Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation.
Cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily that functions as a chloride channel. Previous work has suggested that the external side of the sixth transmembrane segment (TM6) plays an important role in governing chloride permeation, but the function of the internal side remains relatively obscure. Here, on a cysless background, we performed cysteine-scanning mutagenesis and modification to screen the entire TM6 with intracellularly applied thiol-specific methanethiosulfonate reagents. Single-channel amplitude was reduced in seven cysteine-substituted mutants, suggesting a role of these residues in maintaining the pore structure for normal ion permeation. The reactivity pattern of differently charged reagents suggests that the cytoplasmic part of TM6 assumes a secondary structure of an alpha helix, and that reactive sites (341, 344, 345, 348, 352, and 353) reside in two neighboring faces of the helix. Although, as expected, modification by negatively charged reagents inhibits anion permeation, interestingly, modification by positively charged reagents of cysteine thiolates on one face (344, 348, and 352) of the helix affects gating. For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. These results corroborate the idea that a helix rotation of TM6, which has been proposed to be part of the molecular motions during transport cycles in other ABC transporters, is associated with gating of the CFTR pore.


Forelimb Myology of Carnivorous Marsupials (Marsupialia: Dasyuridae): Implications for the Ancestral Body Plan of the Australidelphia.
Carnivorous marsupials of the family Dasyuridae represent a more generalized anatomical condition of both craniodental and postcranial features in comparison to other groups of Australidelphian marsupials. Plesiomorphic characters include polyprotodont dentition, didactylous (rather than syndactylous) pedal morphology, the retention of clavicles and epipubic bones, and an unossified patelloid. In light of the anatomy of the postcranial skeleton, we hypothesized that the muscular anatomy of the Dasyuridae would likely display a range of plesiomorphic traits. We performed gross anatomical dissection on the forelimbs of four species of dasyurid marsupials to produce anatomical descriptions and muscle origin and insertion maps for Dasyurus geoffroii, D. hallucatus, and Phascogale tapoatafa, together with comparative notes for Antechinus flavipes. These new descriptions were then compared with those of other marsupials from the published literature in order to establish the principal patterns in forelimb muscular anatomy. In nearly all aspects of anatomy, we found that the arrangement of the muscular origins and insertions, and the relative degree of separation between muscle bellies among dasyurids, provide a natural starting point from which the anatomies of other Australidelphian marsupial groups can be derived. Anat Rec, 300:1589-1608, 2017. © 2017 Wiley Periodicals, Inc.


Predisposing and precipitating factors for delirium in hospitalized older patients.
Delirium is a common and serious problem for older hospitalized patients. This investigation proposes a multifactorial model of delirium etiology, involving a complex interrelationship of predisposing (vulnerability) factors and precipitating factors (acute insults). An overview of risk factors for delirium identified in 14 studies published since 1980 is provided. Although these studies identify key risk factors for delirium, they do not allow the examination of the interrelationship of predisposing and precipitating factors. Thus, we present two prospective cohort studies by our group which empirically examine: (1) predisposing (vulnerability) factors, (2) precipitating factors, and (3) the interrelationship of predisposing and precipitating factors. Understanding these risk factors is the key to developing appropriate preventive strategies and to target intermediate and high risk patients for intervention efforts.


The golden hour of prehospital reperfusion with triple antiplatelet therapy: a sub-analysis from the Ongoing Tirofiban in Myocardial Evaluation 2 (On-TIME 2) trial early initiation of triple antiplatelet therapy.
It is known that the efficacy of thrombolytic therapy in ST-segment elevation myocardial infarction (STEMI) is highly time dependent with the best efficacy when given within the so-called golden hour. This analysis from the On-TIME 2 trial evaluated the efficacy of triple antiplatelet therapy on initial patency and ST-segment resolution (STR) in relation to time from symptom onset to first medical contact. The On-TIME 2 trial included 1,398 consecutive STEMI patients referred for primary percutaneous coronary intervention (PCI). Patients were randomized to dual (500 mg aspirin and 600 mg clopidogrel) or triple antiplatelet (500 mg aspirin, 600 mg clopidogrel, and tirofiban 25 μg/kg bolus and 0.15 μg/kg per minute maintenance infusion for 18 hours) pretreatment in the ambulance. Primary outcome of this sub-analysis was initial patency of the infarct-related vessel and STR before PCI according to time from symptom onset to first medical contact in quartiles. In addition, the incidence of aborted myocardial infarction, defined as the absence of a rise in creatinine kinase, was assessed. Initial patency, STR before PCI, and the incidence of aborted myocardial infarction gradually increased with shorter time from symptom onset to first medical contact. Initial Thrombolysis in Myocardial Infarction flow was present in 21.2% in the total population and 26.2%, 21.5%, 18.1%, and 18.8% in the time quartiles, respectively (P for trend=.01). The incidence of complete STR pre-angiography was 16.6% in the total population and 23.4%, 18.2%, 14.7%, and 9.9% in the 4 quartiles, respectively (P for trend<.001). This was largely driven by the effect of triple antiplatelet therapy, which further improved initial patency and STR and led to a significantly higher incidence of aborted myocardial infarction (13.2% vs 8.7%, P=.011), especially in the patients with short duration of symptoms. Antiplatelet pretreatment before primary PCI, including a glycoprotein IIb/IIIa blocker, seems to be most effective when given shortly after symptom onset. Further studies should be performed to test this hypothesis.


Axis I comorbidity in body dysmorphic disorder.
Although research on body dysmorphic disorder (BDD) has increased in recent years, this disorder's comorbidity has received little empirical attention. Further work in this area is needed, as it appears that most patients with BDD have at least one comorbid disorder. This study examined axis I comorbidity and clinical correlates of comorbidity in 293 patients with DSM-IV BDD, 175 of whom participated in a phenomenology study and 118 of whom participated in treatment studies of BDD. Subjects were evaluated with the Structured Clinical Interview for DSM-III-R (SCID-P) and a semistructured instrument to obtain information on clinical correlates. Comorbidity was common, with a mean of more than two lifetime comorbid axis I disorders in both the phenomenology and treatment groups. In both groups, the most common lifetime comorbid axis I disorders were major depression, social phobia, obsessive compulsive disorder (OCD), and substance use disorders. Social phobia usually began before onset of BDD, whereas depression and substance use disorders typically developed after onset of BDD. A greater number of comorbid disorders was associated with greater functional impairment and morbidity in a number of domains. Thus, axis I comorbidity is common in BDD patients and associated with significant functional impairment.


How HIV Nef Proteins Hijack Membrane Traffic To Promote Infection.
The accessory protein Nef of human immunodeficiency virus (HIV) is a primary determinant of viral pathogenesis. Nef is abundantly expressed during infection and reroutes a variety of cell surface proteins to disrupt host immunity and promote the viral replication cycle. Nef counteracts host defenses by sequestering and/or degrading its targets via the endocytic and secretory pathways. Nef does this by physically engaging a number of host trafficking proteins. Substantial progress has been achieved in identifying the targets of Nef, and a structural and mechanistic understanding of Nef's ability to command the protein trafficking machinery has recently started to coalesce. Comparative analysis of HIV and simian immunodeficiency virus (SIV) Nef proteins in the context of recent structural advances sheds further light on both viral evolution and the mechanisms whereby trafficking is hijacked. This review describes how advances in cell and structural biology are uncovering in growing detail how Nef subverts the host immune system, facilitates virus release, and enhances viral infectivity.


Male and female impairments in odor span are observed in a rat model of PTSD.
Posttraumatic stress disorder (PTSD) is associated with neural and behavioral alterations in response to trauma exposure, including working memory impairments. Rodent models of PTSD have not fully investigated chronic or reactive working memory deficits, despite clinical relevance. The present study uses footshock to induce a posttraumatic stress state in male and female rats and evaluates the effect of footshock and trauma-paired odor cues on working memory performance in the odor span task. Results demonstrate the emergence of chronic deficits in working memory among animals exposed to footshock by 3 wk after traumatic stress. The presentation of a trauma-paired odor cue was associated with further decrement in working memory performance for male animals. Furthermore, anxiety-like behaviors associated with the PTSD-like phenotype could predict the degree of working memory impairment in response to the trauma-paired odor cue. This study enhances validation of an existing rodent model of PTSD through replication of the clinical observations of working memory deficits associated with PTSD and provides novel insight into effects in female rodents. This will facilitate work to probe underlying mechanistic dysregulation of working memory following footshock trauma exposure and future development of novel treatment strategies.


Effects of puppet therapy on palmar sweating of hospitalized children.
The study investigated effects of a puppet presentation on anxiety levels of hospitalized children as measured by the Palmar Sweat Index (PSI); Subjects were 43 children, ages 5-8 years inclusive, allocated randomly to treatment and nontreatment groups. Treatment consisted of a puppet show designed to familiarize patients with hospital routines and with procedures of their operations. Four PSI measures were collected from each subject, and average difference scores of individuals among all measures were compared. Analyses of variance were used to test mean differences with data categorized by treatment, by sex, and by interaction of sex and treatment, The treatment was associated with a significant reduction in anxiety from the time of admission to both (1) the period immediately after the puppet show, and (2) the evening after surgery. In the interim, the measure of anxiety returned to near the level at admission.


A prospective cohort study of community functioning among psychiatric outpatients.
The present study analyzed the community functioning among Chinese people with common mental disorders and their relationships with different factors under the framework of Model of Human Occupation. The research team followed up a stratified random sample of 238 patients in three public psychiatric specialist outpatient clinics in Hong Kong in one year. The patients completed assessments at baseline and 12-month follow-up in four areas of community functioning (i.e., self-care, independent living skills, social skills, and work skills), self-esteem, self-efficacy, physical functioning, behavioral regulation, mental states, family expressed emotion, and perceived social stigma. The data showed that after 12 months, the patients had positive changes in self-care, work skills, and behavioral regulation. Those patients who had higher levels of self-esteem at baseline and reduced their negative reactions to stigma were more likely to improve social skills, while those patients who perceived less discrimination at baseline and enhanced their self-esteem would have a higher likelihood of making improvement in work skills. The findings implied that the rehabilitation services for people with common mental disorders might target on the enhancement of self-esteem and reduction of discrimination experience to facilitate their improvement in social skills and work skills.


