Calcium regulation in tissue-cultured human and bovine lens epithelial cells.
To study calcium regulatory mechanisms in lens cells with particular reference to the relative contributions from the calcium adenosine triphosphatase of plasma and endoplasmic reticulum membranes, respectively. The calcium-sensitive fluorescent dye, Fura 2, was incorporated into tissue-cultured human and bovine epithelial cells and internal calcium was calibrated using the ionomycin (1 microM) method. The dynamics of calcium release from the endoplasmic reticulum were also studied in digitonin-permeabilized bovine cells. Tissue-cultured bovine and human lens cells have very similar resting calcium levels (235 +/- 22 nM and 216 +/- 12 nM, respectively). Thapsigargin caused an increase in cytoplasmic calcium both in the presence and absence of external calcium, but the calmodulin antagonist W7 only initiated an increase in the presence of external Ca2+. The effects of thapsigargin and W7 were additive. Exposing lens cells to Na(+)-free perfusing solutions caused a transient increase in internal Ca2+. Bovine lens cells permeabilized by digitonin-released Ca2+ when exposed to inositol (1,4,5) triphosphate and the effect was maximal at 1 microM. Lens cytoplasmic calcium is controlled by calcium adenosine triphosphatases at the plasma and endoplasmic reticulum membranes. The former is inhibited by W7 and insensitive to thapsigargin whereas the latter is inhibited by thapsigargin, but insensitive to W7. The lens endoplasmic reticulum store is also controlled by an inositol (1,4,5) trisphosphate calcium-release mechanism. Na+/Ca2+ exchange plays a relatively minor role in calcium regulation, at least at resting calcium levels.


Antigen-free adjuvant assists late effector CD4 T cells to transit to memory in lymphopenic hosts.
The events controlling the transition of T cells from effector to memory remain largely undefined. Many models have been put forth to account for the origin of memory precursors, but for CD4 T cells initial studies reported that memory T cells derive from IFN-γ-nonproducing effectors, whereas others suggested that memory emanates from highly activated IFN-γ-producing effectors. In this study, using cell proliferation, expression of activation markers, and production of IFN-γ as a measure of activation, we defined two types of effector CD4 T cells and investigated memory generation. The moderately activated early effectors readily transit to memory, whereas the highly activated late effectors, regardless of their IFN-γ production, develop minimal memory. Boosting with Ag-free adjuvant, however, rescues late effectors from cell death and sustains both survival and IFN-γ cytokine responses in lymphopenic hosts. The adjuvant-mediated memory transition of late effectors involves the function of TLRs, most notably TLR9. These findings uncover the mechanism by which late effector CD4 T cells are driven to transit to memory and suggest that timely boosts with adjuvant may enhance vaccine efficacy.


Prediction Accuracy in Multivariate Repeated-Measures Bayesian Forecasting Models with Examples Drawn from Research on Sleep and Circadian Rhythms.
In study designs with repeated measures for multiple subjects, population models capturing within- and between-subjects variances enable efficient individualized prediction of outcome measures (response variables) by incorporating individuals response data through Bayesian forecasting. When measurement constraints preclude reasonable levels of prediction accuracy, additional (secondary) response variables measured alongside the primary response may help to increase prediction accuracy. We investigate this for the case of substantial between-subjects correlation between primary and secondary response variables, assuming negligible within-subjects correlation. We show how to determine the accuracy of primary response predictions as a function of secondary response observations. Given measurement costs for primary and secondary variables, we determine the number of observations that produces, with minimal cost, a fixed average prediction accuracy for a model of subject means. We illustrate this with estimation of subject-specific sleep parameters using polysomnography and wrist actigraphy. We also consider prediction accuracy in an example time-dependent, linear model and derive equations for the optimal timing of measurements to achieve, on average, the best prediction accuracy. Finally, we examine an example involving a circadian rhythm model and show numerically that secondary variables can improve individualized predictions in this time-dependent nonlinear model as well.


The therapeutic potential of tuftsin-phosphorylcholine in giant cell arteritis.
Tuftsin-PhosphorylCholine (TPC) is a novel bi-specific molecule which links tuftsin and phosphorylcholine. TPC has shown immunomodulatory activities in experimental mouse models of autoimmune diseases. We studied herein the effects of TPC ex vivo on both peripheral blood mononuclear cells (PBMCs) and temporal artery biopsies (TABs) obtained from patients with giant cell arteritis (GCA) and age-matched disease controls. GCA is an immune-mediated disease affecting large vessels. Levels of 18 cytokines in supernatants, PBMC viability, T helper (Th) cell differentiation of PBMCs and gene expression in TABs were analyzed. Treatment ex vivo with TPC decreased the production of IL-1β, IL-2, IL-5, IL-6, IL-9, IL-12(p70), IL-13, IL-17A, IL-18, IL-21, IL-22, IL-23, IFNγ, TNFα, GM-CSF by CD3/CD28 activated PBMCs whereas it negligibly affected cell viability. It reduced Th1 and Th17 differentiation while did not impact Th22 differentiation in PBMCs stimulated by phorbol 12-myristate 13-acetate plus ionomycin. In inflamed TABs, treatment with TPC down-regulated the production of IL-1β, IL-6, IL-13, IL-17A and CD68 gene expression. The effects of TPC were comparable to the effects of dexamethasone, included as the standard of care, with the exception of a greater reduction of IL-2, IL-18, IFNγ in CD3/CD28 activated PBMCs and CD68 gene in inflamed TABs. In conclusion our results warrant further investigations regarding TPC as an immunotherapeutic agent in GCA and potentially other autoimmune and inflammatory diseases.


Opioid peptides and behavioral and physiological responses of dairy cows to social isolation in unfamiliar surroundings.
To test whether endogenous opioid peptides are involved in the behavioral and physiological responses of cattle to stress, 12 Holstein cows were either placed in social isolation in unfamiliar surroundings for 15 min or remained in their home stalls, either with or without naloxone treatment, following a Latin square design. Vocalizations (judged as high or low frequency), defecation/urination, and heart rate were recorded, latency to respond to local thermal stimulation of the leg by means of a laser was measured to detect pain sensitivity, and blood was sampled and assayed for cortisol concentrations. Naloxone in the home stall increased cortisol concentrations and tended to reduce response latencies to the laser but did not induce vocalization. Social isolation increased the incidence of high-frequency vocalization and of defecation/urination, heart rate, cortisol concentrations, and response latencies to the laser. Prior administration of naloxone increased the incidence of low-frequency vocalization in isolation, but it had no effect on heart rate or on responses to the laser and only limited effect on cortisol concentrations when the cows were isolated. Brief periods of social isolation in unfamiliar surroundings seem to be stressful to cows, as indicated by increased heart rate, hypothalamic-pituitary-adrenocortical axis activity, and vocalization. Isolation also reduces pain sensitivity, suggesting a stress-induced analgesia. However, we found no evidence that naloxone-sensitive opioid receptors were involved in these responses.


Morphological and immunophenotypic variations in malignant melanoma.
A variety of cytomorphological features, architectural patterns and stromal changes may be observed in malignant melanomas. Hence, melanomas may mimic carcinomas, sarcomas, benign stromal tumours, lymphomas, plasmacytomas and germ cell tumours. Melanomas may be composed of large pleomorphic cells, small cells, spindle cells and may contain clear, signet-ring, pseudolipoblastic, rhabdoid, plasmacytoid or balloon cells. Various inclusions and phagocytosed material may be present in their cytoplasm. Nuclei may show bi- or multi-nucleation, lobation, inclusions, grooving and angulation. Architectural variations include fasciculation, whorling, nesting, trabeculation, pseudoglandular/pseudopapillary/pseudofollicular, pseudorosetting and angiocentric patterns. Myxoid or desmoplastic changes and very rarely pseudoangiosarcomatous change, granulomatous inflammation or osteoclastic giant cell response may be seen in the stroma. The stromal blood vessels may exhibit a haemangiopericytomatous pattern, proliferation of glomeruloid blood vessels and perivascular hyalinization. Occasionally, differentiation to nonmelanocytic structures (Schwannian, fibro-/myofibroblastic, osteocartilaginous, smooth muscle, rhabdomyoblastic, ganglionic and ganglioneuroblastic) may be observed. Typically melanomas are S100 protein, NKIC3, HMB-45, Melan-A and tyrosinase positive but some melanomas may exhibit an aberrant immunophenotype and may express cytokeratins, desmin, smooth muscle actin, KP1 (CD68), CEA, EMA and VS38. Very rarely, neurofilament protein and GFAP positivity may be seen.


Previously undiagnosed variant angina as a cause of chest pain after coronary artery bypass surgery.
Variant angina was diagnosed after coronary artery bypass surgery in six patients over a 22-month period. Although all six patients had at least occasional angina at rest preoperatively, all but one had predominantly exertional angina. After surgery, rest angina with transient ST-segment elevation appeared in all six after an asymptomatic interval of 1 week to 4 years. In two patients the involved artery had not been bypassed, in two patients it was perfused by a patent graft and in two patients the graft to the involved vessel was occluded. Treatment with calcium antagonist drugs (four cases) or isosorbide dinitrate (one case) eliminated symptoms; one patient spontaneously became asymptomatic. The diagnosis of variant angina should be considered when rest angina occurs after bypass surgery, particularly if exertional angina is absent and grafts are patent.


Unifying Exchange Sensitivity in Transition-Metal Spin-State Ordering and Catalysis through Bond Valence Metrics.
Accurate predictions of spin-state ordering, reaction energetics, and barrier heights are critical for the computational discovery of open-shell transition-metal (TM) catalysts. Semilocal approximations in density functional theory, such as the generalized gradient approximation (GGA), suffer from delocalization error that causes them to overstabilize strongly bonded states. Descriptions of energetics and bonding are often improved by introducing a fraction of exact exchange (e.g., erroneous low-spin GGA ground states are instead correctly predicted as high-spin with a hybrid functional). The degree of spin-splitting sensitivity to exchange can be understood based on the chemical composition of the complex, but the effect of exchange on reaction energetics within a single spin state is less well-established. Across a number of model iron complexes, we observe strong exchange sensitivities of reaction barriers and energies that are of the same magnitude as those for spin splitting energies. We rationalize trends in both reaction and spin energetics by introducing a measure of delocalization, the bond valence of the metal-ligand bonds in each complex. The bond valence thus represents a simple-to-compute property that unifies understanding of exchange sensitivity for catalytic properties and spin-state ordering in TM complexes. Close agreement of the resulting per-metal-organic-bond sensitivity estimates, together with failure of alternative descriptors demonstrates the utility of the bond valence as a robust descriptor of how differences in metal-ligand delocalization produce differing relative energetics with exchange tuning. Our unified description explains the overall effect of exact exchange tuning on the paradigmatic two-state FeO+/CH4 reaction that combines challenges of spin-state and reactivity predictions. This new descriptor-sensitivity relationship provides a path to quantifying how predictions in transition-metal complex screening are sensitive to the method used.


