times a week as an addition to their regular rehabilitation exercise. Passive range of motion, resistance torque, and stiffness were measured in both ankles before and after the interventions. The changes in Achilles tendon length, walking speed, and lower limb function were also evaluated by the same physician or physiotherapist for each participant. Biomechanical measurements before interventions showed significant difference between the experimental group and the control group due to ankle spasticity. For the control group, there was no significant difference in the 3 months with no robotic intervention. But for the experimental group, passive dorsiflexion range of motion increased (p < 0.01), resistance torque under different dorsiflexion angle levels (0°, 10°, and 20°) decreased (p < 0.05, p < 0.001, and p < 0.001, respectively), and quasi-static stiffness under different dorsiflexion angle levels (0°, 10°, and 20°) also decreased (p < 0.01, p < 0.001, and p < 0.001, respectively). Achilles's tendon length shortened (p < 0.01), while its thickness showed no significant change (p > 0.05). The robotic rehabilitation also improved the muscle strength (p < 0.01) and muscle control performance (p < 0.001). In addition, improvements were observed in clinical and functional measurements, such as Timed Up-and-Go (p < 0.05), normal walking speed (p > 0.05), and fast walking speed (p < 0.05). These results indicated that the PNF-based robotic intervention could significantly alleviate lower limb spasticity and improve the motor function in chronic stroke participant. The robotic system could potentially be used as an effective tool in poststroke rehabilitation training.


Systematic assessment of a maxilla of Homo from Hadar, Ethiopia.
The Hadar site in Ethiopia is a prolific source of hominid fossils attributed to the species Australopithecus afarensis, which spans the period 3.4-3.0 million years (myr) in the Sidi Hakoma, Denen Dora and lower Kada Hadar Members of the Hadar Formation. Since 1992 a major focus of field work conducted at Hadar has centered on sediments younger than 3.0 myr, comprising the bulk of the Kada Hadar Member. Witnessing the rise of the "robust" Australopithecus clade(s), the origin of Homo, and the first record of lithic artifacts, the period between 3.0 and 2.0 myr is strategically vital for paleoanthropology. However, in eastern Africa it is a particularly poorly sampled temporal interval. This paper provides a detailed comparative description of a hominid maxilla with partial dentition found at Hadar in 1994. The specimen, A.L. 666-1, derives from a lithic artifact-bearing horizon high in the Kada Hadar Member, 0.8 m below the BKT-3 tephra, dated by the 40Ar/39Ar method to 2.33 +/- 0.07 myr. Our preliminary investigation of the hominid specimen showed unambiguous affinities with early representatives of the Homo clade (Kimbel et al. [1996] J. Hum. Evol. 31:549-561). Further studies on maxillary and dental morphology lead us to attribute A.L. 666-1 to Homo aff. H. habilis. The new Hadar jaw is the first paleontological evidence for the projection of the H. habilis maxillofacial morphotype well back into the Pliocene. It may represent a male of this species, whose maxillary hypodigm consists chiefly of females. A subsidiary finding of our study is that of the three earliest recorded species of Homo (H. habilis, H. rudolfensis, H. erectus), it is H. habilis that exhibits facial morphology closest to that expected in their last common ancestor.


When care situations evoke difficult emotions in nursing staff members: an ethnographic study in two Norwegian nursing homes.
Caring practice in nursing homes is a complex topic, especially the challenges of meeting the basic needs of residents when their behaviour evokes difficult emotions. Cognitive and physical changes related to aging and disability can contribute to behaviours considered to be unacceptable. For example, resident behaviours such as spitting, making a mess with food or grinding teeth are behaviours that most people do not want to see, hear or experience. The aim of this study was to gain a deeper understanding of how nursing home staff members deal with such behaviours in care situations. This article draws on ethnographic data to describe how nursing home staff members manage unpleasant resident behaviours. The study was based on two long-term units in two Norwegian public nursing homes. The Region's Medical Ethics Committee and the Norwegian Social Science Data Services granted approval. In total, 45 participants (37 nursing aides and eight nurses) agreed to participate in this study. Ten of the participants were interviewed at the end of the field study. This study indicates that nursing home staff members experience difficult emotions related to some residents' behaviours. However, they found these feelings difficult to express and rarely verbalized them openly. In addition, they were characterized by a strong obligation to help all residents, despite their own feelings. Therefore, it appears that an inner struggle occurs as a part of everyday practice. Despite these difficult emotions, nursing staff members believed that they needed to manage their responses and continued to offer good care to all residents. These findings extend our understanding of this unarticulated part of nursing home practice.