Ospemifene displays broad-spectrum synergistic interactions with itraconazole through potent interference with fungal efflux activities.
Azole antifungals are vital therapeutic options for treating invasive mycotic infections. However, the emergence of azole-resistant isolates combined with limited therapeutic options presents a growing challenge in medical mycology. To address this issue, we utilized microdilution checkerboard assays to evaluate nine stilbene compounds for their ability to interact synergistically with azole drugs, particularly against azole-resistant fungal isolates. Ospemifene displayed the most potent azole chemosensitizing activity, and its combination with itraconazole displayed broad-spectrum synergistic interactions against Candida albicans, Candida auris, Cryptococcus neoformans, and Aspergillus fumigatus (ΣFICI = 0.05-0.50). Additionally, in a Caenorhabditis elegans infection model, the ospemifene-itraconazole combination significantly reduced fungal CFU burdens in infected nematodes by ~75-96%. Nile Red efflux assays and RT-qPCR analysis suggest ospemifene interferes directly with fungal efflux systems, thus permitting entry of azole drugs into fungal cells. This study identifies ospemifene as a novel antifungal adjuvant that augments the antifungal activity of itraconazole against a broad range of fungal pathogens.


Pharmacotherapy of suicidal behaviour in major depression, schizophrenia and bipolar disorder.
The psychopathological dynamics in suicidality overcomes actual diagnostic distribution therefore pharmacotherapy has restricted role in overall prevention of suicidal behaviour among mentally ill and is demanding for clinician. This role is achieved through reduction and alleviation of suicidal risk with rational and individual pharmacotherapeutic approach emphasising effective, safe and tolerable treatment. The genetic and epigenetic factors, dysfunction of neurotransmitter, neuroendocrine system and stress response system has been determining for neurobiology of suicidality. Therefore, pharmacotherapeutic approach should be focused, not only on prevention and reduction of suicidality, but adjusted for general and diagnosis-specific risk factors. Suicidality represents trans-diagnostic issue, however making the correct diagnosis is of great importance. Identical group of psychiatric medications or even the same drug, could be palliating for suicidal behaviour in one diagnostic category and in other aggravating concerning suicidal ideations. Clinician should be reserved towards epidemiological studies about reducing suicidal rate due to increased consumption of antidepressants. Detailed data analysis showed there is no relevancy which antidepressants were given to specific patient, in what age and phase of illness. The FDA has issued warnings about possible increased risk of suicidal behaviour in children and adolescents when given antidepressant therapy. In general, serotoninergic drugs have neutral or mildly protective effect on potential suicidal behaviour while noradrenergic drugs may have activating effect or could even worsen suicidal ideation in certain phase of the illness. When given in appropriate dose and the right time, dual or noradrenergic antidepressants, could also have good protective impact on specific patient. In patients with bipolar disorder, antidepressive drug could be trigger for suicidal behaviour. Greater susceptibility when diagnosing bipolar disorder and broader usage of mood stablizing medications, alone or combined with other psychopharmacotherapy, has the significant role in suppression and elimination of suicidal behaviour. The lithium and sodium valproate are found to be particularly suitable for prevention and elimination of suicidal behaviour along with some other mood stabilizers. Pharmacotherapy of suicidality in patients with schizophrenia represents specific problem. Confirmed drug with anti-suicidal effect, clozapine, is not first choice medication and does not represent general solution for suicidality in schizophrenia. For clinician, the pharmacotherapy of suicidal behaviour consists of skilled individual and rational drug administration accompanied with appropriate psychotherapeutic support.


Parker Solar Probe Enters the Magnetically Dominated Solar Corona.
The high temperatures and strong magnetic fields of the solar corona form streams of solar wind that expand through the Solar System into interstellar space. At 09:33 UT on 28 April 2021 Parker Solar Probe entered the magnetized atmosphere of the Sun 13 million km above the photosphere, crossing below the Alfvén critical surface for five hours into plasma in casual contact with the Sun with an Alfvén Mach number of 0.79 and magnetic pressure dominating both ion and electron pressure. The spectrum of turbulence below the Alfvén critical surface is reported. Magnetic mapping suggests the region was a steady flow emerging on rapidly expanding coronal magnetic field lines lying above a pseudostreamer. The sub-Alfvénic nature of the flow may be due to suppressed magnetic reconnection at the base of the pseudostreamer, as evidenced by unusually low densities in this region and the magnetic mapping.


JNK signalling controls remodelling of the segment boundary through cell reprogramming during Drosophila morphogenesis.
Segments are fundamental units in animal development which are made of distinct cell lineages separated by boundaries. Although boundaries show limited plasticity during their formation for sharpening, cell lineages make compartments that become tightly restricted as development goes on. Here, we characterize a unique case of breaking of the segment boundary in late drosophila embryos. During dorsal closure, specific cells from anterior compartments cross the segment boundary and enter the adjacent posterior compartments. This cell mixing behaviour is driven by an anterior-to-posterior reprogramming mechanism involving de novo expression of the homeodomain protein Engrailed. Mixing is accompanied by stereotyped local cell intercalation, converting the segment boundary into a relaxation compartment important for tension-release during morphogenesis. This process of lineage switching and cell remodelling is controlled by JNK signalling. Our results reveal plasticity of segment boundaries during late morphogenesis and a role for JNK-dependent developmental reprogramming in this process.


Publishing in Hematology Journals: A Scientometric and Economic Evaluation.
Herein, we aimed to compare the scientometric data of hematology journals, and compare the publication models, especially the scientometric data of journals with all-open access (OA) and hybrid-OA publication models. Data were obtained from Scimago Journal & Country Rank and Clarivate Analytics InCites websites. Fifty-four journals indexed in Science Citation Index (SCI) and SCI-Expanded were evaluated. Bibliometric data and impact factor (IF), scientific journal rank (SJR), eigenfactor score (ES), and Hirsch (h)-index of the journals were obtained. United States dollar (USD) was used as the requested article publishing charge (APC). Statistics Package for the Social Sciences (SPSS, IBM Corp., Armonk, NY) version 23.0 was used for data analysis. As a publication model, Hybrid-OA was the most common. One journal had subscription-only, and two journals had a free-OA model. Nine journals had a mandatory OA with the APC model and 42 journals used a hybrid model. The Median OA fee was 3400 USD. Hybrid-OA journals had a significantly higher median h-index (72 vs. 40, p=0.03) compared to all-OA journals. Other scientometric indexes were similar. When APCs were compared, all-OA journals were median 900 USD lower than hybrid-OA journals (2490 vs. 3400 USD, p=0.019). There is a widespread use of the OA publication model in hematology journals. Although hybrid OA journals have higher h-index, other scientometric indexes are similar. All-OA journals are more economically feasible considering a lower median APC. Further scientometric studies for journals in the field of hematology, randomized to follow citation per publication according to the OA model would better shed light on the data in this area.


Epigenetic modulations and lineage plasticity in advanced prostate cancer.
Prostate cancer is the most common cancer and second leading cause of cancer-related death in American men. Antiandrogen therapies are part of the standard of therapeutic regimen for advanced or metastatic prostate cancers; however, patients who receive these treatments are more likely to develop castration-resistant prostate cancer (CRPC) or neuroendocrine prostate cancer (NEPC). In the development of CRPC or NEPC, numerous genetic signaling pathways have been under preclinical investigations and in clinical trials. Accumulated evidence shows that DNA methylation, chromatin integrity, and accessibility for transcriptional regulation still play key roles in prostate cancer initiation and progression. Better understanding of how epigenetic change regulates the progression of prostate cancer and the interaction between epigenetic and genetic modulators driving NEPC may help develop a better risk stratification and more effective treatment regimens for prostate cancer patients.


Radiographic, biomechanical, and pathologic effects of hemoglobin glutamer-200 in dogs undergoing cemented total hip arthroplasty.
To determine whether use of hemoglobin glutamer-200 (bovine) as a partial blood volume replacement in dogs undergoing cemented total hip replacement caused any deleterious effects on the bone-cement or cement-prosthesis interface, exerted any deleterious effects on body organs, or caused any complications during the anesthetic, immediate recovery, or long-term recovery period. 9 adult dogs. Dogs were anesthetized, and 15% of the blood volume was removed. Simultaneously, lactated Ringer's solution was infused, and 6 dogs were given hemoglobin glutamer (1 g/kg of body weight, IV). Unilateral total hip replacement was performed. Limb use was assessed visually, and force-plate and radiographic evaluations were performed before, and 8 weeks after, surgery. Eight weeks after surgery, dogs were euthanatized, necropsies were performed, and prosthetic component pullout forces were determined. There were no significant differences between treated and control dogs in regard to biomechanical (visual assessment of gait, force-plate analysis, femoral and acetabular component pullout forces) and pathologic evaluations (physical examination, CBC, serum biochemical analyses, necropsy, and histologic evaluations). Radiographic signs of loosening of the femoral component were seen in 4 dogs treated with hemoglobin glutamer. Administration of hemoglobin glutamer as a blood substitute did not appear to have any deleterious effects in dogs undergoing total hip arthroplasty. The radiographic findings, which were discordant with the biomechanical results, merit further investigation.


Zebrafish as a Model Organism for the Development of Drugs for Skin Cancer.
Skin cancer, which includes melanoma and squamous cell carcinoma, represents the most common type of cutaneous malignancy worldwide, and its incidence is expected to rise in the near future. This condition derives from acquired genetic dysregulation of signaling pathways involved in the proliferation and apoptosis of skin cells. The development of animal models has allowed a better understanding of these pathomechanisms, with the possibility of carrying out toxicological screening and drug development. In particular, the zebrafish (Danio rerio) has been established as one of the most important model organisms for cancer research. This model is particularly suitable for live cell imaging and high-throughput drug screening in a large-scale fashion. Thanks to the recent advances in genome editing, such as the clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) methodologies, the mechanisms associated with cancer development and progression, as well as drug resistance can be investigated and comprehended. With these unique tools, the zebrafish represents a powerful platform for skin cancer research in the development of target therapies. Here, we will review the advantages of using the zebrafish model for drug discovery and toxicological and phenotypical screening. We will focus in detail on the most recent progress in the field of zebrafish model generation for the study of melanoma and squamous cell carcinoma (SCC), including cancer cell injection and transgenic animal development. Moreover, we will report the latest compounds and small molecules under investigation in melanoma zebrafish models.