N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: influence of isomeric substitution on radiolabeling and target cell residualization.
N-succinimidyl 4-guanidinomethyl-3-[(*)I]iodobenzoate ([(*)I]SGMIB) has shown promise for the radioiodination of monoclonal antibodies (mAbs) and other proteins that undergo extensive internalization after receptor binding, enhancing tumor targeting compared to direct electrophilic radioiodination. However, radiochemical yields for [(131)I]SGMIB synthesis are low, which we hypothesize is due to steric hindrance from the Boc-protected guanidinomethyl group ortho to the tin moiety. To overcome this, we developed the isomeric compound, N-succinimidyl 3-guanidinomethyl-5-[(131)I]iodobenzoate (iso-[(131)I]SGMIB) wherein this bulky group was moved from ortho to meta position. Boc2-iso-SGMIB standard and its tin precursor, N-succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-(trimethylstannyl)benzoate (Boc2-iso-SGMTB), were synthesized using two disparate routes, and iso-[*I]SGMIB synthesized from the tin precursor. Two HER2-targeted vectors - trastuzumab (Tras) and a nanobody 5F7 (Nb) - were labeled using iso-[(*)I]SGMIB and [(*)I]SGMIB. Paired-label internalization assays in vitro with both proteins, and biodistribution in vivo with trastuzumab, labeled using the two isomeric prosthetic agents were performed. When the reactions were performed under identical conditions, radioiodination yields for the synthesis of Boc2-iso-[(131)I]SGMIB were significantly higher than those for Boc2-[(131)I]SGMIB (70.7±2.0% vs 56.5±5.5%). With both Nb and trastuzumab, conjugation efficiency also was higher with iso-[(131)I]SGMIB than with [(131)I]SGMIB (Nb, 33.1±7.1% vs 28.9±13.0%; Tras, 45.1±4.5% vs 34.8±10.3%); however, the differences were not statistically significant. Internalization assays performed on BT474 cells with 5F7 Nb indicated similar residualizing capacity over 6h; however, at 24h, radioactivity retained intracellularly for iso-[(131)I]SGMIB-Nb was lower than for [(125)I]SGMIB-Nb (46.4±1.3% vs 56.5±2.5%); similar results were obtained using Tras. Likewise, a paired-label biodistribution of Tras labeled using iso-[(125)I]SGMIB and [(131)I]SGMIB indicated an up to 22% tumor uptake advantage at later time points for [(131)I]SGMIB-Tras. Given the higher labeling efficiency obtained with iso-SGMIB, this residualizing agent might be of value for use with shorter half-life radiohalogens.


The healing Buddha.
The iconography of the healing Buddha embraces two healing traditions, symbolized by the healing stone lapis lazuli from Central Asia and by the myrobalan fruit from the ayurvedic medicine of ancient India. The first mention of the healing Buddha is in Buddhist texts of the first century BC, and the earliest extant icons date from the fourth century AD. This suggests the cult of the healing Buddha was a relatively late development in the history of Buddhism. Worshippers sought his help in alleviating spiritual, mental and physical suffering, as well as for medical cures. In China followers believed he was also a cosmic Buddha, to whom one appealed for longevity and protection from disasters. This form of faith-based healing remains vibrant in China, Japan and Tibet to this day.


The CAT-1 is out of the bag: endothelial Cationic Amino Acid Transporter-1 is a critical player in cardiorenal syndrome type 2.
Although the numbers of patients affected by cardiorenal syndrome keeps increasing, we lack a complete understanding of the molecular pathways involved in its development and progression. Nitric oxide synthase (NOS) may play a role in cardiorenal syndrome, particularly cardiorenal syndrome type 2 (CRS2). However, complexities and paradoxical clinical findings have limited translation. In the current Clinical Science, Giam et al. (Clinical Science (2020) 134, 2755-2769) highlight the role of a key NOS substrate transporter, the cationic amino acid transporter-1, in preserving renal function in CRS2. In this commentary, we introduce the cardiorenal syndrome and the putative role that nitric oxide (NO) may play in the development of this disease and discuss the exciting findings of Giam et al. (Clinical Science (2020) 134, 2755-2769) and their tantalizing translational implications.


AlphaFold Protein Structure Database in 2024: providing structure coverage for over 214 million protein sequences.
The AlphaFold Database Protein Structure Database (AlphaFold DB, https://alphafold.ebi.ac.uk) has significantly impacted structural biology by amassing over 214 million predicted protein structures, expanding from the initial 300k structures released in 2021. Enabled by the groundbreaking AlphaFold2 artificial intelligence (AI) system, the predictions archived in AlphaFold DB have been integrated into primary data resources such as PDB, UniProt, Ensembl, InterPro and MobiDB. Our manuscript details subsequent enhancements in data archiving, covering successive releases encompassing model organisms, global health proteomes, Swiss-Prot integration, and a host of curated protein datasets. We detail the data access mechanisms of AlphaFold DB, from direct file access via FTP to advanced queries using Google Cloud Public Datasets and the programmatic access endpoints of the database. We also discuss the improvements and services added since its initial release, including enhancements to the Predicted Aligned Error viewer, customisation options for the 3D viewer, and improvements in the search engine of AlphaFold DB.


[Immunomorphology in the diagnosis of orbital rhabdomyosarcoma].
Immunohistochemistry (IHC) using monoclonal antibodies is presently the most reliable method to verify tumor tissue belonging. The histological diagnosis of rhabdomyosarcoma (RMS) was immunohistochemically confirmed in 7 of 10 orbital neoplasms in children. In 3 cases the diagnosis of RMS was established after IHC study that revealed the atypical tumor cell expression of the muscular antigens--myoglobin, protein Myo D1, actin HHF-35, which is indicative of the rhabdomyogenic genesis of a tumor. IHC data indicated that, no matter what the histological pattern, orbital RMS is characterized by the expression of cytospecific proteins--myoglobin, Myo D1, actin HHF-35, and tissue-specific (intermediate filaments)--vimentin and desmin. The use of appropriate antibodies offers a means of making a histogenetic diagnosis even in cases of the atypical morphological pattern.


In vivo evaluation of antidiarrhoeal activity of Rhus semialata fruit extract in rats.
Rhus semialata Murr. (Anacardiaceae) is a deciduous tree of north eastern India. The fruit of this plant is traditionally used to control diarrhoea and dysentery. The Present study was undertaken to evaluate anti-diarrhoeal potency of methanol extract of fruits of R. semialata using Wister albino rats to substantiate folklore claims. The extract at graded doses (100, 200, 400 and 600 mg/kg body weight) was investigated for anti-diarrhoeal activity in term of reduction in the rate of defecation in castor oil induced diarrhoea. To understand the mechanism of its antidiarrhoeal activity, the gastrointestinal transit and PGE(2)-induced intestinal fluid accumulation (enteropooling) were further evaluated. At graded doses, the extract showed a remarkable anti-diarrhoeal activity evidenced by the reduction in the rate of defecation up to 80.70% of control diarrhoeal animals at the dose of 600 mg/kg body weight. Results are comparable to that of standard drug diphenoxylate (50 mg/kg body weight). Extract produced profound decrease in intestinal transit (8.02-47.05%) at selected doses comparable to that of single intraperitoneal injection of standard drug atropine sulphate at doses of 0.1 mg/kg body weight. It significantly inhibited PGE(2)-induced enteropooling (21.98-56.03%). The results indicated that the methanol extract of the fruits of R. semialata possesses significant anti-diarrhoeal effect and substantiated the use of this herbal remedy as a non-specific treatment for diarrhoea in folk medicine.


Universal Health Coverage and the Pacific Islands: An Overview of Senior Leaders' Discussions, Challenges, Priorities and Solutions, 2015-2020.
In 1995, Pacific Health Ministers articulated their vision of a healthy Pacific as 'a place where children are nurtured in body and mind; environments invite learning and leisure; people work and age with dignity; where ecological balance is a source of pride; and where the ocean is protected.' Central to this vision is the achievement of universal health coverage (UHC). To provide an indication of the UHC-related priorities of Pacific health authorities and promote alignment of domestic and international investments in health sector development, we thematically analyzed the discussion, resolutions, and recommendations from 5 years (2015-2020) of senior-level Pacific health meetings. Five main themes emerged: (i) the Healthy Islands vision has (and continues to have) a unifying influence on action for UHC; (ii) adoption of appropriate service delivery models that support integrated primary health care at the community level are needed; (iii) human resources for health are critical if efforts to achieve UHC are to be successful; (iv) access to reliable health information is core to health sector improvement; and (v) while not a panacea for all challenges, digital health offers many opportunities. Small and isolated populations, chronic workforce limitations, weak governance arrangements, ageing and inadequate health facilities, and supply chain and logistics difficulties (among other issues) interact to challenge primary health care delivery across the Pacific Islands. We found evidence that the Healthy Islands vision is a tool that garners support for UHC; however, to realize the vision, a realistic understanding of needed political, human resource, and economic investments is required. The significant disruptive effect of COVID-19 and the uncertainty it brings for implementation of the medium- to long-term health development agenda raises concern that progress may stagnate or retreat.


The utilization of gene targeting models during in preclinical study of drug discovery process--example of phenotypic and functional analysis of Cacna1beta gene product.
Using gene knockout mice of particular genes is one of the most effective methods in conducting successful study on the mode of action of target gene products in targeted organs. So called the knockout technology is now a powerful tool that can lead us to find clear understanding on difficult questions such as the effects of full antagonist against target molecules. Cacna1b (alpha(1B)) gene knockout mouse was generated to study mechanisms of N-type calcium (Ca(2+)) channel. The model was able to overcome physiological obstacles in studies of N-type Ca(2+) channel selective blockers, such as unspecific binding to structurally similar molecules, and failed distribution to targeted organs. In the case of N-type Ca(2+) channel studies, knockout technology was successfully applied to various cardiovascular, sympathetic, nociceptive, sleep-awake cycles, metabolic and neurodegenerative experiments using homozygous mutants of the alpha(1B) gene that turned out to be viable. These studies were able to confirm not only the predicted phenotypes, but were able to present completely unexpected phenotypes that are great interest for future study. Thus the outputs from the knockout mouse studies lead to gain the proof of concept as a drug for specific inhibitors of the gene products and enabled us to make further prediction of side-effects of these inhibitors in the drug discovery and development process.