Calcyon, a novel partner of clathrin light chain, stimulates clathrin-mediated endocytosis.
In the central nervous system, clathrin-mediated endocytosis is crucial for efficient synaptic transmission. Clathrin-coated vesicle assembly and disassembly is regulated by some 30 adaptor and accessory proteins, most of which interact with clathrin heavy chain. Using the calcyon cytosolic domain as bait, we isolated clathrin light chain in a yeast two-hybrid screen. The interaction domain was mapped to the heavy chain binding domain and C-terminal regions of light chain. Further, the addition of the calcyon C terminus stimulated clathrin self-assembly in a dose-dependent fashion. Calcyon, which is a single transmembrane protein predominantly expressed in brain, localized to vesicular compartments within pre- and postsynaptic structures. There was a high degree of overlap in the distribution of LC and calcyon in neuronal dendrites, spines, and cell bodies. Co-immunoprecipitation studies further suggested an association of calcyon with the clathrin-mediated endocytic machinery. Compared with controls, HEK293 cells overexpressing calcyon exhibited significantly enhanced transferrin uptake but equivalent levels of recycling. Conversely, transferrin uptake was largely abolished in neocortical neurons obtained from mice homozygous for a calcyon null allele, whereas recycling proceeded at wild type levels. Collectively, these data indicate a role for calcyon in clathrin-mediated endocytosis in brain.


The South Africa Stress and Health Study: rationale and design.
The South Africa Stress and Health Study (SASH) is a large psychiatric epidemiological survey that is currently underway in South Africa. It is a part of the World Health Organization's World Mental Health (WMH) 2000 initiative and seeks to complete interviews with a nationally representative sample of 5000 adults. The WMH initiative is obtaining population-based data on the prevalence and severity of specific psychiatric disorders, demographic and psychosocial correlates of these diagnoses, and the levels and adequacy of mental health service utilization. SASH is using the fully structured pencil and paper version of the WHO Composite International Diagnostic Interview (CIDI) to assess lifetime and 12-month rates of mental disorders using both the DSM-IV and the ICD-10 diagnostic systems. In addition, the SASH seeks to collect information on the prevalence of exposure to physical and psychological torture in South Africa and to assess the association between such traumas and specific psychiatric disorders. It will also assess a broad range of risk factors and resources that may modify the association between exposure to human rights violations and mental health.


Interleukin 2-induced proliferation of murine natural killer cells in vivo.
The growth factor, IL-2, was administered to mice to evaluate the in vivo responsiveness of NK cells to this factor. The immediate effects of this factor on NK cells were determined by examining cytotoxic activity at 18-24 h after a single treatment with rIL-2. Although moderate doses of rIL-2 (3 x 10(4) U) could be shown to activate existing cytotoxic cells on a per cell basis, higher doses (10(6) U) were required to elicit blast size killer cells. The elicited killer cells were characterized as NK cells by the following criteria: (a) they were readily induced in athymic mice; (b) they mediated killing of NK-sensitive YAC-1 target cells but not NK-resistant P815 target cells; and (c) they expressed the NK cell determinants asialo ganglio-n-tetraosylceramide and NK1.1, but not the T cell determinants CD3, L3T4, or Lyt-2. High-dose IL-2 treatment induced not only the appearance of blast size NK cells, but also the expansion of this population. After treatments, the number of large granular lymphocytes and the number of NK1.1+ cells were increased at least twofold. Analysis of DNA content within the NK1.1+ cell subset demonstrated that IL-2 preferentially drove NK1.1+ cells into S and G2/M phases of the cell cycle. The in vivo elicited blast lymphocytes were examined by Northern blot analysis and in situ hybridization for expression of the IL-2-R p55 alpha chain gene. As previous work from this laboratory has demonstrated that NK cells proliferate in response to IFNs and IFN inducers in vivo, blast lymphocytes were also prepared after IFN treatments. The NK cells were not induced to express detectable levels of the alpha chain gene under any of the conditions examined. Blast T lymphocytes, isolated at times during viral infections when IL-2 production can be demonstrated in vitro, were induced to transcribe the alpha chain gene. Treatments of euthymic mice with high-dose IL-2 also induced transcription of the alpha chain gene in 41% of the non-B blast lymphocytes, but only background percentages of the NK1.1+ cells expressed the alpha chain gene. Transcription of the alpha chain gene was not induced in the NK cell-abundant athymic mice after IL-2 treatment. All of the in vivo elicited blast lymphocytes were induced to express IFN-gamma. Taken together, these data definitively demonstrate that IL-2 can induce NK cell proliferation and expansion in vivo. They also show that exposure to IL-2 in vivo, either by administration or endogenous production of the factor, induces transcription of the IL-2-R alpha chain gene in populations of cells containing T cell subsets. The results suggest, however, that murine NK cells are not induced to express high levels of the alpha chain gene in response to IL-2 in vivo.