Association between chronic fatigue syndrome and the corticosteroid-binding globulin gene ALA SER224 polymorphism.
Chronic fatigue syndrome (CFS) is characterized by idiopathic fatigue of greater than 6 months' duration with postexertional exacerbation and many other symptoms. A trend toward relative hypocortisolism is described in CFS. Twin and family studies indicate a substantial genetic etiologic component to CFS. Recently, severe corticosteroid-binding globulin (CBG) gene mutations have been associated with CFS in isolated kindreds. Human leukocyte elastase, an enzyme important in CBG catabolism at inflammatory sites, is reported to be elevated in CFS. We hypothesized that CBG gene polymorphisms may act as a genetic risk factor for CFS. A total of 248 patients with CFS defined by Centers for Disease Control criteria, and 248 controls were recruited. Sequencing and restriction enzyme testing of the CBG gene coding region allowed detection of severe CBG gene mutations and a common exon 3 polymorphism (c.825G-->T, Ala-Ser224). Plasma CBG levels were measured in 125 CFS patients and 198 controls by radioimmunoassay. Total and free (calculated and measured) cortisol levels were ascertained in single samples between 8-10 a.m. The age of onset (mid 30s) and gender ratio (2.2:1, female:male) of the patients were similar to those reported in U.S. epidemiologic studies. A trend toward a preponderance of serine224 homozygosity among the CFS patients was noted, compared with controls (chi2 = 5.31, P = 0.07). Immunoreactive-CBG (IR-CBG) levels were higher in Serine/Alanine (Ser/Ala) than Ala/Ala subjects and higher again in Ser/Ser subjects, this effect was strongest in controls; Ser/Ser: 46.1+/-1.8 (n = 31, P = 0.03) vs. Ser/Ala: 42.4+/-1.0 (n = 56, P = 0.05) vs. Ala/Ala: 40.8+/-1.7 microg/mL (n = 21). Despite higher CBG levels, there was a nonsignificant trend toward lower total and free plasma cortisol in serine allele positive patients, total cortisol: Ser/Ser: 13.3+/-1.4 (n = 34) vs. Ser/Ala: 14.0+/-0.7 (n = 66) vs. Ala/Ala: 15.4+/-1.0 (n = 23). Homozygosity for the serine allele of the CBG gene may predispose to CFS, perhaps due to an effect on hypothalamic-pituitary-adrenal axis function related to altered CBG-cortisol transport function or immune-cortisol interactions.


Species-area relationships always overestimate extinction rates from habitat loss.
Extinction from habitat loss is the signature conservation problem of the twenty-first century. Despite its importance, estimating extinction rates is still highly uncertain because no proven direct methods or reliable data exist for verifying extinctions. The most widely used indirect method is to estimate extinction rates by reversing the species-area accumulation curve, extrapolating backwards to smaller areas to calculate expected species loss. Estimates of extinction rates based on this method are almost always much higher than those actually observed. This discrepancy gave rise to the concept of an 'extinction debt', referring to species 'committed to extinction' owing to habitat loss and reduced population size but not yet extinct during a non-equilibrium period. Here we show that the extinction debt as currently defined is largely a sampling artefact due to an unrecognized difference between the underlying sampling problems when constructing a species-area relationship (SAR) and when extrapolating species extinction from habitat loss. The key mathematical result is that the area required to remove the last individual of a species (extinction) is larger, almost always much larger, than the sample area needed to encounter the first individual of a species, irrespective of species distribution and spatial scale. We illustrate these results with data from a global network of large, mapped forest plots and ranges of passerine bird species in the continental USA; and we show that overestimation can be greater than 160%. Although we conclude that extinctions caused by habitat loss require greater loss of habitat than previously thought, our results must not lead to complacency about extinction due to habitat loss, which is a real and growing threat.


Purification and Biophysical Characterization of the Mre11-Rad50-Nbs1 Complex.
The Mre11-Rad50-Nbs1 (MRN) complex coordinates the repair of DNA double-strand breaks, replication fork restart, meiosis, class-switch recombination, and telomere maintenance. As such, MRN is an essential molecular machine that has homologs in all organisms of life, from bacteriophage to humans. In human cells, MRN is a >500 kDa multifunctional complex that encodes DNA binding, ATPase, and both endonuclease and exonuclease activities. MRN also forms larger assemblies and interacts with multiple DNA repair and replication factors. The enzymatic properties of MRN have been the subject of intense research for over 20 years, and more recently, single-molecule biophysics studies are beginning to probe its many biochemical activities. Here, we describe the methods used to overexpress, fluorescently label, and visualize MRN and its activities on single molecules of DNA.


Structural overshoot of tree growth with climate variability and the global spectrum of drought-induced forest dieback.
Ongoing climate change poses significant threats to plant function and distribution. Increased temperatures and altered precipitation regimes amplify drought frequency and intensity, elevating plant stress and mortality. Large-scale forest mortality events will have far-reaching impacts on carbon and hydrological cycling, biodiversity, and ecosystem services. However, biogeographical theory and global vegetation models poorly represent recent forest die-off patterns. Furthermore, as trees are sessile and long-lived, their responses to climate extremes are substantially dependent on historical factors. We show that periods of favourable climatic and management conditions that facilitate abundant tree growth can lead to structural overshoot of aboveground tree biomass due to a subsequent temporal mismatch between water demand and availability. When environmental favourability declines, increases in water and temperature stress that are protracted, rapid, or both, drive a gradient of tree structural responses that can modify forest self-thinning relationships. Responses ranging from premature leaf senescence and partial canopy dieback to whole-tree mortality reduce canopy leaf area during the stress period and for a lagged recovery window thereafter. Such temporal mismatches of water requirements from availability can occur at local to regional scales throughout a species geographical range. As climate change projections predict large future fluctuations in both wet and dry conditions, we expect forests to become increasingly structurally mismatched to water availability and thus overbuilt during more stressful episodes. By accounting for the historical context of biomass development, our approach can explain previously problematic aspects of large-scale forest mortality, such as why it can occur throughout the range of a species and yet still be locally highly variable, and why some events seem readily attributable to an ongoing drought while others do not. This refined understanding can facilitate better projections of structural overshoot responses, enabling improved prediction of changes in forest distribution and function from regional to global scales.


ras mutations are associated with aggressive tumor phenotypes and poor prognosis in thyroid cancer.
ras oncogenic activation has long been demonstrated in thyroid carcinomas of follicular cell derivation, but no consistent relationship has been shown between mutations and clinicopathologic features. We analyzed H-, K-, and N-ras mutations by polymerase chain reaction-single-strand conformational polymorphism followed by DNA sequencing in 125 thyroid carcinoma specimens from 107 patients, to include tumors covering the entire spectrum of thyroid tumor differentiation. Mutations were identified in four (8.2%) of 49 well-differentiated carcinomas (WDCs; two [6.7%] of 30 of the tumors were papillary carcinomas, two [10.5%] of 19 of them were follicular carcinomas), in 16 (55.2%) of 29 poorly differentiated carcinomas (PDCs), and in 15 (51.7%) of 29 undifferentiated carcinomas, with a significant association between ras mutation and poorly or undifferentiated tumors (P <.001). Twenty-six (74.3%) of 35 patients with ras-mutated tumors died as a result of disease as opposed to 23 (31.9%) of 72 patients with tumors lacking the mutations. Among patients with differentiated thyroid carcinomas (WDC and PDC), 11 (55.0%) of 20 patients with mutated tumors died as a result of disease as opposed to nine (15.5%) of 58 patients with wild-type ras tumors, and the correlation was independent of tumor differentiation and stage (P =.016). K-ras codon 13 mutations (all with G-A nucleotide transitions resulting in Gly>Asp substitution) and single activating mutations in any of the ras genes were also independent predictors of poor survival in differentiated thyroid carcinomas (P =.027 and P =.007, respectively). These findings demonstrate that ras mutations are a marker for aggressive cancer behavior and indicate a possible role of ras genotyping to identify thyroid carcinoma subsets associated with poor prognosis.


Gigahertz dynamics of a strongly driven single quantum spin.
Two-level systems are at the core of numerous real-world technologies such as magnetic resonance imaging and atomic clocks. Coherent control of the state is achieved with an oscillating field that drives dynamics at a rate determined by its amplitude. As the strength of the field is increased, a different regime emerges where linear scaling of the manipulation rate breaks down and complex dynamics are expected. By calibrating the spin rotation with an adiabatic passage, we have measured the room-temperature "strong-driving" dynamics of a single nitrogen vacancy center in diamond. With an adiabatic passage to calibrate the spin rotation, we observed dynamics on sub-nanosecond time scales. Contrary to conventional thinking, this breakdown of the rotating wave approximation provides opportunities for time-optimal quantum control of a single spin.


p53 autoantibodies in patients with pancreatitis and pancreatic carcinoma.
In human pancreatic carcinoma (PCa) mutations in the p53 tumor suppressor gene are present in up to 50% of cases. Conformational change and cellular accumulation, together with subsequent release of mutant and normal p53 protein from transformed cells, may initiate a B-cell response with generation of circulating autoantibodies to p53 protein (anti-p53). In the present study we analyzed the sera of 85 consecutive patients with acute pancreatitis (N = 19), chronic pancreatitis (N = 33), and PCa (N = 33) to evaluate the specificity of autoantibodies to p53 protein as a serological marker for PCa. Detection of anti-p53 was performed using an enzyme-linked immunosorbent assay system with immobilized recombinant wild-type p53 protein. Autoantibodies to p53 were detectable in 1 of 19 patients with acute (5.3%) and in 4 of 33 patients with chronic pancreatitis (12.1%). All anti-p53-positive patients with acute or chronic pancreatitis were carefully examined and no underlying malignant disease was found. During follow-up (range, 281-647 days; mean, 472 days) none of these patients showed any evidence for subsequent development of PCa or any other malignant disease. In patients with PCa, anti-p53 was detected in 6 of 33 cases, resulting in a sensitivity of 18.2% with a specificity of 90.4%. In contrast to anti-p53, detection of serum carbohydrate antigen (CA 19-9) resulted in a sensitivity and specificity of 69.7 and 71.2% (CA 19-9, > 37 U/ml) and 51.5 and 96.2% (CA 19.9, > 100 U/ml) for the detection of PCa, respectively. Taken together, the sensitivity of anti-p53 formation was low in patients with PCa (18.2%). Furthermore, the detection of anti-p53 was not specific for malignancy, indicating that severe inflammatory processes may also induce anti-p53 formation.