Overall lack of genotoxic activity among five common low- and no-calorie sweeteners: A contemporary review of the collective evidence.
Low- and no-calorie sweeteners (LNCS) are food additives that have been widely consumed for many decades. Their safety has been well established by authoritative bodies globally and is re-evaluated periodically. The objective herein was to survey and summarize the genotoxicity potential of five commonly utilized LNCS: acesulfame potassium (Ace-K), aspartame, saccharin, steviol glycosides and sucralose. Data from peer-reviewed literature and the ToxCast/Tox21 database were evaluated and integrated with the most recent weight-of-evidence evaluations from authoritative sources. Emphasis was placed on assays most frequently considered for hazard identification and risk assessment: mutation, clastogenicity and/or aneugenicity, and indirect DNA damage, such as changes in DNA repair mechanisms or gene expression data. These five sweeteners have been collectively evaluated in hundreds of in vivo or in vitro studies that employ numerous testing models, many of which have been conducted according to specific testing guidelines. The weight-of-evidence demonstrates overall negative findings across assay types for each sweetener when considering the totality of study design, reliability and reporting quality, as well as the lack of carcinogenic responses (or lack of responses relevant to humans) in animal cancer bioassays as well as observational studies in humans. This conclusion is consistent with the opinions of authoritative sources that have consistently determined that these sweeteners lack mutagenic and genotoxic potential.


[Induction of apoptosis of splenic lymphocytes in mice by accelerated carbon ions].
To assess the capacity of heavy ions to induce apoptosis in lymphocytes, mice have been irradiated with accelerated carbon ions (95 MeV/nucleon) at doses ranging from 0.1 to 4 Gy. Their spleens were removed 24 h later and gently dissociated to prepare a single cell suspension. Mononuclear cells were then maintained in culture at 37 degrees C, and the occurrence of apoptosis in these cells was analysed 24 h later. Lymphocytes were also irradiated in vitro, in the presence of Ac-DEVD-CHO, a potent caspase-3 and -7 inhibitor. Results from three experiments performed at the Grand Accelerateur National d'Ions Lourds (GANIL, Caen, France) are reported here. They indicate that carbon ions induce a marked, dose-dependent, reduction of the spleen weight and cellularity. However, in sharp contrast with spleen cells prepared from X-ray irradiated mice, only a slight increase of apoptosis is evidenced in cultured lymphocytes from mice irradiated with heavy ions. The significance of such results is discussed. So far, few data exist concerning the biological effects of heavy ions, in particular their capacity to induce apoptosis in lymphocytes; the present study provides useful clues for further investigations.


[Violent relationship in young people and STD/AIDS risk].
To verify whether affective relationships involving violence are associated with increased risk of sexually transmitted diseases (STDs), including AIDS, we conducted a survey among youth 14 to 22 years of age residing in two low-income communities in Rio de Janeiro, Brazil. We used a qualitative methodology with focal groups and individual interviews. Violence is part of the routine among these youth in both their community and families. The following factors were associated with violence in interpersonal relations: lack of money, unemployment, drug and alcohol use, jealousy, and infidelity. The young people reported that condom use is not negotiated with violent partners, resulting in increased risk of STD/AIDS. The results indicate that violence is multi-factorial, and when present in interpersonal relationships it intervenes negatively in relation to protection against STD/AIDS.


The epidemiology of schoolboy rugby injuries.
During one 18-week season, in which players from 26 high schools played 3,350 rugby matches, 495 injuries prevented players from participating in rugby for at least 1 week; 71% occurred during matches and 29% during practices. Injury was more common during the first 4 weeks of the season and again in the same time period after the mid-season vacation. At all ages, A-team players suffered the greatest number of injuries. The safest playing positions were tight-forward and scrum-half; the most dangerous loose-forward and in the back-line excluding the scrum-half. Overall, eightmen were the most often injured players. Of all injuries 55% occurred while the player was tackling or being tackled and 18% during the loose scrum/maul. The lower limb (37%), the head and neck (29%) and the upper limb (20%) were most commonly injured, and fractures (27%), ligament/tendon injuries (25%) and muscle injuries (17%) were commonest. However, concussion injuries were underreported in 19 of the 26 schools. This study shows: that monitoring rugby injuries through correspondence results in underreporting of injuries; that rugby injuries show specific trends with age, team level, playing position, time of the season and phase of play; and that players in the different positions suffer specific injuries in predictable phases of the game. Speed of play may be the most important aetiological factor in the majority of rugby injuries.


Management of HPV-positive women in cervical screening using results from two consecutive screening rounds.
We studied whether triage of human papillomavirus (HPV)-positive women participating in an HPV-based screening programme can be improved by including the HPV result at the previous screen in the triage algorithm. We analyzed data of a subgroup of 366 women from the POBASCAM trial, screened by cytology and HPV cotesting. Women were included if they tested HPV-positive in the second HPV-based screening round. We evaluated the clinical performance of 16 strategies, consisting of cytology, HPV genotyping, and/or previous screen HPV result. The clinical endpoint was cervical precancer or cancer (CIN3+). The current Dutch triage testing policy for HPV-positive women is to refer women for colposcopy if they have abnormal cytology at baseline or after 6-18 months. In the second HPV-based screening round, this strategy yielded a negative predictive value (NPV) of 95.8% (95% confidence interval: 91.9-98.2) and colposcopy referral rate of 37.6% (32.3-43.2%). Replacing repeat cytology by the previous screen HPV result yielded a similar NPV (96.9%, 93.3-98.9) and colposcopy referral rate (38.8%, 33.4-44.4). A higher NPV (99.2%, 96.3-100%) at the cost of a higher colposcopy referral rate (49.2%, 43.6-54.8) was achieved when cytology was combined with HPV16/18 genotyping. The other 13 triage strategies yielded a lower NPV, a higher colposcopy referral rate or performed similarly but required additional testing. HPV-positive women in the second HPV-based screening round can be suitably managed by cytology, HPV16/18 genotyping and the HPV result at the previous screen, obviating the need for repeat testing of HPV-positive, cytology negative women.


Structure and receptor-binding properties of an airborne transmissible avian influenza A virus hemagglutinin H5 (VN1203mut).
Avian influenza A virus continues to pose a global threat with occasional H5N1 human infections, which is emphasized by a recent severe human infection caused by avian-origin H7N9 in China. Luckily these viruses do not transmit efficiently in human populations. With a few amino acid substitutions of the hemagglutinin H5 protein in the laboratory, two H5 mutants have been shown to obtain an air-borne transmission in a mammalian ferret model. Here in this study one of the mutant H5 proteins developed by Kawaoka's group (VN1203mut) was expressed in a baculovirus system and its receptor-binding properties were assessed. We herein show that the VN1203mut had a dramatically reduced binding affinity for the avian α2,3-linkage receptor compared to wild type but showed no detectable increase in affinity for the human α2,6-linkage receptor, using Surface Plasmon Resonance techonology. Further, the crystal structures of the VN1203mut and its complexes with either human or avian receptors demonstrate that the VN1203mut binds the human receptor in the same binding manner (cis conformation) as seen for the HAs of previously reported 1957 and 1968 pandemic influenza viruses. Our receptor binding and crystallographic data shown here further confirm that the ability to bind the avian receptor has to decrease for a higher human receptor binding affinity. As the Q226L substitution is shown important for obtaining human receptor binding, we suspect that the newly emerged H7N9 binds human receptor as H7 has a Q226L substitution.


One evidence base; three stories: do opioids relieve chronic breathlessness?
The efficacy of low-dose systemic opioids for chronic breathlessness was questioned by the recent Cochrane review by Barnes et al We examined the reasons for this conflicting finding and re-evaluated the efficacy of systemic opioids. Compared with previous meta-analyses, Barnes et al reported a smaller effect and lower precision, but did not account for matched data of crossover trials (11/12 included trials) and added a risk-of-bias criterion (sample size). When re-analysed to account for crossover data, opioids decreased breathlessness (standardised mean differences -0.32; -0.18 to -0.47; I2=44.8%) representing a clinically meaningful reduction of 0.8 points (0-10 numerical rating scale), consistent across meta-analyses.


Graphene visualizes the first water adlayers on mica at ambient conditions.
The dynamic nature of the first water adlayers on solid surfaces at room temperature has made the direct detection of their microscopic structure challenging. We used graphene as an atomically flat coating for atomic force microscopy to determine the structure of the water adlayers on mica at room temperature as a function of relative humidity. Water adlayers grew epitaxially on the mica substrate in a layer-by-layer fashion. Submonolayers form atomically flat, faceted islands of height 0.37 +/- 0.02 nanometers, in agreement with the height of a monolayer of ice. The second adlayers, observed at higher relative humidity, also appear icelike, and thicker layers appear liquidlike. Our results also indicate nanometer-scale surface defects serve as nucleation centers for the formation of both the first and the second adlayers.


Spectroscopy and dynamics of double proton transfer in formic acid dimer.
We present the isolated gas phase infrared spectra of formic acid dimer, (HCOOH)2, and its deuterated counterpart formic-d acid, (DCOOH)2, at room temperature. The formic acid dimer spectrum was obtained by spectral subtraction of a spectrum of formic acid vapor recorded at low pressure from that recorded at a higher pressure. The spectra of formic acid vapor contain features from both formic acid monomer and formic acid dimer, but at low and high pressures of formic acid, the equilibrium is pushed towards the monomer and dimer, respectively. A similar approach was used for the formic-d acid dimer. Building on the previous development of the Molecular Mechanics with Proton Transfer (MMPT) force field for simulating proton transfer reactions, molecular dynamics (MD) simulations were carried out to interpret the experimental spectra in the OH-stretching region. Within the framework of MMPT, a combination of symmetric single and double minimum potential energy surfaces (PESs) provides a good description of the double proton transfer PES. In a next step, potential morphing together with electronic structure calculations at the B3LYP and MP2 level of theory was used to align the computed and experimentally observed spectral features in the OH-stretching region. From this analysis, a barrier for double proton transfer between 5 and 7 kcal mol(-1) was derived, which compares with a CCSD(T)/aug-cc-pVTZ calculated barrier of 7.9 kcal mol(-1). Such a combination of experimental and computational techniques for estimating barriers for proton transfer in gas phase systems is generic and holds promise for further improved PESs and energetics of these important systems. Additional MD simulations at the semi-empirical DFTB level of theory agree quite well for the center band position but underestimate the width of the OH-stretching band.