An index of barriers for the implementation of personalised medicine and pharmacogenomics in Europe.
Personalised medicine (PM) is an innovative way to produce better patient outcomes by using an individualised or stratified approach to disease and treatment rather than a collective treatment approach for patients. Despite its tangible advantages, the complex process to translate PM into the member states and European healthcare systems has delayed its uptake. The aim of this study is to identify relevant barriers represented by an index to summarise challenging areas for the implementation of PM in Europe. A systematic literature review was conducted, and a gaps-and-needs assessment together with a strengths-weaknesses-opportunities-and-threats analysis were applied to review strategic reports and conduct interviews with key stakeholders. Furthermore, surveys were sent out to representatives of stakeholder groups. The index was constructed based on the priorisation of relevant factors by stakeholders. A need for stakeholder-agreed standards at all levels of implementation of PM exists, from validating biomarkers to definitions of 'informed consent'. The barriers to implement PM are identified in 7 areas, namely, stakeholder involvement, standardisation, interoperable infrastructure, European-level policy making, funding, data and research, and healthcare systems. Challenges in the above-mentioned areas can and must be successfully tackled if we are to create a healthier Europe through PM. In order to create an environment in which PM can thrive for the patients' best outcomes, there is an urgent need for systematic actions to remove as many barriers as possible.


Ferroelectric polarization induces electric double layer bistability in electrolyte-gated field-effect transistors.
The dense surface charges expressed by a ferroelectric polymeric thin film induce ion displacement within a polyelectrolyte layer and vice versa. This is because the density of dipoles along the surface of the ferroelectric thin film and its polarization switching time matches that of the (Helmholtz) electric double layers formed at the ferroelectric/polyelectrolyte and polyelectrolyte/semiconductor interfaces. This combination of materials allows for introducing hysteresis effects in the capacitance of an electric double layer capacitor. The latter is advantageously used to control the charge accumulation in the semiconductor channel of an organic field-effect transistor. The resulting memory transistors can be written at a gate voltage of around 7 V and read out at a drain voltage as low as 50 mV. The technological implication of this large difference between write and read-out voltages lies in the non-destructive reading of this ferroelectric memory.


BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin.
BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.


Detection of antibody to hepatitis C virus in renal transplant recipients.
Non-A, non-B hepatitis is a significant cause of liver disease among renal allograft recipients. In order to assess the impact and prevalence of hepatitis C in a series of renal allograft recipients, we retrospectively screened 621 consecutive patients transplanted between 1979 and 1989 and 484 cadaver organ donors retrieved in the same interval for serologic evidence of hepatitis C viral (HCV) infection using the enzyme-linked assay for anti-HCV antibody. Of 596 HBsAg negative patients, 180 (30%) were anti-HCV positive at the time of transplant. One-year posttransplant, 117 (22%) had detectable levels of anti-HCV antibody. Chemically significant hepatitis developed in 52/234 (22%) anti-HCV positive patients, and 26 of these followed a clinical course consistent with chronic hepatitis. Significantly more males and patients with antibody to HCV detectable at 1 year posttransplant were in the group experiencing an increase in liver enzymes. Ten-year patient and graft survival was 78% and 50%, respectively, for the anti-HCV positive patients who had an elevation of alanine aminotransferase, and 76% and 57% for the cohort maintaining normal liver function (P = NS). There were also no differences in patient and graft survival among the anti-HCV positive group and the consistently sero-negative patients. Of 484 cadaver organ donors with serum available for analysis (out of 1200 retrieved), 67 (14%) were anti-HCV positive at the time of organ donation. Among 23 anti-HCV negative kidney recipients who received a kidney from an HCV antibody positive donor, only one had seroconverted at 1 year posttransplant. Antibody to HCV appears to be widespread among renal transplant recipients and cadaver organ donors. We were unable to demonstrate any evidence of long-term adverse effects on patient and graft survival among anti-HCV positive patients employing the first generation anti-HCV assay.