[Food-dependent Cushing syndrome of long standing with mild clinical features].
A 40-year-old woman complained of weight gain for 10 years. Because of this, she yearly went on a excessive weight reduction diet with weight loss of up to 12 kg. Nevertheless, a total weight gain of 15 kg as well as changing degrees of hypertension, muscular weakness and severe depression were registered during this long period. For 5 years ecchymosis and for 2 years oligomenorrhoea as well as oedema had become evident. Recently large macronodular adrenal glands were found on routine abdominal ultrasound. At the time of clinical investigation early this year she presented with relatively mild Cushing features, such as moon face, central obesity, hirsutism, ecchymosis and hypertension. Endocrine testing demonstrated suppressed plasma ACTH, low to normal plasma cortisol after overnight fasting and an inverse diurnal rhythm. During 24 hours of fasting plasma cortisol levels and free urinary cortisol excretion were within normal range, but highly elevated under normal food intake. Plasma cortisol levels increased dramatically in response to a lipid-rich meal (5.6-fold), to a protein-rich meal (6.6-fold) and to oral glucose (3.7-fold). Increases in plasma cortisol were closely correlated to increases of gastric inhibitory polypeptide (GIP) concentrations. Subcutaneous injection of octreotide completely inhibited the plasma cortisol and GIP response to oral glucose. Plasma ACTH and cortisol levels did not increase after CRH administration, but fasting plasma cortisol promptly increased after ACTH stimulation. Non-fasting plasma cortisol levels and free urinary cortisol excretion were not suppressed by high doses of dexamethasone. Abdominal computed tomography showed macronodular enlargement of both adrenal glands. The patient underwent bilateral adrenalectomy. Postoperatively, she was treated with replacement doses of hydrocortisone and fludrocortisone. All Cushing features have regressed, a normal menstrual cycle and normal plasma ACTH concentrations have become evident. This case report not only presents a very rare cause of Cushing's syndrome--food-dependent or gastric inhibitory peptide (GIP)-dependent hypercortisolism--but it also demonstrates a special course of this disease of relatively long duration. Severe adrenal hyperplasia contrasted with mild clinical symptoms obviously caused by frequent periods of weight reduction diet. Considering this moderate clinical course it might well be that food-dependent hypercortisolism is more frequent than supposed, but not diagnosed owing to relatively mild clinical symptoms.


Description of Komagataella phaffii sp. nov. and the transfer of Pichia pseudopastoris to the methylotrophic yeast genus Komagataella.
The new methanol-assimilating yeast species Komagataella phaffii Kurtzman sp. nov. (type strain NRRL Y-7556(T)=CBS 2612(T)) is described. Of the four known strains of this species, two were isolated from black oak trees in California, USA, one from an Emory oak in Arizona, USA, and one from an unidentified source in Mexico. The species forms hat-shaped ascospores in deliquescent asci and appears to be homothallic. Analysis of nucleotide sequences from domains D1/D2 of large-subunit (26S) rDNA separates the new species from Komagataella pastoris, the type species of the genus, and from Pichia pseudopastoris, which is here renamed Komagataella pseudopastoris (Dlauchy, Tornai-Lehoczki, Fulop & Peter) Kurtzman comb. nov. (type strain NRRL Y-27603(T)=CBS 9187(T)=NCAIM Y 01541(T)). On the basis of D1/D2 26S rDNA sequence analysis, the three species now assigned to the genus Komagataella represent a clade that is phylogenetically isolated from other ascomycetous yeast genera.


Celastrol Protects RPE Cells from Oxidative Stress-Induced Cell Death via Activation of Nrf2 Signaling Pathway.
Oxidative stress to retinal pigment epithelial (RPE) cells and inflammation are closely related to the pathogenesis of age-related macular degeneration (AMD). Celastrol is a natural compound isolated from the root of Tripterygium wilfordii. Celastrol has been shown to have potent anti-inflammatory and anti-tumor effects in multiple disease models. The objective of this study was to test the anti-oxidative effects of celastrol in RPE cells and to investigate the underlying mechanisms. ARPE-19 cells were treated with hydrogen peroxide (H2O2) and menadione alone or in combination with celastrol. Cell viability and apoptosis were examined by CCK-8 and TUNEL assay, respectively. The expression of Nrf2 and its target genes, such as GCLM and HO-1 was determined by Western blotting. The knockdown of Nrf2 was done by transfecting ARPE-19 cells with lentivirus encoding shRNA against Nrf2. The knockdown efficiency was determined by real-time quantitative PCR and Western blotting. Treatment of ARPE-19 cells with celastrol significantly attenuated the toxic effects of both H2O2 and menadione. Treatment with celastrol enhanced the expression of transcription factor Nrf2 and its targets, GCLM and HO-1. Knockdown of Nrf2 expression by shRNA partially abolished the protective effects of celastrol. Chemical inhibition of glutathione synthesis by L-buthionine-S,R-sulfoximine (BSO) completely abolished the protective effects of celastrol against H2O2 and menadione-induced damage. However, chemical inhibition of HO-1 activity by ZnPPIX did not reduce the protective effects of celastrol. This study provides evidence that treatment of RPE cells with celastrol shows potent protective effects against oxidative insults via activation of Nrf2 signaling pathway and upregulation of GCLM expression. This finding suggests that celastrol might be used as a potential therapeutic agent for oxidative stress-related eyes diseases, such as AMD.


Funding source and primary outcome changes in clinical trials registered on ClinicalTrials.gov are associated with the reporting of a statistically significant primary outcome: a cross-sectional study.
We and others have shown a significant proportion of interventional trials registered on ClinicalTrials.gov have their primary outcomes altered after the listed study start and completion dates. The objectives of this study were to investigate whether changes made to primary outcomes are associated with the likelihood of reporting a statistically significant primary outcome on ClinicalTrials.gov. A cross-sectional analysis of all interventional clinical trials registered on ClinicalTrials.gov as of 20 November 2014 was performed. The main outcome was any change made to the initially listed primary outcome and the time of the change in relation to the trial start and end date. 13,238 completed interventional trials were registered with ClinicalTrials.gov that also had study results posted on the website. 2555 (19.3%) had one or more statistically significant primary outcomes. Statistical analysis showed that registration year, funding source and primary outcome change after trial completion were associated with reporting a statistically significant primary outcome . Funding source and primary outcome change after trial completion are associated with a statistically significant primary outcome report on clinicaltrials.gov.


Oral and maxillofacial sclerosing epithelioid fibrosarcoma: report of five cases.
Sclerosing epithelioid fibrosarcoma (SEF) has distinctive morphology and occurs mainly in deep soft tissue of adult extremities. Approximately 59 cases of SEF have been reported, with only 12 previously described in head and neck locations. Lesions involving the oral and maxillofacial region (OMFR) and intraosseous examples are rare. We present five cases of OMFRSEF. The OMF Pathology Department Registry was searched for cases coded from 1990 to the present as "SEF," "fibrosarcoma not otherwise specified" or "neoplasm of uncertain histiogenesis." Inclusion required OMFR location, an abundantly sclerotic sarcoma with epithelioid features, and lack of other phenotype by immunohistochemistry. Five cases of SEF included 3 males and 2 females. The age of the patients were: 19, 22, 35, 47 and 47 years. Tumor location included the infra-temporal fossa, buccal mucosa (recurrence extending into bone), anterior mandible (intraosseous primary, focally extending into soft tissue), and left parotid and submandibular gland (with metaplastic bone) regions. Tumor sizes ranged from 1.0 to 5.7 cm, median 3.5 cm. Histologically, the tumors were well delineated and multinodular, separated by fibrous septae. The spindled to primarily epithelioid tumor cells formed moderately cellular sheets and cords of irregularly contoured medium to large, round to oval, occasionally overlapping nuclei, indistinct nucleoli, wispy eosinophilic (retracting) cytoplasm, and distinctive cytoplasmic borders, embedded in osteoid-like stroma. Hemangiopericytoid (HPC-like) vessels were observed. Despite numerous apoptotic cells, mitoses were generally low; necrosis was present in two cases. Three tumors were graded as 2/3 and two 1/3. Immunohistochemically, the tumor cells were positive for vimentin, 1 case focally for CD34, whereas all cases were negative for S100 protein, keratins, EMA, desmin, and SMA. Wide or radical excision was performed with no adjuvant therapy. Follow-up revealed that 4 cases recurred at a range of 12-120 months. One case had no recurrent/residual disease at 3 months. Metastatic disease was present in 2 cases, to chest wall and lumbar/thoracic spine at 12 and 21 months, respectively. One patient died of disease complications at 15 months. OMFRSEF occur in adults in various locations, but with a common propensity to involve bone; there is recurrent potential and morbidity with higher grade lesions. The differential diagnosis for these tumors in this site includes sclerosing carcinoma, Ewing/PNET, osteosarcoma, osteoblastoma, and benign and malignant myoepithelial salivary gland tumors. The collagen, focal spindle cell features, HPC-like vasculature, and weak focal CD34 reactivity in one case might have raised a possible relationship between OMFRSEF and low grade malignant solitary fibrous tumor, but the intraosseous propensity, epithelioid features and relative lack of CD34 make this a distinctive entity.