The use of cilostazol in patients with peripheral arterial disease: results of a national physician survey.
Although international guidelines recommend cilostazol as first-line therapy for peripheral arterial disease (PAD) because it improves the symptoms and quality of life, it remains an underused agent for amputation-free survival. The objective of this study was to evaluate the practice among Italian physicians of the use of cilostazol in patients suffering from peripheral arterial disease (PAD). For the present study, a cross-sectional survey was carried out. Physicians specialized in the medical and/or surgical treatment of PAD and who prescribe cilostazol regularly were invited to a phone interview. A nationally-representative probability sample of hospital-based physicians who diagnose and treat patients with PAD was randomly selected among the members of the Italian Physicians' Association. Out of a total of 641 physicians contacted, 250 of them (39%) accepted to take part in this survey. Reasons of survey refusal were the non-attitude to prescribe cilostazol (45%), or unspecified motivations (16%). Among the 250 physicians participating the survey: 120 were vascular surgeons, 80 internal medicine doctors, 50 angiologists. Cilostazol was suggested in 79% of patients with symptomatic intermittent claudication, and in 30% of patients who had undergone revascularization. The majority of physicians stated to prescribe cilostazol at recommended dosage of 100 mg bid (46.4%). The principal reason to suggest a reduced drug dosage was to limit early side effect at the time of treatment onset, but the increase to the full dose of cilostazol is suggested by 91.3% of interviewed within 4 weeks. Sixty-three percent of physicians affirmed to prescribe cilostazol as a continuous treatment for a mean of 4 months, while a lifelong treatment was suggested by 17.6% of participants. Among physicians who habitually prescribe cilostazol adherence to the recommended drug dosage and length of treatment is high. The prescription of cilostazol is particularly appreciated in patients with symptomatic intermittent claudication, even before any non-invasive diagnosis of PAD, and before any invasive therapy. Finally a relevant number of physicians regularly prescribe cilostazol also after revascularization, advocating the anti-restenotic properties of the drug.


Protective effects of preserving the posterior complex on the development of adjacent-segment degeneration after lumbar fusion: clinical article.
During the past decades, lumbar fusion has increasingly become a standard treatment for degenerative spinal disorders. However, it has also been associated with an increased incidence of adjacent-segment degeneration (ASD). Previous studies have reported less ASD in anterior fusion surgeries; thus, the authors hypothesized that the integrity of the posterior complex plays an important role in ASD. This study was designed to investigate the effect of the posterior complex on adjacent instability after lumbar instrumentation and the development of ASD. To evaluate different surgical interventions, 120 patients were randomly allocated into 3 groups of 40 patients each who were statistically similar with respect to demographic and clinical data. Patients in Group A were allocated for facet joint resection and L4-5 fusion, Group B for semilaminectomy and fusion, and Group C for complete laminectomy and fusion. All of the patients were followed up for 5-7 years (mean 5.9 years). The disc height, intervertebral disc angle, dynamic intervertebral angular range of motion (ROM), L3-4 slip, and the total lordosis angle were each measured before the operation and at the final follow-up. The Japanese Orthopaedic Association (JOA) score was determined before surgery and at the final follow-up to evaluate the clinical results. Among the 3 groups, no significant differences were detected in all clinical and demographic assessments before surgery. At 3 months after surgery, the JOA score of all groups improved significantly and showed no significant differences among the groups. At the final follow-up, Group C had a significantly (p < 0.05) lower JOA score than the other 2 groups. Moreover, the disc height and total lumbar lordosis in patients of Group C were significantly decreased compared with disc height and total lumbar lordosis in the other 2 groups. In contrast, disc angle, dynamic angular ROM, and listhesis were significantly higher in Group C than in the other 2 groups. Twenty-four patients showed signs of ASD after the operation (3 patients in Group A, 4 in B, and 17 in C). The number of patients in Group C showing ASD was significantly different from that in Groups A and B. During follow-up for 6 years, a significantly higher number of patients with ASD were noted in the complete-laminectomy group. The number of reoperations for treating ASD was much higher in this patient group than in the patients undergoing facet joint resection and L4-5 fusion or semilaminectomy and fusion. Therefore, preserving the posterior complex as much as possible during surgery plays an important role in preventing ASD and in reducing the reoperation rate.


Development and construct validation of the Client-Centredness of Goal Setting (C-COGS) scale.
Client-centred philosophy is integral to occupational therapy practice and client-centred goal planning is considered fundamental to rehabilitation. Evaluation of whether goal-planning practices are client-centred requires an understanding of the client's perspective about goal-planning processes and practices. The Client-Centredness of Goal Setting (C-COGS) was developed for use by practitioners who seek to be more client-centred and who require a scale to guide and evaluate individually orientated practice, especially with adults with cognitive impairment related to acquired brain injury. To describe development of the C-COGS scale and examine its construct validity. The C-COGS was administered to 42 participants with acquired brain injury after multidisciplinary goal planning. C-COGS scores were correlated with the Canadian Occupational Performance Measure (COPM) importance scores, and measures of therapeutic alliance, motivation, and global functioning to establish construct validity. The C-COGS scale has three subscales evaluating goal alignment, goal planning participation, and client-centredness of goals. The C-COGS subscale items demonstrated moderately significant correlations with scales measuring similar constructs. Findings provide preliminary evidence to support the construct validity of the C-COGS scale, which is intended to be used to evaluate and reflect on client-centred goal planning in clinical practice, and to highlight factors contributing to best practice rehabilitation.


Strong Country Level Correlation between Syphilis and HSV-2 Prevalence.
Background. Syphilis is curable but Herpes Simplex Virus-2 (HSV-2) is not. As a result, the prevalence of syphilis but not HSV-2 may be influenced by the efficacy of national STI screening and treatment capacity. If the prevalence of syphilis and HSV-2 is found to be correlated, then this makes it more likely that something other than differential STI treatment is responsible for variations in the prevalence of both HSV-2 and syphilis. Methods. Simple linear regression was used to evaluate the relationship between national antenatal syphilis prevalence and HSV-2 prevalence in women in two time periods: 1990-1999 and 2008. Adjustments were performed for the laboratory syphilis testing algorithm used and the prevalence of circumcision. Results. The prevalence of syphilis was positively correlated with that of HSV-2 for both time periods (adjusted correlations, 20-24-year-olds: 1990-99: R (2) = 0.54, P < 0.001; 2008: R (2) = 0.41, P < 0.001 and 40-44-year-olds: 1990-99: R (2) = 0.42, P < 0.001; 2008: R (2) = 0.49, P < 0.001). Conclusion. The prevalence of syphilis and HSV-2 is positively correlated. This could be due to a common set of risk factors underpinning both STIs.


[Rigid spine syndrome in children and adolescents].
The syndrome of the rigid spine is described in two patients aged 15 and 12 years. A total of 8 patients with this syndrome were studied. The disease was characterized by the myopathic syndrome, scoliosis, failure to bend the head and body, cardiopathy and occasional contractures of the proximal joints. The clinical, biochemical, roentgenological and morphological findings are presented. A multimodality treatment suggested should be carried out by a team comprising an orthopedist, a neuropathologist, and a pediatrician.


Viral haemorrhagic disease of rabbits: vaccination and immune response.
Results are presented for the large-scale use of a tissue vaccine, inactivated with beta-propiolactone and containing aluminium hydroxide adjuvant, against viral haemorrhagic disease of rabbits. The kinetics of haemagglutination-inhibiting antibodies over eighteen months, the response of vaccinated animals to challenge infection with field virus between six and fifteen months after vaccination, the serological response to revaccination, and the immunological mechanisms involved in primary vaccination and revaccination were investigated.


Axon collaterals of supraoptic neurones: anatomical and electrophysiological evidence for their existence in the lateral hypothalamus.
The magnocellular neurones of the supraoptic nucleus which synthesize and secrete vasopressin and oxytocin have been commonly regarded as simple "output" neurones in that they receive an input, generate an action potential and in turn release hormone from their terminals in the posterior pituitary. Three lines of evidence are presented which suggest that rat supraoptic nucleus neurons also have axon collaterals which terminate in the hypothalamus close to the nucleus. Small injections of horseradish peroxidase were made directly into the nucleus in hypothalamic slices, allowing visualization of the axons of supraoptic neurones. Collaterals of these axons could be observed in regions both dorsal and dorsolateral to the supraoptic nucleus. In a separate series of experiments, sections of perfusion-fixed hypothalamus were stained for vasopressin and oxytocin using specific antisera. Peptide-containing collaterals of both types were observed near the supraoptic nucleus, in a region similar to that seen after horseradish peroxidase injections. Finally, electrophysiological studies were carried out on hypothalamic slices containing the supraoptic nucleus. A small concentric bipolar stimulating electrode was placed directly into the nucleus and activity of lateral hypothalamic neurones within 0.1-1 mm of the nucleus was recorded. Of 68 neurones studied, 52 were excited by supraoptic stimulation via a synaptic pathway that could be blocked by Ca2+ -free solutions containing 18 mM Mg2+. These studies suggest that supraoptic neurones communicate via axon collaterals with other neurones in the lateral hypothalamus, in addition to their previously well characterised functional role in neurosecretion.


The Logic of Condom Use in Female Sex Workers in Bali, Indonesia.
Studies on human immunodeficiency virus (HIV) prevention practices of female sex workers often examine the use of condom as a single behaviour: using or not using condom. This study explores typologies of the logic of condom use as part of exercising meaningful identities from female sex workers' perspectives. We employed in-depth interviews with a purposely selected 35 female sex workers in Bali, Indonesia. Information from the in-depth interviews was analysed using thematic framework analysis to develop typologies of female sex workers' experiences on the logic of condom use and its relation to the construction of identity. We identified two main logics for not using condom: the prioritising of financial stability and romantic relationships over condom use. The main logic for using condom was to protect their health in order to improve their future economic security. Embedded within these logics, women chose to practise agency and negotiate meaningful identities consistent with their ideals of being responsible mothers, successful migrant workers and loyal partners. Our study concluded that female sex workers had clear logics for both the use and non-use of condom with their clients, highlighting the rational nature of female sex workers decision making.