Genetic diversity and different cross-neutralization capability of porcine circovirus type 2 isolates recently circulating in South Korea.
Porcine circovirus type 2 (PCV2) is a small single-stranded DNA virus and a primary cause of PCV-associated diseases (PCVAD) that result insubstantial economic loss for swine farms. Between 2016 and 2018, PCV2 field viruses were isolated from PCVAD-affected swine farms in South Korea and investigated for genetic and antigenic heterogeneity. The genetic analysis of ORF2 showed that the genotype of the Korean PCV2 field isolates has been rapidly shifted from PCV2a or 2b to mutant PCV2b known as PCV2d with 82.6 to 100% amino acid sequence similarity. PCV2-specific monoclonal antibodies (mAbs) demonstrated variable antigen-binding activity to four representative Korean PCV2 field isolates [QIA215 (PCV2a), QIA418 (PCV2b), QIA169 (PCV2d), and QIA244 (PCV2d)] without genotype specificity, and one mAb showed neutralization activity to QIA215. In a cross-virus neutralization assay using anti-PCV2 sera of pigs and guinea pigs injected with a commercial vaccine and the Korean PCV2 field isolates, the anti-porcine sera of a commercial vaccine had high neutralization activity against QIA215 and QIA418 with statistically lower activity against PCV2d viruses. Anti-guinea pig sera of QIA215, QIA418, QIA169, and a commercial vaccine had high neutralization activity against all of the viruses with significantly lower activity against QIA244. Importantly, anti-guinea pig sera of QIA244 had high neutralization activity against all of the viruses. This study confirmed genetic and antigenic diversity among recent PCV2 field isolates in Korean swine farms, and the strain-based difference in virus neutralization capability should be considered for more effective control by vaccination.


Liver fat content in type 2 diabetes: relationship with hepatic perfusion and substrate metabolism.
Hepatic steatosis is common in type 2 diabetes. It is causally linked to the features of the metabolic syndrome, liver cirrhosis, and cardiovascular disease. Experimental data have indicated that increased liver fat may impair hepatic perfusion and metabolism. The aim of the current study was to assess hepatic parenchymal perfusion, together with glucose and fatty acid metabolism, in relation to hepatic triglyceride content. Fifty-nine men with well controlled type 2 diabetes and 18 age-matched healthy normoglycemic men were studied using positron emission tomography to assess hepatic tissue perfusion, insulin-stimulated glucose, and fasting fatty acid metabolism, respectively, in relation to hepatic triglyceride content, quantified by proton magnetic resonance spectroscopy. Patients were divided into two groups with hepatic triglyceride content below (type 2 diabetes-low) or above (type 2 diabetes-high) the median of 8.6%. Type 2 diabetes-high patients had the highest BMI and A1C and lowest whole-body insulin sensitivity (ANOVA, all P < 0.001). Compared with control subjects and type 2 diabetes-low patients, type 2 diabetes-high patients had the lowest hepatic parenchymal perfusion (P = 0.004) and insulin-stimulated hepatic glucose uptake (P = 0.013). The observed decrease in hepatic fatty acid influx rate constant, however, only reached borderline significance (P = 0.088). In type 2 diabetic patients, hepatic parenchymal perfusion (r = -0.360, P = 0.007) and hepatic fatty acid influx rate constant (r = -0.407, P = 0.007) correlated inversely with hepatic triglyceride content. In a pooled analysis, hepatic fat correlated with hepatic glucose uptake (r = -0.329, P = 0.004). In conclusion, type 2 diabetic patients with increased hepatic triglyceride content showed decreased hepatic parenchymal perfusion and hepatic insulin mediated glucose uptake, suggesting a potential modulating effect of hepatic fat on hepatic physiology.


Climate adaptive urban planning and design with water in Dutch polders.
The existing water management in Dutch polders is based on independent water systems for each polder. These are featuring artificial stabilized ground and surface water levels. As a result of the local climate the water levels in the polders are not continuously at a constant level. To maintain a stable water table in the polders, the surplus of relatively clean rainwater has to be pumped away during the cold seasons into canals or rivers, which are located on a higher level. During the summer relatively polluted water from these waterways is led into the polders to top up the declining water levels. This procedure leads to various problems regarding water quantity and water quality. The described existing system is not adaptable to climate change and includes the risk of flooding, particularly from torrential rain. Therefore it is crucial to develop, preferably self-sufficient, rainwater management systems in the polders. They should allow the fluctuation of the water levels inside the polders for seasonal storage and flood control. The described concept is adopted in the present water policy in the Netherlands as well as in research and recent urban development projects in Dutch polders.