Immune modulatory effects of Prunella vulgaris L.
Prunella vulgaris L. (Labiatae) is a perennial plant known as 'self-heal' in Western herbal medicine. It has a wide array of biological effects exhibiting numerous therapeutic potentials. Its anti-microbial effects including anti-viral and anti-bacterial effects are, presently, receiving increasing attention. While its anti-viral effects are attributed mainly to the inhibition of virus replication, the biological mechanisms of its anti-bacterial effects or actions remain unknown. In view of the fact that polysaccharides isolated from medicinal herbs often function as biological response modifier of body immunity, we hypothesized that the anti-microbial effect of polysaccharides isolated from P. vulgaris is probably also mediated via immune modulation. We have isolated four polysaccharides containing fractions from P. vulgaris, one of the fractions, PV2, could markedly stimulate the production of superoxide and nitrite representing nitric oxide from murine macrophage RAW264.7 and brain macrophage BV2 cells. The amount of nitrite and superoxide produced after PV2 stimulation was as high as that seen in stimulation using bacterial endotoxin lipopolysaccharide (LPS), and this stimulatory response is dose-dependent. In addition to monocyte/macrophage, PV2 also stimulated the proliferation of splenocytes. In this study, we have shown that the polysaccharides isolated from P. vulgaris have marked immune stimulatory effects, which may bring about the anti-microbial effects of P. vulgaris.


Formation of the antithrombin heterodimer in vivo and the onset of thrombosis.
Antithrombin is shown to undergo a slow spontaneous conversion to its inactive latent conformation with readily discernible amounts present in plasma on incubation at 37 degrees C for 72 hours. More rapid conversion occurs on incubation of isolated antithrombin at 41 degrees C or 50 degrees C, but the appearance on electrophoresis of free latent antithrombin is preceded by the formation, in reciprocal proportions, of a new slow band. This slow component is shown to be a heterodimer of active and latent antithrombin. It can be isolated as a single stable band either by incubation of antithrombin or by mixing equimolar proportions of active and latent antithrombin under the same conditions that give overnight crystallization of the active/latent antithrombin heterodimer. Similarly, equimolar addition of latent antithrombin to plasma results electrophoretically in a quantitative shift to the slower heterodimer mobility. Clinically, the presence of latent antithrombin is potentially deleterious, because its linkage to form the heterodimer results in inactivation of the otherwise normal molecule linked to the latent antithrombin. In the case of alpha-antithrombin, because the dimer readily dissociates, there is only a 11% additive loss of activity, but with beta-antithrombin the dimer appears more stable, with the additive loss of activity from the normal beta component being 21%, increasing to 33% on stabilization of the dimer with heparin. This linked and selective loss of activity of beta-antithrombin provides an explanation for the unexpected severity of thrombotic episodes in heterozygotes with conformationally unstable antithrombins.


The shipboard Beirut terrorist bombing experience: a historical account and recommendations for preparedness in events of mass neurological injuries.
On a Sunday morning at 06:22 on October 23, 1983, in Beirut, Lebanon, a semitrailer filled with TNT sped through the guarded barrier into the ground floor of the Civilian Aviation Authority and exploded, killing and wounding US Marines from the 1st Battalion 8th Regiment (2nd Division), as well as the battalion surgeon and deployed corpsmen. The truck bomb explosion, estimated to be the equivalent of 21,000 lbs of TNT, and regarded as the largest nonnuclear explosion since World War II, caused what was then the most lethal single-day death toll for the US Marine Corps since the Battle of Iwo Jima in World War II. Considerable neurological injury resulted from the bombing. Of the 112 survivors, 37 had head injuries, 2 had spinal cord injuries, and 9 had peripheral nerve injuries. Concussion, scalp laceration, and skull fracture were the most common cranial injuries.Within minutes of the explosion, the Commander Task Force 61/62 Mass Casualty Plan was implemented by personnel aboard the USS Iwo Jima. The wounded were triaged according to standard protocol at the time. Senator Humphreys, chairman of the Preparedness Committee and a corpsman in the Korean War, commented that he had never seen such a well-executed evolution. This was the result of meticulous preparation that included training not only of the medical personnel but also of volunteers from the ship's company, frequent drilling with other shipboard units, coordination of resources throughout the ship, the presence of a meticulous senior enlisted man who carefully registered each of the wounded, the presence of trained security forces, and a drilled and functioning communication system.Viewed through the lens of a neurosurgeon, the 1983 bombings and mass casualty event impart important lessons in preparedness. Medical personnel should be trained specifically to handle the kinds of injuries anticipated and should rehearse the mass casualty event on a regular basis using mock-up patients. Neurosurgery staff should participate in training and planning for events alongside other clinicians. Training of nurses, corpsmen, and also nonmedical personnel is essential. In a large-scale evolution, nonmedical personnel may monitor vital signs, work as scribes or stretcher bearers, and run messages. It is incumbent upon medical providers and neurosurgeons in particular to be aware of the potential for mass casualty events and to make necessary preparations.


Neural responses to faces reflect social personality traits.
Faces are a developmentally primary and critically important source of social information, and they are processed differently from most other visual percepts. Studies of brain electrophysiology reveal a face-sensitive component, the N170, which is typically enhanced to faces relative to other stimuli. Research in social disabilities suggests that an atypical N170 response in this population may stem from decreased developmental exposure to faces secondary to reduced social interest. Here we examined the relationship between neural responses to faces and social personality characteristics in a normative sample. Participants were pre-screened to identify individuals scoring high on extraversion or introversion. Both groups were presented with upright and inverted face stimuli. An inversion effect, a marker of expertise for faces, was observed in people with high extraversion but not in those with high introversion. These findings suggest that, within typically developing populations, social attitudes are reflected in the neural correlates of face perception.


Family life 1 year after infantile colic.
To determine the persistence of family psychological characteristics associated with infantile colic. A 1-year follow-up survey of families with colicky infants was carried out. The family interview was used to evaluate the psychological factors of colicky and control families. A total of 59 families with a colicky infant and 58 control families were enrolled in the initial evaluation in the population-based study. On the basis of a structured diary of the infant's crying, filled in by the parents, 3 groups of families were formed: severe colic group (n = 36), moderate colic group (n = 23), and control group (n = 58). The families were interviewed when the infants were 2 and 12 months old. The Beavers-Timberlawn Family Evaluation Scale and the Oulu Family Assessment Scale were used. Thirty-two families (89%) from the severe colic group, 17 (74%) from the moderate colic group, and 49 (84%) from the control group participated in the 1-year assessment. At the 1-year assessment, the structural profile of the families did not differ between the groups. However, families in the severe colic group had more difficulties in communication, more unresolved conflicts, more dissatisfaction, and greater lack of empathy than families in the other groups. Furthermore, there was less flexibility in both colic groups than in the control group. During the follow-up period, coalition between parents in the moderate colic group became stronger compared with the first assessment, and the atmosphere improved significantly in all 3 study groups. However, the amount of empathy decreased in the severe colic group. Our study shows that certain family characteristics associated with severe infantile colic persist. Families in the moderate colic group coped nearly as well as families in the control group. The family mood had improved a lot in all families when the infant was 1 year old.


Effects of extracellular potassium on ventricular automaticity and evidence for a pacemaker current in mammalian ventricular myocardium.
Automaticity was induced in isolated guinea pig and cat papillary muscles by application of depolarizing constant current pulses. Increasing extracellular potassium from 1 to 15 mM caused a shift of pacemaker-like activity to less negative diastolic potentials and a decrease in maximum phase 4 slope. Membrane resistance, estimated from the relation of applied current to maximum diastolic potential, decreased when extracellular potassium was increased. Voltage clamps of cat papillary muscle demonstrated that action potentials activate a time-dependent outward current which has a reversal potential of -79.1 mV (+/- 0.99 SE, n = 20) at an extracellular potassium concentration of 5 mM. The reversal potential of this current varies with extracellular K+ with a slope of 50-60 mV per 10-fold concentration change. The current is activated by voltage clamps or action potential plateaus in the range of -30 to +30 mV. It has a time constant of deactivation which increases from approximately 100 to over 400 msec as clamp potential is increased from -90 to -60 mV. It is proposed that this current is equivalent to Ix1 demonstrated in other cardiac tissues and is responsible, in combination with inward currents, for automaticity in ventricular fibers.


The current perspective on tick-borne encephalitis awareness and prevention in six Central and Eastern European countries: report from a meeting of experts convened to discuss TBE in their region.
Tick-borne encephalitis (TBE) is a potentially life-threatening disease in humans and is caused by a flavivirus spread by infected ticks (Ixodes ricinus and Ixodes persulcatus). TBE is endemic across much of Central and Eastern Europe and the incidence is increasing, with numbers estimated to be as many as 8755 cases per year. The reasons for this increase are multi-faceted and may involve improvements in diagnosis and reporting of TBE cases, increases in recreational activities in areas inhabited by infected ticks and changes in climatic conditions affecting tick habitats. Vaccination is the most effective method of preventing TBE; following a successful nationwide vaccination campaign in Austria, the annual number of TBE cases fell to about 10% of those reported in the pre-vaccination era. This report describes the findings of a group of leading experts from six Central and Eastern European countries who convened to discuss TBE in their region during the 28th Annual Meeting of the European Society for Paediatric Infectious Diseases (ESPID) Nice, France, 4-8 May 2010.


Effects of magnesium sulfate administration in attenuating chronic postsurgical pain in rats.
Chronic postsurgical pain (CPSP) is a serious issue for many postoperative patients. Though there are numerous treatment options for the prevention of CPSP, none of them is optimal as the mechanisms of the transition from acute to chronic postoperative pain have not been elucidated. Ketamine and opioids have been administered for chronic postoperative pain treatment but induce severe adverse reactions and/or physical dependency. Here, we examined whether pre-administration of the nonselective N-methyl-d-aspartate (NMDA) receptor antagonist magnesium sulfate attenuates CPSP behavior and alters the expression of glutamate ionotropic receptor NMDA type subunit 1a (Grin1 mRNA) in a rat skin/muscle incision and retraction (SMIR) model. We assessed the effects of a single subcutaneous magnesium sulfate injection on nociceptive behaviors including guarding pain, mechanical hyperalgesia, and heat hypersensitivity in rats after SMIR surgery. We used reverse transcription-quantitative PCR (RT-qPCR) to evaluate Grin1 mRNA expression in the dorsal horn of the spinal cord on postoperative day 14. Compared with the vehicle, magnesium sulfate administration before SMIR surgery reduced mechanical hyperalgesia for 17 d Grin1 gene expression was significantly higher on the ipsilateral side than the contralateral side (P = 0.001) on postoperative day 14. The magnesium sulfate injection prevented Grin1 mRNA upregulation in the spinal cord dorsal horn. A single magnesium sulfate injection mitigated SMIR-induced mechanical hyperalgesia possibly by modulating Grin1 expression. Preoperative magnesium sulfate administration could prove to be a simple and safe CPSP treatment.