Identification of putative CLE peptide receptors involved in determinate nodulation on soybean.
CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides tightly control the balance between stem cell proliferation and differentiation in several plant developmental processes. Transmission of the CLE peptide signal has been shown to be rather complex. Despite their recent identification, little is known about the receptors by which nodulation-specific CLE peptides, which were identified in soybean, are perceived. Genetic analysis has indicated that the leucine-rich repeat receptor-like kinase NARK of soybean (Glycine max) and its orthologs in other legumes are possible candidates. However, more receptors need to be identified because CLE peptides are often detected by heteromultimeric complexes. Here, we identified two additional putative CLE peptide receptor pairs in the soybean genome with a nodulation-related expression pattern, GmRLK1-GmRLK2 and GmRLK3-GmRLK4, and discuss their role in CLE peptide perception during nodulation.


Spatially global representations in human primary visual cortex during working memory maintenance.
Recent studies suggest that visual features are stored in working memory (WM) via sensory recruitment or sustained stimulus-specific patterns of activity in cortical regions that encode memoranda. One important question concerns the spatial extent of sensory recruitment. One possibility is that sensory recruitment is restricted to neurons that are retinotopically mapped to the positions occupied by the remembered items. Alternatively, specific feature values could be represented via a spatially global recruitment of neurons that encode the remembered feature, regardless of the retinotopic position of the remembered stimulus. Here, we evaluated these alternatives by requiring subjects to remember the orientation of a grating presented in the left or right visual field. Functional magnetic resonance imaging and multivoxel pattern analysis were then used to examine feature-specific activations in early visual regions during memory maintenance. Activation patterns that discriminated the remembered feature were found in regions of contralateral visual cortex that corresponded to the retinotopic position of the remembered item, as well as in ipsilateral regions that were not retinotopically mapped to the position of the stored stimulus. These results suggest that visual details are held in WM through a spatially global recruitment of early sensory cortex. This spatially global recruitment may enhance memory precision by facilitating robust population coding of the stored information.


Neoplastic pancreaticobiliary duct obstruction: evaluation with breath-hold MR cholangiopancreatography.
The purpose of this study was to investigate the use of breath-hold single-shot fast spin-echo MR cholangiopancreatography in neoplastic pancreaticobiliary duct obstruction. Breath-hold MR cholangiopancreatography was performed for preoperative examination of 32 consecutive patients with pathologically confirmed neoplastic obstruction of the biliary tract or pancreatic duct using a single-shot fast spin-echo sequence. Two observers, unaware of clinical or pathologic findings, independently reviewed the MR cholangiopancreatograms to assess level of obstruction and site of underlying tumor. Pathologic diagnoses, based on surgical or CT-guided biopsy specimens, were pancreatic tumor (n = 12), gallbladder cancer (n = 9), intrahepatic cancer (n = 9), and ampullary cancer (n = 2). On the basis of conventional cholangiography, CT, and surgical findings, 20 patients had isolated bile duct obstruction, 11 patients had combined pancreatic and bile duct obstruction, and one patient had isolated pancreatic duct obstruction. Isolated bile duct obstruction was classified as lobar (n = 5), hilar (n = 12), or distal (n = 3). These levels of obstruction were correctly identified in 27 (84%) and 28 (88%) of the 32 cases by the two observers. The site of the underlying tumor was identified in 27 (84%) and 29 (91%) cases by the two observers. Good interobserver agreement was reached for both level of obstruction (kappa = .70) and identification of tumor site (kappa = .75). Breath-hold single-shot fast spin-echo MR cholangiopancreatography is accurate in identifying the level of obstruction and the site of underlying tumor in neoplastic pancreaticobiliary duct obstruction, with good interobserver agreement.


Food patterns and socioeconomic indicators of food consumption amongst Inuvialuit in the Canadian Arctic.
Inuvialuit in the Canadian Arctic have been experiencing a nutrition transition resulting in a decrease in nutrient-dense food consumption, which may, in part, explain this population's increasing chronic disease rates. Because the available literature is limited, the present study aimed to document the extent of this transition by examining current dietary patterns and socioeconomic factors affecting food group consumption. This cross-sectional study was conducted in three Inuvialuit communities in the Northwest Territories between 2007 and 2008. A validated food frequency questionnaire determined intake frequency of fruit and vegetables (FV), traditional foods (TF) and non-nutrient-dense foods (NNDF). Socioeconomic status (SES) was assessed by questions on education, ownership of items in working condition used to create a Material Style of Life (MSL) scale and residents in household employed/on income support. Daily intake frequencies were compared by gender and age group using Wilcoxon rank sum test. SES association with food group intake was determined using logistic regression. The response rate was 65-85%. One hundred and seventy-five participants were female and 55 were male, aged 19-84 years [mean (SD) 44 (14)]. Mean frequencies of FV and TF consumption were 1.6 (1.5) and 1.6 (1.7) times per day, respectively. NNDF were reported 9.2 (3.0) times per day. The highest MSL score (>12) was significantly associated with higher fruit (≥0.7 times per day) and higher TF intake (≥1.1 times per day) compared with the lowest score (≤7). An intermediate MSL score (8-12) was related to higher vegetable consumption (≥0.4 times per day). NNDF were consumed approximately seven times more frequently than TF in the present study, indicating that the dietary transition is well underway amongst Inuvialuit. Participants with higher SES were more likely to consume nutrient-dense foods, suggesting possible cost barriers.


Spin in organics: a new route to spintronics.
New developments in the nascent field of organic spintronics are discussed. Two classes of phenomena can be discerned. In hybrid organic spin valves (OSVs), an organic semiconducting film is sandwiched between two ferromagnetic (FM) thin films, aiming at magnetoresistive effects as a function of the relative alignment of the respective magnetization directions. Alternatively, organic magnetoresistance (OMAR) is achieved without any FM components, and is an intrinsic property of the organic semiconductor material. Some of the exciting characteristics of OMAR, in both electrical conductance and photoconductance, are presented. A systematic, combined experimental-theoretical study of sign changes between positive and negative magnetoresistance is shown to provide important insight about the underlying mechanisms of OMAR. A simple explanation of experimental observations is obtained by combining a 'spin-blocking' mechanism, an essential ingredient in the recently proposed bipolaron model, with specific features of the device physics of space charge limited current devices in the bipolar regime. Finally, we discuss possible links between the physics relevant for OMAR and that for OSVs. More specifically, weak hyperfine fields from the hydrogen atoms in organic materials are thought to be crucial for a proper understanding of both types of phenomena.


KIP THORNE: The Shaman of Space and Time.
A generation of physicists probing the extremes of gravity can trace its scientific heritage to one man: Kip Thorne of the California Institute of Technology. A recent symposium to mark Thorne's 60th birthday brought together nearly 200 experts on gravity at its strongest and strangest: the domains of black holes, colliding neutron stars, and other exotic deep-space objects. Participants came to honor their mentor, who has led the way in converting Albert Einstein's General Theory of Relativity from a purely theoretical science into an astrophysical and observational one.


Macrophages feel their age in macular degeneration.
Macular degeneration, during which the posterior part of the eye known as the macula suffers from thinning, atrophy, and bleeding caused by abnormal angiogenesis (blood vessel formation), predominantly affects elderly adults and results in the loss of central vision. In this issue of the JCI, Kelly et al. investigate the regulation of innate immune cells, specifically macrophages, in ocular neovascularization following eye injury in mice (see the related article beginning on page 3421). They found that, as the mice aged, increased expression of IL-10 by senescent macrophages and changes in their expression of other cytokines altered the ability of these cells to restrain trauma-induced angiogenesis in the eye. These data provide insight into the effect of senescence on macrophage function and angiogenesis and have important implications for age-related diseases such as macular degeneration.


The effect of drying method temperature, collection method, and marker type on apparent ileal amino acid digestibility in 21-day-old broilers fed corn-soybean meal-barley based diet.
For accurate estimation of nutrient digestibility, an ideal drying and sampling method is required to preserve the quality of the digesta. A standard corn-soybean meal (corn-SBM) broiler starter diet was fed from d 0 to 10 before birds were placed on the experimental diets until d 21. One hundred and sixty-eight male Cobb 500 broiler chicks were used to evaluate the effect of two drying methods (freeze-dryer vs. forced air-oven) and two drying temperatures (40 vs. 55°C) (Exp 1), while ninety-six chicks were used to evaluate the effect of flushing and squeezing as well as marker types (titanium vs. chromium) on apparent ileal DM, N, Ca, P, and AA digestibility (Exp 2). There were seven (Exp 1) or eight (Exp 2) replicate cages per treatment with 6 birds/cage. Digesta from the distal two thirds of the ileum was obtained from birds following euthanasia on d 21 by squeezing (Exp 1) and squeezing or flushing (Exp 2). Samples collected were stored in the freezer at -20°C until they were either freeze-dried (FD) or oven-dried (OD) at 40 or 55°C. There were no interactions between the drying methods and drying temperatures (Exp 1) on apparent ileal DM, N, and AA digestibility. Met had the highest (92.3%) while Cys had the lowest (73.8%) digestibility value. In Exp 2, no interaction between sampling methods and marker types was observed. The effect of sampling methods was not significant except for Arg and Met where squeezing resulted in higher (P < 0.05) digestibility values. Furthermore, apparent ileal His, Ile, Cys, Ser, and Tyr digestibility tended to be higher (P < 0.1) in squeezed digesta compared to the flushed digesta. Results from these studies showed that OD ileal digesta at 40 or 55°C had no negative effect on apparent ileal AA digestibility. Likewise, marker type did not influence apparent ileal AA digestibility values.