Chronic sinonasal disease in patients with inflammatory bowel disease.
The objective of this study was to explore the possible relationship between inflammatory bowel disease (IBD) and chronic sinonasal disease. A cross-sectional study was undertaken of 241 patients with Crohn's disease (CD) or ulcerative colitis from a tertiary medical center IBD clinic. Patient demographic data and information regarding IBD diagnosis and management, sinonasal disease diagnosis and management, and complications related to these diagnoses were gathered by retrospective chart review and a standardized patient survey. One hundred sixty surveys (67%) were returned and analyzed. Overall 48% of patients with IBD reported chronic sinonasal disease symptoms. Patients with CD had a higher prevalence of sinonasal disease than patients with ulcerative colitis (53% versus 32%; p < 0.02). The subgroup of CD patients with obstructive bowel complications had the highest prevalence of sinonasal disease (68% versus 27%; p = < 0.001), with 23% reporting chronic rhinosinusitis, 13% reporting chronic rhinitis, and an additional 32% reporting chronic nasal or sinus symptoms. The prevalence of chronic sinonasal disease is elevated in patients with IBD, occurring in approximately one-half of patients followed at a tertiary IBD center. Patients with CD experiencing obstructive complications had significantly increased rates of sinonasal disease. The relationship between chronic sinonasal disease and obstructive CD is not defined, but several hypotheses are generated.


Cyclin-binding motifs are essential for the function of p21CIP1.
The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.


Rad59 regulates association of Rad52 with DNA double-strand breaks.
Homologous recombination among repetitive sequences is an important mode of DNA repair in eukaryotes following acute radiation exposure. We have developed an assay in Saccharomyces cerevisiae that models how multiple DNA double-strand breaks form chromosomal translocations by a nonconservative homologous recombination mechanism, single-strand annealing, and identified the Rad52 paralog, Rad59, as an important factor. We show through genetic and molecular analyses that Rad59 possesses distinct Rad52-dependent and -independent functions, and that Rad59 plays a critical role in the localization of Rad52 to double-strand breaks. Our analysis further suggests that Rad52 and Rad59 act in multiple, sequential processes that determine genome structure following acute exposure to DNA damaging agents.


Nkx3.2-mediated repression of Runx2 promotes chondrogenic differentiation.
Runx2, a transcription factor known to be essential for osteoblast maturation and skeletogenesis, is also expressed in pre-cartilaginous mesenchymal condensations in the developing embryo. It is therefore necessary to understand the control and consequential regulatory activity of the Runx2 gene within the context of chondrogenic differentiation of a mesenchymal progenitor cell. We identify the homeodomain protein Nkx3.2 as a potent sequence-specific repressor of the Runx2 promoter that acts through a regulatory element 0.1 kb upstream from the site of transcriptional initiation. The biological significance of this repression is established by utilizing bone morphogenic protein 2 (BMP-2)-induced chondrogenic differentiation of pluripotent C3H10T1/2 cells as a model for the initial events of mesenchymal chondrogenesis. We demonstrate that induction of the chondrogenic phenotype and endogenous Nkx3.2 expression is accompanied by a repression of Runx2 gene activity. Bypassing Runx2 repression by adenoviral-mediated introduction of Runx2 into C3H10T1/2 cells can prevent the induction of chondrogenesis, but cannot reverse the chondrogenic phenotype once it has been initiated, as evidenced by Sox9 and type II collagen expression and extracellular matrix deposition. Our results demonstrate that Runx2 is a direct transcriptional target of Nkx3.2, and that repression of Runx2 at the onset of chondrogenesis is a prerequisite for the activation of a chondrocyte-specific program of gene expression. We postulate that Runx2 is a critical link in BMP-2-mediated initiation of mesenchymal chondrogenesis that results in activation of Sox9 at least in part through the Nkx3.2-dependent repression of Runx2.


Metal mobilization under alkaline conditions in ash-covered tailings.
The aim of this study was to determine element mobilization and accumulation in mill tailings under alkaline conditions. The tailings were covered with 50 cm of fly ash, and above a sludge layer. The tailings were geochemically and mineralogically investigated. Sulfides, such as pyrrhotite, sphalerite and galena along with gangue minerals such as dolomite, calcite, micas, chlorite, epidote, Mn-pyroxene and rhodonite were identified in the unoxidized tailings. The dissolution of the fly ash layer resulted in a high pH (close to 12) in the underlying tailings. This, together with the presence of organic matter, increased the weathering of the tailings and mobilization of elements in the uppermost 47 cm of the tailings. All primary minerals were depleted, except quartz and feldspar which were covered by blurry secondary carbonates. Sulfide-associated elements such as Cd, Fe, Pb, S and Zn and silicate-associated elements such as Fe, Mg and Mn were released from the depletion zone and accumulated deeper down in the tailings where the pH decreased to circum-neutral. Sequential extraction suggests that Cd, Cu, Fe, Pb, S and Zn were retained deeper down in the tailings and were mainly associated with the sulfide phase. Calcium, Cr, K and Ni released from the ash layer were accumulated in the uppermost depletion zone of the tailings.