Computer-aided designed/computer-assisted manufactured composite resin versus ceramic single-tooth restorations: a 3-year clinical study.
No clinical evidence has been provided to suggest that metal-free all-composite resin indirect restorations are a functional and esthetic alternative to all-ceramic restorations. The aim of this study was to evaluate the clinical performance of single-tooth computer-aided design/computer-assisted manufacturing (CAD/CAM)-generated all-composite resin and all-ceramic crowns after 3 years of function. In a prospective trial, 200 all-composite resin and all-ceramic crowns were rated over a 3-year period. Restorations were evaluated at 3 weeks and 1 and 3 years after insertion by the California Dental Association quality evaluation index, the patient's self-assessment, marginal fit, periodontal parameters, volume loss, and wear patterns of the veneering material. Statistical analysis was performed using t tests (a = .05). Cumulative survival and success rates after 3 years were 87.9% and 55.6% for all-composite resin and 97.2% and 81.2% for all-ceramic crowns, respectively (P < .05 for success rates). Restoration loosening occurred exclusively for all-composite resin crowns cemented on a cast post. All-ceramic restorations demonstrated satisfactory esthetic results. All-composite resin crowns resulted in significantly more mean total volume loss and mean vertical wear at occlusal contact areas after 6 months and 3 years of function. The clinical performance of the CAD/CAM-generated all-ceramic crowns used in this study was similar to that of other all-ceramic CAD/CAM systems. For up to 3 years of function, all-composite resin single-tooth restorations have inferior success rates compared to all-ceramic restorations. Due to the inferior esthetics and wear resistance of all-composite resin crowns, all-ceramic crowns remain the preferred treatment for CAD/CAM-generated metal-free single-tooth restorations.


Development of a centrifugal bioreactor for rapid expansion of CD8 cytotoxic T cells for use in cancer immunotherapy.
One of the current difficulties limiting the use of adoptive cell therapy (ACT) for cancer treatment is the lack of methods for rapidly expanding T cells. As described in the present report, we developed a centrifugal bioreactor (CBR) that may resolve this manufacturing bottleneck. The CBR operates in perfusion by balancing centrifugal forces with a continuous feed of fresh medium, preventing cells from leaving the expansion culture chamber while maintaining nutrients for growth. A bovine CD8 cytotoxic T lymphocyte (CTL) cell line specific for an autologous target cell infected with a protozoan parasite, Theileria parva, was used to determine the efficacy of the CBR for ACT purposes. Batch culture experiments were conducted to predict how CTLs respond to environmental changes associated with consumption of nutrients and production of toxic metabolites, such as ammonium and lactate. Data from these studies were used to develop a kinetic growth model, allowing us to predict CTL growth in the CBR and determine the optimal operating parameters. The model predicts the maximum cell density the CBR can sustain is 5.5 × 107 cells/mL in a single 11-mL conical chamber with oxygen being the limiting factor. Experimental results expanding CTLs in the CBR are in 95% agreement with the kinetic model. The prototype CBR described in this report can be used to develop a CBR for use in cancer immunotherapy.


Effects of a dorsal column lesion on temporal processing within the somatosensory system of primates.
A dorsal column (DC) lesion has lasting effects on behavioral tasks that require temporal processing of tactile information (e.g., frequency and duration discrimination). The present experiments describe physiological correlates of these deficits in temporal discrimination. Compound action potentials evoked by electrocutaneous stimulation were recorded from the major white matter subdivisions of the spinal cord in anesthetized monkeys, and relationships between stimulation frequency and evoked potential (EP) amplitude were determined for the ascending pathways. At 10 pulses per second (Hz) EPs recorded in the lateral spinal columns were attenuated slightly (by 15% or less, relative to 1.5 Hz), whereas potentials recorded from the DCs were not attenuated. The attenuation increased with stimulation frequencies up to 50 Hz, reaching 80% for the anterolateral column and 38% for the dorsolateral column, but only 15% for the DC. Epidural EPs were recorded, before and after interruption of the contralateral DC, from awake animals with electrodes chronically implanted over primary somatosensory cortex (SI). Following the lesion. EP responses to 1.5-Hz stimulation were 46% of preoperative responses to the same stimulus. At 10 Hz, EP amplitudes were attenuated even more, to 27% of the preoperative amplitude at 1.5 Hz. Principal components analysis was employed to quantify alterations in EP conformation and stimulus frequency was varied from 1.5 to 10 Hz, before and after a DC lesion. Interruption of the DC resulted in a significant decrease in the information provided by the EP about changes in stimulus frequency. EPs were also recorded from different locations along the anterior-posterior dimension of the hindlimb region of SI in lightly anesthetized animals. Principal components analysis revealed that there was less information present in the EP about changes in stimulus frequency (1.5-10 Hz) at all recording locations in animals with a DC lesion, compared with the cortex of normal animals. The DC lesion significantly decreased the amplitude of cortical EPs evoked by repetitive stimulation. At 10 Hz the EP was nearly buried in noise, consistent with behavioral deficits in discrimination of the duration of 10 Hz stimulation following interruption of the DC. Also, significantly less information was present in the cortical EPs about changes in stimulus frequency in the absence of intact DCs, which is consistent with deficits in frequency discrimination. This reduction could be explained in part by a lesser capacity of spinal pathways in the lateral column to follow repetitive stimulation above 10 Hz. However, more rostral manifestations of a DC lesion, at either the thalamus or the cortex, are likely to contribute to the reduced capacity of animals with DC lesions to make temporal discriminations.


Hydrogen sulfide [corrected] increases survival during sepsis: protective effect of CHOP inhibition.
Sepsis is a major cause of mortality, and dysregulation of the immune response plays a central role in this syndrome. H2S, a recently discovered gaso-transmitter, is endogenously generated by many cell types, regulating a number of physiologic processes and pathophysiologic conditions. We report that H2S increased survival after experimental sepsis induced by cecal ligation and puncture (CLP) in mice. Exogenous H2S decreased the systemic inflammatory response, reduced apoptosis in the spleen, and accelerated bacterial eradication. We found that C/EBP homologous protein 10 (CHOP), a mediator of the endoplasmic reticulum stress response, was elevated in several organs after CLP, and its expression was inhibited by H2S treatment. Using CHOP-knockout (KO) mice, we demonstrated for the first time, to our knowledge, that genetic deletion of Chop increased survival after LPS injection or CLP. CHOP-KO mice displayed diminished splenic caspase-3 activation and apoptosis, decreased cytokine production, and augmented bacterial clearance. Furthermore, septic CHOP-KO mice treated with H2S showed no additive survival benefit compared with septic CHOP-KO mice. Finally, we showed that H2S inhibited CHOP expression in macrophages by a mechanism involving Nrf2 activation. In conclusion, our findings show a protective effect of H2S treatment afforded, at least partially, by inhibition of CHOP expression. The data reveal a major negative role for the transcription factor CHOP in overall survival during sepsis and suggest a new target for clinical intervention, as well potential strategies for treatment.


Type VIII Si based clathrates: prospects for a giant thermoelectric power factor.
Although clathrate materials are known for their small thermal conductivity, they have not shown a large thermoelectric power factor so far. We present the band structures of type VIII Si, Ge, and Sn clathrates as well as the alkali and alkaline-earth intercalated type VIII Si clathrates. Our calculations revealed that this group of materials has potentially large power factors due to the existence of a large number of carrier pockets near their band edges. In particular, we calculated the charge carrier transport properties of Si46-VIII both for n-type and p-type materials. The exceptionally high multi-valley band structure of Si46-VIII near the Fermi energy due to the high crystallographic symmetry resulted in a giant power factor in this material. It was shown that the intercalation of Si46-VIII with alkali and alkaline-earth guest atoms shifts the Fermi energy close to the conduction band edge and, except for Be8Si46 and Mg8Si46, they weakly influence the band structure of Si46. Among these clathrate systems, Ca8Si46, Sr8Si46, and Ba8Si46 showed negative formation energy, which should facilitate their synthesis. Our results imply that the intercalation affects the conduction band of Si46-VIII more than its valence band. Also, interestingly, the type VIII clathrates of Si46 and its derivatives (except Be8Si46 and Mg8Si46), Sn46, and Ge46 all have 26 carrier pockets near their valence band edge. Among the different derivatives of Si46-VIII, Rb8Si46 and Ba8Si46 have the highest number of electron pockets near their band edges. The thermoelectric power factor was predicted using a multiband Boltzmann transport equation linked with parameters extracted from density functional calculations. It was shown that both the increment of charge mobility and the existence of multiple band extrema contribute to the enhancement of the thermoelectric power factor considerably. Such a large power factor along with their inherently low thermal conductivity can make this group of clathrates promising thermoelectric materials.


Nuclear expression of lysyl oxidase enzyme is an independent prognostic factor in rectal cancer patients.
Emerging evidence has implicated a pivotal role for lysyl oxidase (LOX) in cancer progression and metastasis. Whilst the majority of work has focused on the extracellular matrix cross-linking role of LOX, the exact function of intracellular LOX localisation remains unclear. In this study, we analysed the LOX expression patterns in the nuclei of rectal cancer patient samples and determined the clinical significance of this expression. Nuclear LOX expression was significantly increased in patient lymph node metastases compared to their primary tumours. High nuclear LOX expression in tumours was correlated with a high rate of distant metastasis and increased recurrence. Multivariable analysis showed that high nuclear LOX expression was also correlated with poor overall survival and disease free survival. Furthermore, we are the first to identify LOX enzyme isoforms (50 kDa and 32 kDa) within the nucleus of colon cancer cell lines by confocal microscopy and Western blot. Our results show a powerful link between nuclear LOX expression in tumours and patient survival, and offer a promising prognostic biomarker for rectal cancer patients.