The effect of relevance appraisal on the emotional response.
Many models of emotion assume that the emotional response is preceded by an assessment of a stimulus' relevance to the perceiver's goals. Although widely assumed, experimentally controlling and, hence, empirically testing the effect of a stimulus' relevance on the emotional response has proven challenging. In this study, we used stimuli with high ecological validity and manipulated their relevance while holding constant the perceptual features of the stimuli. In the experiment, participants were given the result of their Israeli Psychometric Entrance Test (PET). The PET score is highly relevant to most participants, as, at the time of the experiment, it is the only unknown about whether they shall be admitted to their major of choice at the university. Relevance of the information was experimentally controlled both binarily by manipulating whether the presented score is the participant's or belongs to another unfamiliar participant and parametrically by manipulating the probability that a presented score is their actual PET score. We found a substantial effect for manipulated relevance on self-report, electrodermal activity, and heart rate. The results provide evidence that information about a stimulus' relevance modulates the emotional response to it. (PsycINFO Database Record (c) 2019 APA, all rights reserved).


Transient receptor potential canonical 3 (TRPC3) is required for IgG immune complex-induced excitation of the rat dorsal root ganglion neurons.
Chronic pain may accompany immune-related disorders with an elevated level of serum IgG immune complex (IgG-IC), but the underlying mechanisms are obscure. We previously demonstrated that IgG-IC directly excited a subpopulation of dorsal root ganglion (DRG) neurons through the neuronal Fc-gamma receptor I (FcγRI). This might be a mechanism linking IgG-IC to pain and hyperalgesia. The purpose of this study was to investigate the signaling pathways and transduction channels activated downstream of IgG-IC and FcγRI. In whole-cell recordings, IgG-IC induced a nonselective cation current (I(IC)) in the rat DRG neurons, carried by Ca(2+) and Na(+). The I(IC) was potentiated or attenuated by, respectively, lowering or increasing the intracellular Ca(2+) buffering capacity, suggesting that this current was regulated by intracellular calcium. Single-cell RT-PCR revealed that transient receptor potential canonical 3 (TRPC3) mRNA was always coexpressed with FcγRI mRNA in the same DRG neuron. Moreover, ruthenium red (a general TRP channel blocker), BTP2 (a general TRPC channel inhibitor), and pyrazole-3 (a selective TRPC3 blocker) each potently inhibited the I(IC). Specific knockdown of TRPC3 using small interfering RNA attenuated the IgG-IC-induced Ca(2+) response and the I(IC). Additionally, the I(IC) was blocked by the tyrosine kinase Syk inhibitor OXSI-2, the phospholipase C (PLC) inhibitor neomycin, and either the inositol triphosphate (IP(3)) receptor antagonist 2-aminoethyldiphenylborinate or heparin. These results indicated that the activation of neuronal FcγRI triggers TRPC channels through the Syk-PLC-IP(3) pathway and that TRPC3 is a key molecular target for the excitatory effect of IgG-IC on DRG neurons.


Insulin-secreting islet cell tumors: establishing a diagnosis and the clinical course for 25 dogs.
Twenty-five dogs with insulin-secreting neoplasms of the pancreas were studied. The diagnosis in each case was determined by histologic evaluation of pancreatic tissue obtained at surgery. The breed distribution revealed that German Shepherd Dogs, Irish Setters, and Collies were most commonly represented. Physical examination, complete blood counts, serum biochemical analysis, and urinalysis were of little diagnostic value, aside from the finding of hypoglycemia in 21 of 25 dogs. Radiographs of the thorax and abdomen were noncontributory to the ultimate diagnosis. Prior to surgery, fasting immunoreactive insulin concentrations and blood glucose concentrations were studied. Insulin:glucose ratios, glucose:insulin ratios, and amended insulin:glucose ratios were determined from the insulin and glucose concentrations in a single blood sample in each of 28 trials. In addition, glucagon tolerance tests were performed on 12 dogs. The amended insulin:glucose ratios proved to be the most reliable for diagnosis. Pancreatic masses were evident at surgery in 23 of 25 dogs; the remaining 2 dogs had microscopic evidence of an islet cell tumor. Nineteen of the islet cell tumors were carcinomas and 6 were simply described as "islet cell tumors." The mean life expectancy after surgery was 12.3 months. Treatment for malignant islet cell tumours included frequent feeding glucocorticoids, and diazoxide.


TGFBI mutational analysis in a New Zealand population of inherited corneal dystrophy patients.
The corneal dystrophies represent a group of clinically and genetically heterogeneous, inherited diseases, often resulting in bilateral opacification of the cornea, and may require penetrating keratoplasty. Mutations in the transforming growth factor beta-induced (TGFBI) gene segregate with a wide range of phenotypically heterogeneous corneal dystrophies. Many of the other dystrophies remain without molecular characterisation. This study aimed to characterise the molecular basis for corneal disease in a New Zealand population. Nineteen unrelated individuals affected with a corneal dystrophy (granular, fleck, lattice, posterior polymorphous) and their family members were recruited, a pedigree obtained and their dystrophy extensively phenotyped. After informed consent, samples were taken for DNA extraction. PCR and sequencing of all coding exons of TGFBI was undertaken. All five patients with granular dystrophy had the R555W mutation, and H626P was identified in an intermediate dystrophy of Bowman layer pedigree. No other mutations were detected including in the stromal dystrophy cases. Mutational analysis of TGFBI in a small population has identified sequence changes consistent with previously identified genotype-phenotype correlations. A new genotype-phenotype association was also characterised. No mutations were identified in some individuals/pedigrees suggesting greater genetic heterogeneity than is currently known in this group of disorders.


An Immunofluorescence-Assisted Microfluidic Single Cell Quantitative Reverse Transcription Polymerase Chain Reaction Analysis of Tumour Cells Separated from Blood.
Circulating tumour cells (CTCs) are important indicators of metastatic cancer and may provide critical information for individualized treatment. As CTCs are usually very rare, the techniques to obtain information from very small numbers of cells are crucial. Here, we propose a method to perform a single cell quantitative reverse transcription polymerase chain reaction (qPCR) analysis of rare tumour cells. We utilized a microfluidic immunomagnetic assay to separate cancer cells from blood. A combination of detailed immunofluorescence and laser microdissection enabled the precise selection of individual cells. Cancer cells that were spiked into blood were successfully separated and picked up for a single cell PCR analysis. The breast cancer cell lines MCF7, SKBR3 and MDAMB231 were tested with 10 different genes. The result of the single cell analysis matched the results from a few thousand cells. Some markers (e.g., ER, HER2) that are commonly used for cancer identification showed relatively large deviations in expression levels. However, others (e.g., GRB7) showed deviations that are small enough to supplement single cell disease profiling.


Children, but not great apes, respect ownership.
Access to and control of resources is a major source of costly conflicts. Animals, under some conditions, respect what others control and use (i.e. possession). Humans not only respect possession of resources, they also respect ownership. Ownership can be viewed as a cooperative arrangement, where individuals inhibit their tendency to take others' property on the condition that those others will do the same. We investigated to what degree great apes follow this principle, as compared to human children. We conducted two experiments, in which dyads of individuals could access the same food resources. The main test of respect for ownership was whether individuals would refrain from taking their partner's resources even when the partner could not immediately access and control them. Captive apes (N = 14 dyads) failed to respect their partner's claim on food resources and frequently monopolized the resources when given the opportunity. Human children (N = 14 dyads), tested with a similar apparatus and procedure, respected their partner's claim and made spontaneous verbal references to ownership. Such respect for the property of others highlights the uniquely cooperative nature of human ownership arrangements.


Inflammation and atherosclerosis: the value of the high-sensitivity C-reactive protein assay as a risk marker.
C-reactive protein (CRP) is a prototypic marker of inflammation. Numerous prospective studies in healthy volunteers have confirmed that high-sensitivity CRP (hsCRP) predicts cardiovascular events (CVEs), and hsCRP seems additive to an elevated total cholesterol level and a total/high-density lipoprotein cholesterol ratio in men and women in predicting risk. In smokers and people with metabolic syndrome, hsCRP levels are elevated; in elderly people, there seems to be a relationship between hsCRP and CVEs and mortality. Several properties of CRP make it proatherogenic; however; pending further studies, it should be considered as a risk marker. In people with acute coronary syndromes, hsCRP measurement may be valuable. Elevated levels in the highest quantile seem to predict greater mortality and poorer prognosis in patients with unstable angina and myocardial infarction (MI). While hsCRP is a strong independent predictor of risk of future MI, stroke, peripheral arterial disease, and vascular death, the validity of hsCRP as a risk marker needs to be assessed in all populations. Weight loss, statin drugs, aspirin, and high-dose alpha tocopherol therapy could affect hsCRP. It has its greatest validity as an adjunctive measure in the primary prevention of cardiovascular disease.


Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions.
Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects.


Providing quality skin and wound care for the bariatric patient: an overview of clinical challenges.
Obesity, (defined as body mass index [BMI] ≥30), and especially morbid obesity (defined as BMI ≥40), has a profound impact on the health and integrity of the patient's integumentary system and on the caregivers who strive to provide care for larger, heavy patients. The purpose of this overview is to address some common skin and wound care issues faced by bariatric patients in order to inform clinicians, patients, and caregivers and enable them to optimize care. For bariatric patients, extra attention must be paid to skin care, cleanliness, skin fold management, perigenital care, odor management, and effective pressure redistribution. Despite these interventions, the multifactorial challenges presented by morbid obesity increase patient risk for serious skin diseases and wound conditions. Implications for practice include how best to educate patients and caregivers for optimal problem prevention. Future research should target improving bariatric care equipment and decreasing risk indices.


Salvia miltiorrhiza: A source for anti-Alzheimer's disease drugs.
Alzheimer's disease (AD) is a devastating neurodegenerative disorder that affects millions of elderly people worldwide. However, no efficient therapeutic method for AD has yet been developed. Recently, Salvia miltiorrhiza Bunge (Lamiaceae), a well-known traditional Chinese medicine which is widely used for treating cardio-cerebrovascular, exerts multiple neuroprotective effects and is attracting increased attention for the treatment of AD. The objective of this study is to discuss the neuroprotective effects and neurogenesis-inducing activities of S. miltiorrhiza components. A detailed search using major electronic search engines (such as Pubmed, ScienceDirect, and Google Scholar) was undertaken with the search terms: Salvia miltiorrhiza, the components of S. miltiorrhiza such as salvianolic acid B, salvianolic acid A, danshensu, tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone, and neuroprotection. Salvia miltiorrhiza components exert multiple neuroprotective potentials relevant to AD, such as anti-amyloid-β, antioxidant, anti-apoptosis, acetylcholinesterase inhibition, and anti-inflammation. Moreover, S. miltiorrhiza promotes neurogenesis of neural progenitor cells/stem cells in vitro and in vivo. The properties of S. miltiorrhiza indicate their therapeutic potential in AD via multiple mechanisms. In addition, S. miltiorrhiza provides lead compounds for developing new drugs against AD.


Identification of tobacco-specific carcinogen in the cervical mucus of smokers and nonsmokers.
In 1996, an estimated 15,700 new cases of cancer of the uterine cervix and 4,900 deaths from this disease were expected to occur in the United States. In a recent international study, human papillomavirus DNA was found in more than 90% of cervical tumor specimens examined, irrespective of the nationality of the patients from whom the samples were obtained. Although infection with human papillomavirus is the major known risk factor for the development of cervical cancer, it alone is not sufficient. Other etiologic factors that have been associated with this disease include deficiencies in micronutrients, lower socioeconomic status, oral contraceptive use, and cigarette smoking. Several compounds from cigarette smoke (nicotine and its major metabolite, cotinine) have been identified in cervical mucus, and the occurrence of smoking-related DNA damage in the cervical epithelium has been documented. This investigation was conducted to determine for the first time whether carcinogenic tobacco-specific N-nitrosamines are present in the cervical mucus of cigarette smokers and of nonsmokers (most likely as a result of environmental exposure). Cervical mucus specimens from 15 smokers and 10 nonsmokers were subjected to supercritical fluid extraction with the use of carbon dioxide that contained 10% methanol, and the resultant extracts were analyzed for tobacco-specific nitrosamines by use of a very sensitive method that involved gas chromatography and mass spectroscopy analyses. In a total of 16 samples obtained from 15 women who were current smokers (two samples from the same woman), we detected the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at concentrations that ranged from 11.9 to 115.0 ng/g of mucus. Only one of a total of 10 cervical mucus specimens obtained from 10 women who claimed to be nonsmokers did not contain detectable NNK, and NNK concentrations ranged from 4.1 to 30.8 ng/g of mucus in the specimens from the remaining nine women. The concentrations of NNK in specimens from cigarette smokers were significantly higher than those from nonsmokers (mean +/- standard deviation: 46.9 +/- 32.5 ng/g of mucus versus 13.0 +/- 9.3 ng/g of mucus; two-tailed Student's t test, P = .004). The cervical mucus of cigarette smokers contains measurable amounts of the potent carcinogen NNK. This compound represents the first tobacco-specific carcinogen identified in this physiologic fluid of women who smoke cigarettes. The presence of NNK in the cervical mucus of nonsmokers is likely due to environmental exposure or to the fact that some of the subjects in this study may not have revealed that they occasionally smoked cigarettes. The presence of NNK in human cervical mucus further strengthens the association between cervical cancer and tobacco smoking.


The Biology and Targeting of FLT3 in Pediatric Leukemia.
Despite remarkable improvement in treatment outcomes in pediatric leukemia over the past several decades, the prognosis for high-risk groups of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), as well as for relapsed leukemia, remains poor. Intensification of chemotherapy regimens for those at highest risk has improved success rates, but at the cost of significantly increased morbidity and long-term adverse effects. With the success of imatinib in Philadelphia-chromosome-positive leukemia and all-trans retinoic acid in acute promyelocytic leukemia, the quest to find additional molecularly targeted therapies has generated much excitement over recent years. Another such possible target in pediatric acute leukemia is FMS-like tyrosine kinase 3 (FLT3). FLT3 aberrations are among the most frequently identified transforming events in AML, and have significant clinical implications in both high-risk pediatric AML and in certain high-risk groups of pediatric ALL. Therefore, the successful targeting of FLT3 has tremendous potential to improve outcomes in these subsets of patients. This article will give an overview of the molecular function and signaling of the FLT3 receptor, as well as its pathogenic role in leukemia. We review the discovery of targeting FLT3, discuss currently available FLT3 inhibitors in pediatric leukemia and results of clinical trials to date, and finally, consider the future promise and challenges of FLT3 inhibitor therapy.


Roles of Hedgehog pathway components and retinoic acid signalling in specifying zebrafish ventral spinal cord neurons.
In mouse, Hedgehog (Hh) signalling is required for most ventral spinal neurons to form. Here, we analyse the spinal cord phenotype of zebrafish maternal-zygotic smoothened (MZsmo) mutants that completely lack Hh signalling. We find that most V3 domain cells and motoneurons are lost, whereas medial floorplate still develops normally and V2, V1 and V0v cells form in normal numbers. This phenotype resembles that of mice that lack both Hh signalling and Gli repressor activity. Ventral spinal cord progenitor domain transcription factors are not expressed at 24 hpf in zebrafish MZsmo mutants. However, pMN, p2 and p1 domain markers are expressed at early somitogenesis stages in these mutants. This suggests that Gli repressor activity does not extend into zebrafish ventral spinal cord at these stages, even in the absence of Hh signalling. Consistent with this, ectopic expression of Gli3R represses ventral progenitor domain expression at these early stages and knocking down Gli repressor activity rescues later expression. We investigated whether retinoic acid (RA) signalling specifies ventral spinal neurons in the absence of Hh signalling. The results suggest that RA is required for the correct number of many different spinal neurons to form. This is probably mediated, in part, by an effect on cell proliferation. However, V0v, V1 and V2 cells are still present, even in the absence of both Hh and RA signalling. We demonstrate that Gli1 has a Hh-independent role in specifying most of the remaining motoneurons and V3 domain cells in embryos that lack Hh signalling, but removal of Gli1 activity does not affect more dorsal neurons.


Influence of Pregnancy on Hemorrhage Risk in Women With Cerebral and Spinal Cavernous Malformations.
Pregnancy as a hemorrhage risk factor in women with cavernous malformations (CMs) is controversial. We describe prospective hemorrhage risk in women who become pregnant after an established CM diagnosis. Beginning in 2015, we recruited consecutive patients with radiologically confirmed CM of the brain or spinal cord to participate in a prospective registry. Participants underwent a baseline and annual medical record review, imaging review, and annual surveys to ascertain pregnancies, deliveries, and neurological complications. We collected prospective hemorrhage data on reproductive age women. We calculated prospective hemorrhage rates on childbearing age patients based on the number of hemorrhages occurring post-CM diagnosis during pregnancy and nonpregnancy, divided by the years of follow-up and censored at last follow-up, age 46, or surgery in sporadic-form CM. The hemorrhage rates were compared and the 95% CI presented with a P value of <0.05. Of 160 women, 90 presented with CM under the age of 46 (average age 31.6 years; 25.6% familial form; 46.7% with hemorrhage; 24.4% brain stem location). These 90 patients had 136 pregnancies before CM diagnosis; 36 pregnancies occurred at or after the diagnosis of CM. Four patients had a hemorrhage while pregnant or postpartum leading to the first CM diagnosis. During 402.6 years of follow-up while not pregnant, 42 hemorrhages occurred prospectively yielding a 10.4% per year (95% CI, 7.5-14.0) risk of hemorrhage while not pregnant. No hemorrhages occurred during 32 prospective pregnancies (26 live births including 1 twinset and 7 nonviable fetuses) after CM diagnosis during 26.9 years of pregnancy time yielding a rate of 0% per year (95% CI, 0-13.6). We found no statistical difference in the rates of hemorrhage during pregnant and nonpregnant time (P=0.09). No hemorrhage occurred during delivery. Our prospective data suggest that pregnancy does not increase the risk of hemorrhage in women with a known brain or spinal cord CM and that vaginal delivery is safe in appropriate candidates.


siRNA-mediated silencing of survivin inhibits proliferation and enhances etoposide chemosensitivity in acute myeloid leukemia cells.
Overexpression of survivin, a known inhibitor of apoptosis, is associated with tumor progression and drug resistance in numerous malignancies, including leukemias. The aim of this study was to investigate the effect of a specific survivin small interference RNA (siRNA) on proliferation and the sensitivity of HL-60 acute myeloid leukemia (AML) cells to the chemotherapeutic drug etoposide. The cells were transfected with siRNAs using LipofectamineTM2000 transfection reagent. Relative survivin mRNA and protein levels were measured by quantitative real-time PCR and Western blotting, respectively. Trypan blue exclusion assays were performed to monitor tumor cell proliferation after siRNA transfection. The cytotoxic effects of etoposide and survivin siRNA, alone and in combination, on leukemic cells were determined using MTT assay. Apoptosis was assessed by ELISA cell death assay. Survivin siRNA markedly reduced both mRNA and protein expression levels in a time-dependent manner, leading to distinct inhibition of cell proliferation and increased spontaneous apoptosis. Surprisingly, survivin siRNA synergistically increased the cell toxic effects of etoposide. Moreover, survivin down-regulation significantly enhanced its induction of apoptosis. Our study suggests that down-regulation of survivin by siRNA can trigger apoptosis and overcome drug resistance of leukemia cells. Therefore, survivin siRNA may be an effective adjuvant in AML chemotherapy.


Pathways for utilization of carbon reserves in Desulfovibrio gigas under fermentative and respiratory conditions.
The sulfate-reducing bacterium Desulfovibrio gigas accumulates large amounts of polyglucose as an endogenous carbon and energy reserve. In the absence of exogenous substrates, the intracellular polysaccharide was utilized, and energy was conserved in the process (H. Santos, P. Fareleira, A. V. Xavier, L. Chen, M.-Y. Liu, and J. LeGall, Biochem. Biophys. Res. Commun. 195:551-557, 1993). When an external electron acceptor was not provided, degradation of polyglucose by cell suspensions of D. gigas yielded acetate, glycerol, hydrogen, and ethanol. A detailed investigation of the metabolic pathways involved in the formation of these end products was carried out, based on measurements of the activities of glycolytic enzymes in cell extracts, by either spectrophotometric or nuclear magnetic resonance (NMR) assays. All of the enzyme activities associated with the glycogen cleavage and the Embden-Meyerhof pathway were determined as well as those involved in the formation of glycerol from dihydroxyacetone phosphate (glycerol-3-phosphate dehydrogenase and glycerol phosphatase) and the enzymes that catalyze the reactions leading to the production of ethanol (pyruvate decarboxylase and ethanol dehydrogenase). The key enzymes of the Entner-Doudoroff pathway were not detected. The methylglyoxal bypass was identified as a second glycolytic branch operating simultaneously with the Embden-Meyerhof pathway. The relative contribution of these two pathways for polyglucose degradation was 2:3. 13C-labeling experiments with cell extracts using isotopically enriched glucose and 13C-NMR analysis supported the proposed pathways. The information on the metabolic pathways involved in polyglucose catabolism combined with analyses of the end products formed from polyglucose under fermentative conditions provided some insight into the role of NADH in D. gigas. In the presence of electron acceptors, NADH resulting from polyglucose degradation was utilized for the reduction of sulfate, thiosulfate, or nitrite, leading to the formation of acetate as the only carbon end product besides CO2. Evidence supporting the role of NADH as a source of reducing equivalents for the production of hydrogen is also presented.