Directed forgetting of faces: the role of response criterion.
In two experiments, we examined recognition for faces following item method directed forgetting. During testing, participants reported whether the face was a new face or, if they thought it was a studied face, they identified the instruction paired with the face during study. In both experiments, the proportion of new faces falsely recognized and classified as forget faces exceeded those falsely recognized and classified as remember faces. Despite the use of different response criteria during testing, participants showed greater discrimination accuracy when identifying remember faces than when identifying forget faces. Taken together, these data patterns indicate that participants employed a strength-based criterion when responding. Specifically, participants responding to new faces were more likely to classify those faces as forget faces from study rather than as remember faces from study.


Gene-environment dependencies lead to collider bias in models with polygenic scores.
The application of polygenic scores has transformed our ability to investigate whether and how genetic and environmental factors jointly contribute to the variation of complex traits. Modelling the complex interplay between genes and environment, however, raises serious methodological challenges. Here we illustrate the largely unrecognised impact of gene-environment dependencies on the identification of the effects of genes and their variation across environments. We show that controlling for heritable covariates in regression models that include polygenic scores as independent variables introduces endogenous selection bias when one or more of these covariates depends on unmeasured factors that also affect the outcome. This results in the problem of conditioning on a collider, which in turn leads to spurious associations and effect sizes. Using graphical and simulation methods we demonstrate that the degree of bias depends on the strength of the gene-covariate correlation and of hidden heterogeneity linking covariates with outcomes, regardless of whether the main analytic focus is mediation, confounding, or gene × covariate (commonly gene × environment) interactions. We offer potential solutions, highlighting the importance of causal inference. We also urge further caution when fitting and interpreting models with polygenic scores and non-exogenous environments or phenotypes and demonstrate how spurious associations are likely to arise, advancing our understanding of such results.


Antibody-dependent cellular cytotoxicity-inducing antibodies enhance the natural killer cell anti-cancer response against patient-derived pancreatic cancer organoids.
Pancreatic cancer is associated with poor prognosis, and limited treatment options are available for the majority of patients. Natural killer (NK) cells in combination with antibodies inducing antibody-dependent cell-mediated cytotoxicity (ADCC) could be a highly effective new therapeutic option in pancreatic cancer. Accurate predictive preclinical models are needed to develop successful NK cell immunotherapy. Tumor organoids, in vitro 3D organ-like structures that retain important pathophysiological characteristics of the in vivo tumor, may provide such a model. In the current study, we assessed the cytotoxic potential of adoptive NK cells against human pancreatic cancer organoids. We hypothesized that NK cell anti-tumor responses could be enhanced by including ADCC-triggering antibodies. We performed cytotoxicity assays with healthy donor-derived IL-2-activated NK cells and pancreatic cancer organoids from four patients. A 3D cytotoxicity assay using live-cell-imaging was developed and enabled real-time assessment of the response. We show that NK cells migrate to and target pancreatic cancer organoids, resulting in an increased organoid death, compared to the no NK cell controls (reaching an average fold change from baseline of 2.1±0.8 vs 1.4±0.6). After 24-hours of co-culture, organoid 2D growth increased. Organoids from 2 out of 4 patients were sensitive to NK cells, while organoids from the other two patients were relatively resistant, indicating patient-specific heterogeneity among organoid cultures. The ADCC-inducing antibodies avelumab (anti-PD-L1) and trastuzumab (anti-HER2) increased NK cell-induced organoid cell death (reaching an average fold change from baseline of 3.5±1.0 and 4.5±1.8, respectively). Moreover, combination therapy with avelumab or trastuzumab resulted in complete disintegration of organoids. Finally, inclusion of ADCC-inducing antibodies was able to overcome resistance in NK-organoid combinations with low or no kill. These results support the use of organoids as a relevant and personalized model to study the anti-tumor response of NK cells in vitro and the potential of ADCC-inducing antibodies to enhance NK cell effector function.


Atrial natriuretic peptides negatively and positively modulate circulating endothelin in humans.
The present investigation was designed to examine the effect of four atrial peptide hormones with vasodilatory properties on the circulating immunoreactive (ir) levels of the vasoconstrictive peptide endothelin (ET) in 36 healthy human subjects. Circulating levels of human ET and cyclic guanosine monophosphate ([cGMP], a potential mediator of the effects of atrial peptides), were measured every 30 minutes during 1-hour preinfusion, 1-hour infusion, and 3-hour postinfusion periods. Atrial natriuretic factor ([ANF] amino acid (aa) 99 to 126 of the 126-aa ANF prohormone) and kaliuretic peptide (aa 79 to 98 of this same prohormone) significantly (P<.05) decreased circulating ET concentrations. Kaliuretic peptide effects were early and ANF effects were delayed until kaliuretic peptide effects began to wane. Long-acting natriuretic peptide (LANP), consisting of aa 1 to 30 of the ANF prohormone, on the other hand, significantly (P<.05) increased ET circulating concentrations during a 1-hour infusion period. The increase in ET in the circulation secondary to LANP became nonsignificant, although it was still elevated, within 30 minutes of ceasing LANP infusion. Vessel dilator, consisting of aa 31 to 67 of the ANF prohormone, and infusion of vehicle alone did not significantly change circulating levels of ET during the 5 hours of this investigation. ANF infusion increased plasma cGMP sevenfold, but plasma cGMP had decreased to only onefold above normal during the period that ANF had an effect on circulating ET levels. There was not any significant increase or decrease in plasma cGMP concentrations secondary to the other atrial peptide hormones. These data suggest that kaliuretic peptide and ANF negatively modulate circulating ET concentrations, while LANP, which is released simultaneously with ANF in response to physiologic stimuli, positively modulates circulating ET concentrations to help maintain circulating ET within a narrow normal range. The data from the present investigation would further suggest that circulating cGMP levels do not mediate the various atrial peptide effects on circulating ET levels.


[Diagnostic utility of tyrosine hydroxylase in peripheral neuroblastic tumors].
Objective: To investigation the diagnostic utility of tyrosine hydroxylase (TH) immunohistochemically as a marker of peripheral neuroblastic tumors(pNT). Methods: The study included 1 024 cases, 643 primary and metastatic pNT cases, 381 non-pNT cases, including small round cell tumors such as primitive neuroectodermal tumor (PNET), rhabdomyosarcoma, lymphoma, nephroblastoma, as well as other more common tumors (medulloblastoma, hepatoblastoma, pleuropulmonary blastoma, renal clear cell sarcoma, Langerhans cell histiocytosis, lipoblatoma etc). Tissue slides of these 1 024 cases were stained with TH and other common antibodies to evaluate whether TH was useful to identify pNT. Results: TH showed cytoplasmic staining in 100.0% (643/643) of pNT and 3.9% (15/381) of non-pNT. TH expression was seen in some poorly differentiated and undifferentiated tumors with focal or scattered patterns. In some cases, more scattering was seen in neuroblastoma with poor differentiation. The sensitivity, specificity, positive predictive value and negative predictive value of TH was 100.0% (643/643), 96.1%(366/381), 97.7%(643/658) and 100.0% (366/366), respectively. There was no TH expression in the 248 cases of other small round cell tumors (PNET, rhabdomyosarcoma, lymphoma, etc), thus suggesting TH was useful to differentiate between PNET and neuroblastoma. As CD99 was positive in 95% PNET, some PNET would be CD99 negative. In those cases, particularly with diffuse rosettes forming, TH immunostaining would be useful. Conclusion: TH is sensitive and specific for pNT in the differential diagnosis from other tumors of childhood and shows high diagnostic utility.


Presence of Legionellaceae in warm water supplies and typing of strains by polymerase chain reaction.
Outbreaks of Legionnaire's disease present a public health challenge especially because fatal outcomes still remain frequent. The aim of this study was to describe the abundance and epidemiology of Legionellaceae in the human-made environment. Water was sampled from hot-water taps in private and public buildings across the area of Göttingen, Germany, including distant suburbs. Following isolation, we used polymerase chain reaction in order to generate strain specific banding profiles of legionella isolates. In total, 70 buildings were examined. Of these 18 (26%) had the bacterium in at least one water sample. Legionella pneumophila serogroups 1, 4, 5 and 6 could be identified in the water samples. Most of the buildings were colonized solely by one distinct strain, as proven by PCR. In three cases equal patterns were found in separate buildings. There were two buildings in this study where isolates with different serogroups were found at the same time.


Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1.
We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C(16) species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1.


A Minimally Invasive Approach for Placing Sew-On Epicardial Leads in the Child.
Traditionally, pacing leads are placed transvenously, although smaller pediatric patients who require permanent pacemakers may benefit from delaying tranvenous lead placement until they are larger. Alternative, minimally invasive atrioventricular pacing options have not previously existed for this patient population, leaving many of these children with large sternotomies or thoracotomies. Using three port sites and an adjustable shaft dual-needle suturing device, we placed a steroid-eluting, sew-on epicardial lead on the right atrium of a 9-year-old patient. This is one of the earliest reported cases of a minimally invasive technique for sew-on epicardial lead placement on the atrium of a child. Although based on a single case, we believe that this approach is safe, reliable, and reproducible and that it can be used to place leads on any aspect of the heart. Adoption of this technique will allow for earlier atrioventricular pacing, which may decrease the incidence of pacemaker syndrome, and thus improve overall outcomes in this patient population.


Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily.
The ARO8 and ARO9 genes of Saccharomyces cerevisiae were isolated by complementation of the phenylalanine/tyrosine auxotrophy of an aro8 and aro9 double-mutant strain that is defective in aromatic aminotransferase I (aro8) and II (aro9). The genes were sequenced, and deletion mutants were constructed and analysed. The expression of ARO8 and ARO9 was studied. The deduced amino acid sequences of Aro8p and Aro9p suggest that the former is a 500-residue, 56168-Da polypeptide and the latter a 513-residue, 58516-Da polypeptide. They correspond, respectively, to Ygl202p and Yhr137p, two putative proteins of unknown function revealed by systematic sequencing of the yeast genome. We show that aromatic aminotransferases I and II are homologous proteins, members of aminotransferase subgroup I, and, together with three other proteins, they constitute within the subgroup a new subfamily of enzymes specialised for aromatic amino acid and alpha-aminoadipate transamination. ARO8 expression is subject to the general control of amino acid biosynthesis. ARO9 expression is induced when aromatic amino acids are present in the growth medium and also in aro8 mutants grown on minimal ammonia medium. An autonomously replicating sequence (ARS) element is located between the ARO8 gene and YGL201c which encodes a protein of the minichromosome maintenance family.