HTLV-III exposure among drug users.
The Centers for Disease Control hierarchical classification of those at risk for acquired immunodeficiency syndrome (AIDS) underestimates the number of cases in which illicit drug use may play a role in the exposure to human T-cell leukemia virus type III. The immunosuppressive effects of nitrite inhalants are not sufficiently documented to elucidate their role as a cofactor in the development of AIDS. The currently available data on the immunosuppressive effects of self-administered parenteral drugs and their diluents indicate an associated elevation of immunoglobulin M, depressed helper/suppression T-cell ratios, and even damage to DNA. Illicit psychoactive drugs and their diluents may influence the virulence of the virus among parenteral drug users. An association between parenteral drug use and prostitution is not unexpected. Female prostitutes who use parenteral drugs may be at high risk for exposure to the virus and thus may transmit this infectious agent to their clients and their families.


Psychrophilic and psychrotolerant fungi on bats and the presence of Geomyces spp. on bat wings prior to the arrival of white nose syndrome.
Since 2006, Geomyces destructans, the causative agent of white nose syndrome (WNS), has killed over 5.7 million bats in North America. The current hypothesis suggests that this novel fungus is an invasive species from Europe, but little is known about the diversity within the genus Geomyces and its distribution on bats in the United States. We documented the psychrophilic and psychrotolerant fungal flora of hibernating bats prior to the arrival of WNS using culture-based techniques. A total of 149 cultures, which were obtained from 30 bats in five bat hibernacula located in four caves and one mine, were sequenced for the entire internal transcribed spacer (ITS) nuclear ribosomal DNA (nrDNA) region. Approximately 53 operational taxonomic units (OTUs) at 97% similarity were recovered from bat wings, with the community dominated by fungi within the genera Cladosporium, Fusarium, Geomyces, Mortierella, Penicillium, and Trichosporon. Eleven Geomyces isolates were obtained and placed in at least seven distinct Geomyces clades based on maximum-likelihood phylogenetic analyses. Temperature experiments revealed that all Geomyces strains isolated are psychrotolerant, unlike G. destructans, which is a true psychrophile. Our results confirm that a large diversity of fungi, including several Geomyces isolates, occurs on bats prior to the arrival of WNS. Most of these isolates were obtained from damaged wings. Additional studies need to be conducted to determine potential ecological roles of these abundant Geomyces strains isolated from bats.


Combined effects of water activity, solute, and temperature on the growth of Vibrio parahaemolyticus.
Vibrio parahaemolyticus was grown at 36 C in tryptic soy broth (pH 7.8) containing added levels of NaCl ranging from 0.5 to 7.9% (wt/wt). The fastest generation time was 16.4 min in tryptic soy broth containing 2.9% NaCl (TSBS) which corresponded to a water activity (a(w)) of 0.992 (+/-0.005). Tryptic soy broth containing lower or higher levels of NaCl resulted in higher or lower a(w), respectively, and slower generation times. Growth was measured turbidimetrically at 36 C in TSBS containing added amounts of NaCl, KCl, glucose, sucrose, glycerol, or propylene glycol. The solutes used to reduce a(w) to comparable levels resulted in extended lag times of varied magnitude, dissimilar growth rates, and different cell numbers. Reduction of a(w) with glycerol was less inhibitory to growth than similar a(w) reductions with NaCl and KCl. Sucrose, glucose, and propylene glycol generally had the greatest effect on extending the lag times of V. parahaemolyticus when the addition of these solutes was made to establish similar a(w) levels lower than 0.992. Minimal a(w) for growth at 15, 21, 29, and 36 +/- 0.2 C for each of four strains of V. parahaemolyticus was tested in TSBS containing added solutes. Reduced a(w) was generally most tolerable at 29 C, whereas higher minimal a(w) for growth was required at 15 C. Solutes added to TSBS to achieve reduction in a(w), minimal a(w) for growth after 20 days, and incubation temperatures were as follows: glycerol, 0.937, 29 C; KCl, 0.945, 29 C; NaCl, 0.948, 29 C; sucrose, 0.957, 29 and 36 C; glucose, 0.983, 21 C; and propylene glycol, 0.986, 29 C. Each of the four strains tested responded similarly to investigative conditions. It appears that minimal a(w) for growth of V. parahaemolyticus depends upon the solute used to control a(w).


Okadaic acid mimics a nuclear component required for cyclin B-cdc2 kinase microinjection to drive starfish oocytes into M phase.
G2-arrested oocytes contain cdc2 kinase as an inactive cyclin B-cdc2 complex. When a small amount of highly purified and active cdc2 kinase, prepared from starfish oocytes at first meiotic metaphase, is microinjected into Xenopus oocytes, it induces activation of the inactive endogenous complex and, as a consequence, drives the recipient oocytes into M phase. In contrast, the microinjected kinase undergoes rapid inactivation in starfish oocytes, which remain arrested at G2. Endogenous cdc2 kinase becomes activated in both nucleated and enucleated starfish oocytes injected with cytoplasm taken from maturing oocytes at the time of nuclear envelope breakdown, but only cytoplasm taken from nucleated oocytes becomes able thereafter to release second recipient oocytes from G2 arrest, and thus contains M phase-promoting factor (MPF) activity. Both nucleated and enucleated starfish oocytes produce MPF activity when type 2A phosphatase is blocked by okadaic acid. If type 2A phosphatase is only partially inhibited, neither nucleated nor enucleated oocytes produce MPF activity, although both do so if purified cdc2 kinase is subsequently injected as a primer to activate the endogenous kinase. The nucleus of starfish oocytes contains an inhibitor of type 2A phosphatase, but neither active nor inactive cdc2 kinase. Microinjection of the content of a nucleus into the cytoplasm of G2-arrested starfish oocytes activates endogenous cdc2 kinase, produces MPF activity, and drives the recipient oocytes into M phase. Together, these results show that the MPF amplification loop is controlled, both positively and negatively, by cdc2 kinase and type 2A phosphatase, respectively. Activation of the MPF amplification loop in starfish requires a nuclear component to inhibit type 2A phosphatase in cytoplasm.


Oligoribonucleotides: improvement in chromatographic separation.
The separation of mixed oligoribonucleotides obtained from enzymatic digestion of soluble RNA into fractions containing mixed oligonucleotides of identical chain length is improved by absence of terminal phosphate. Each fraction may be separated into individual components by ionexchange (Dowex) chromatography. Combination of these two chromatographic procedures yields maximum information on primary structure of the oligonucleotides.


Toward Practice-Based Continuing Education Protocols: Using Testing to Help Physicians Update Their Knowledge.
Using assessment to facilitate learning is a well-established priority in education but has been associated with variable effectiveness for continuing professional development. What factors modulate the impact of testing in practitioners are unclear. We aimed to improve capacity to support maintenance of competence by exploring variables that influence the value of web-based pretesting. Family physicians belonging to a practice-based learning program studied two educational modules independently or in small groups. Before learning sessions they completed a needs assessment and were assigned to either sit a pretest intervention or read a relevant review article. After the learning session, they completed an outcome test, indicated plans to change practice, and subsequently documented changes made. One hundred twelve physicians completed the study, 92 in small groups. The average lag between tests was 6.3 weeks. Relative to those given a review article, physicians given a pretest intervention: (1) reported spending less time completing the assigned task (16.7 versus 25.7 minutes); (2) performed better on outcome test questions that were repeated from the pretest (65.9% versus 58.7%); and (3) when the learning module was completed independently, reported making a greater proportion of practice changes to which they committed (80.0% versus 45.0%). Knowledge gain was unrelated to physicians' stated needs. Low-stakes formative quizzes, delivered with feedback, can influence the amount of material practicing physicians remember from an educational intervention independent of perceptions regarding the need to engage in continuing professional development on the particular topic.


Effects of Intraoperative Infusion of Esmolol on Systemic and Pulmonary Inflammation in a Porcine Experimental Model of Lung Resection Surgery.
Lung resection surgery (LRS) is associated with systemic and pulmonary inflammation, which can affect postoperative outcomes. Activation of β-adrenergic receptors increases the expression of proinflammatory and anti-inflammatory mediators, and their blockade may attenuate the systemic inflammatory response. The aim of this study was to analyze the effect of a continuous perioperative intravenous perfusion of esmolol on postoperative pulmonary edema in an experimental model of LRS requiring periods of one-lung ventilation (OLV). Twenty-four large white pigs were randomly assigned to 3 groups: control (CON), esmolol (ESM), and sham. The ESM group received an intravenous esmolol bolus (0.5 mg/kg) and then an esmolol infusion (0.05 mg·kg·minute) throughout the procedure. The CON group received the same volume of 0.9% saline solution as the ESM group plus a continual infusion of saline. The sham group underwent a left thoracotomy without LRS or OLV. At the end of the LRS, the animals were awakened, and after 24 hours, they underwent general anesthesia again. Lung biopsies and plasma samples were obtained to analyze the levels and expression of inflammatory mediators, and the animals also received a bronchoalveolar lavage. At 24 hours after the operation, the ESM group had less lung edema and lower expression of the proinflammatory biomarkers tumor necrosis factor (TNF) and interleukin (IL)-1 compared to the CON group for both lung lobes. For the mediastinal lobe biopsies, the mean difference and 95% confidence interval (CI) between the groups for edema, TNF, and IL-1 were 14.3 (95% CI, 5.6-23.1), P = .002; 0.19 (95% CI, 0.07-0.32), P = .002; and 0.13 (95% CI, 0.04-0.22), P = .006, respectively. In the left upper lobe, the mean differences for edema, TNF, and IL-1 were 12.4 (95% CI, 4.2-20.6), P = .003; 0.25 (95% CI, 0.12-0.37), P < .001; and 0.3 (95% CI, 0.08-0.53), P = .009. Our results suggest that esmolol reduces lung edema and inflammatory responses in the intraoperative and postoperative periods in animals that underwent LRS with OLV.