Long-term effects of bullying.
Bullying is the systematic abuse of power and is defined as aggressive behaviour or intentional harm-doing by peers that is carried out repeatedly and involves an imbalance of power. Being bullied is still often wrongly considered as a 'normal rite of passage'. This review considers the importance of bullying as a major risk factor for poor physical and mental health and reduced adaptation to adult roles including forming lasting relationships, integrating into work and being economically independent. Bullying by peers has been mostly ignored by health professionals but should be considered as a significant risk factor and safeguarding issue.


Optic chiasm involvement in AQP-4 antibody-positive NMO and MOG antibody-associated disorder.
Optic neuritis (ON) is often the presenting symptom in inflammatory central nervous system demyelinating disorders. To compare the frequency and pattern of optic chiasm involvement in patients with aquaporin-4-immunoglobulin G (AQP4-IgG)-associated ON to patients with myelin oligodendrocyte glycoprotein-immunoglobulin G (MOG-IgG)-associated ON. Retrospective review of all patients evaluated at Mayo Clinic, Stanford University and Ramathibodi Hospital who were found to have: (1) ON, (2) either MOG-IgG or AQP4-IgG by cell-based assay, and (3) magnetic resonance imaging (MRI) at the time of ON. MRI was reviewed for contrast enhancement of the optic chiasm and the pattern of involvement. One hundred and fifty-four patients (74 AQP4-IgG and 80 MOG-IgG) were included. Among patients with AQP4-IgG-ON, 20% had chiasmal involvement, compared with 16% of patients with MOG-IgG-ON (p = 0.66). In patients with chiasmal involvement, longitudinally extensive optic nerve enhancement (from orbit extending to chiasm) was identified in 54% of MOG-IgG-ON patients, compared with 7% of AQP4-IgG-ON patients (p = 0.01). Chiasmal involvement of MOG-IgG-ON and AQP4-IgG-ON occur at more similar frequencies than previously reported. Furthermore, MOG-IgG-ON chiasmal involvement is more likely to be part of a longitudinally extensive optic nerve lesion.


Effect of different diets on proteolytic enzyme activity, trypsinogen gene expression and dietary carbon assimilation in Senegalese sole (Solea senegalensis) larvae.
The effect of diet on larval growth, anionic trypsinogen gene expression (ssetryp1), and trypsin (EC 22.214.171.124) and chymotrypsin (EC 184.108.40.206) activities was assessed in Solea senegalensis. Changes in larval carbon stable isotope (δ(13)C) composition were used to estimate carbon assimilation. Diets were supplied for 20days to fish held in larval rearing tanks and consisted of live rotifers, Artemia sp. nauplii, rotifers followed by Artemia sp., rotifers co-fed with inert diet and inert diet alone. Growth was significantly faster in larvae fed only Artemia and those fed rotifers and Artemia (k=0.381-0.387day(-1)). Trypsin and chymotrypsin activities increased from 3 to 4days after hatching (DAH) in all dietary treatments, while ssetryp1 transcripts increased at 4-5 DAH only in larvae fed live prey. ssetryp1 gene expression was activated later in larvae fed only Artemia and this corresponded with Artemia δ(13)C values being reflected in larval tissue. Larval δ(13)C values also indicated greater selection and/or assimilation of rotifers in relation to the inert diet. Results demonstrate that during early larval development of sole, diet modulates ssetryp1 gene expression. The rapid and intense response to diets that promoted different growth and survival suggests the suitability of this biomarker as a nutritional status indicator in early sole larvae.


Differential introgression and the maintenance of species boundaries in an advanced generation avian hybrid zone.
Evolutionary processes, including selection and differential fitness, shape the introgression of genetic material across a hybrid zone, resulting in the exchange of some genes but not others. Differential introgression of molecular or phenotypic markers can thus provide insight into factors contributing to reproductive isolation. We characterized patterns of genetic variation across a hybrid zone between two tidal marsh birds, Saltmarsh (Ammodramus caudacutus) and Nelson's (A. nelsoni) sparrows (n = 286), and compared patterns of introgression among multiple genetic markers and phenotypic traits. Geographic and genomic cline analyses revealed variable patterns of introgression among marker types. Most markers exhibited gradual clines and indicated that introgression exceeds the spatial extent of the previously documented hybrid zone. We found steeper clines, indicating strong selection for loci associated with traits related to tidal marsh adaptations, including for a marker linked to a gene region associated with metabolic functions, including an osmotic regulatory pathway, as well as for a marker related to melanin-based pigmentation, supporting an adaptive role of darker plumage (salt marsh melanism) in tidal marshes. Narrow clines at mitochondrial and sex-linked markers also offer support for Haldane's rule. We detected patterns of asymmetrical introgression toward A. caudacutus, which may be driven by differences in mating strategy or differences in population density between the two species. Our findings offer insight into the dynamics of a hybrid zone traversing a unique environmental gradient and provide evidence for a role of ecological divergence in the maintenance of pure species boundaries despite ongoing gene flow.


An integrated approach to meta-analysis in alcohol studies.
The research design and methods utilized by the Collaborative Alcohol-Related Longitudinal Project are described. The project design addresses the critical need to develop procedures to assess the replicability of research results in alcohol studies. Key features of the research plan include: re-analysis of original data from multiple longitudinal studies of drinking behavior in the general population; centralization of all data analyses, developed and implemented by an interdisciplinary core staff; development of the research plan and interpretation of results in co-operation with original investigators of studies included in the project; and use of modeling procedures from meta-analysis to quantify the relative contribution of factors influencing the distribution of effect estimates across studies, including both methodological differences and aggregate level variables. The final section describes statistical methods for meta-analysis used by the project, including procedures for the calculation and combination of estimates of effect magnitude, categorical and continuous modeling procedures for use with effect sizes, and random effects models.


The utility of routine chest radiography in the initial evaluation of adult patients with febrile neutropenia patients undergoing HSCT.
The routine use of chest radiographs (CXRs) in the initial evaluation of asymptomatic patients with febrile neutropenia undergoing hematopoietic stem cell transplant (HSCT) is controversial. The goal of this study was to document the incidence of pneumonia demonstrated on CXR during an initial febrile neutropenic episode in adult patients undergoing HSCT. Clinical records of 1083 adult patients undergoing autologous (n=766), allogeneic (n=269), or umbilical cord blood HSCT (n=48) between October 1, 2009, and December 31, 2012, were retrospectively reviewed. CXRs obtained at the initial febrile neutropenic episode were evaluated for radiologic features of pneumonia. The presence of clinical symptoms, length of stay (LOS), and readmission rates were assessed. A total of 817 (75%) febrile neutropenic episodes were noted. Of the patients with neutropenic fevers, 455 (55%) had CXRs. Of the 76 patients with respiratory symptoms at the time of CXR, 24 (31.6%) had findings suggestive of pneumonia. None of the 379 CXRs performed in the absence of symptoms revealed an infectious process (P=.0001). Moreover, the mean LOS was 23.8 days for patients receiving a CXR compared with 22.2 days (P=.04) in patients without a CXR. Additionally, in patients who had CXRs, 15.7% were readmitted within 30 days compared with 7.4% in those without CXRs (P=.0004). Indiscriminate routine CXR at the time of first neutropenic fever in asymptomatic adults undergoing HSCT is unlikely to reveal an infectious process or change clinical practice, and may be associated with increased LOS and readmission rates.


Influence of track structure and condensed history physics models of Geant4 to nanoscale electron transport in liquid water.
The Geant4 toolkit offers a range of electromagnetic (EM) models for simulating the transport of charged particles down to sub-keV energies. They can be divided to condensed-history (CH) models (like the Livermore and Penelope models) and the track-structure (TS) models included in the Geant4-DNA low-energy extension of Geant4. Although TS models are considered the state-of-the-art for nanoscale electron transport, they are difficult to develop, computationally intensive, and commonly tailored to a single medium (e.g., water) which prohibits their use in a wide range of applications. Thus, the use of CH models down to sub-keV energies is particularly intriguing in the context of general-purpose Monte Carlo codes. The aim of the present work is to compare the performance of the CH models of Geant4 against the recently implemented TS models of Geant4-DNA for nanoscale electron transport. Calculations are presented for two fundamental quantities, the dose-point-kernel and the microdosimetric lineal energy. The influence of user-defined simulation parameters (tracking and production cuts, and maximum step size) on the above calculations is also examined. It is shown that Livermore offers the best performance among the CH models of Geant4 for nanoscale electron transport. However, even under optimally-chosen simulation parameters, the differences between the CH and TS models examined may be sizeable for low energy electrons (<1 keV) and/or nanometer size targets (<100 nm).


Molecular cloning of murine endogenous viral sequences and expression of a newly constructed recombinant murine leukemia virus DNA in transfected mink cells.
In the process of molecularly cloning unintegrated proviral DNA from NIH-3T3 mouse cells infected with Rauscher murine leukemia virus, we observed the occurrence of clones with inserts smaller than the expected Rauscher murine leukemia virus fragments. The insert of one of these clones, lambda.Xe-1, was characterized in more detail. It had a size of 3.5 kilobases. The restriction map was similar but not identical to that of the envelope regions of Moloney and Rauscher murine leukemia viruses. After ligation to previously cloned Moloney murine leukemia viral sequences and transfection of the ligated DNA into mink lung cells a nondefective xenotropic murine leukemia virus, XH-19, was isolated. Restriction mapping of proviral DNA isolated from mink lungs cells chronically infected with XH-19 showed the presence of Moloney murine leukemia virus-derived sequences coupled to xenotropic viral sequences.