Effect of parietal transcranial magnetic stimulation on spatial working memory in healthy elderly persons--comparison of near infrared spectroscopy for young and elderly.
In a previous study, we succeeded in improving the spatial working memory (WM) performance in healthy young persons by applying transcranial magnetic stimulation (TMS) to the parietal cortex and simultaneously measuring the oxygenated hemoglobin (oxy-Hb) level using near-infrared spectroscopy (NIRS). Since an improvement in WM was observed when TMS was applied to the right parietal cortex, the oxy-Hb distribution seemed to support a model of hemispheric asymmetry (HA). In the present study, we used the same study design to evaluate healthy elderly persons and investigated the effect of TMS on WM performance in the elderly, comparing the results with those previously obtained from young persons. The application of TMS did not affect WM performance (both reaction time and accuracy) of 38 elderly participants (mean age = 72.5 years old). To investigate the reason for this result, we conducted a three-way ANOVA examining oxy-Hb in both young and elderly participants. For the right parietal TMS site in the elderly, TMS significantly decreased the oxy-Hb level during WM performance; this result was the opposite of that observed in young participants. An additional three-way ANOVA was conducted for each of the 52 channels, and a P value distribution map was created. The P value maps for the young participants showed a clearly localized TMS effect for both the WM and control task, whereas the P map for the elderly participants showed less significant channels and localization. Further analysis following the time course revealed that right-side parietal TMS had almost no effect on the frontal cortex in the elderly participants. This result can most likely be explained by age-related differences in HA arising from the over-recruitment of oxy-Hb, differentiation in the parietal cortex, and age-related alterations of the frontal-parietal networks.


Xpa deficiency reduces CAG trinucleotide repeat instability in neuronal tissues in a mouse model of SCA1.
Expansion of trinucleotide repeats (TNRs) is responsible for a number of human neurodegenerative disorders. The molecular mechanisms that underlie TNR instability in humans are not clear. Based on results from model systems, several mechanisms for instability have been proposed, all of which focus on the ability of TNRs to form alternative structures during normal DNA transactions, including replication, DNA repair and transcription. These abnormal structures are thought to trigger changes in TNR length. We have previously shown that transcription-induced TNR instability in cultured human cells depends on several genes known to be involved in transcription-coupled nucleotide excision repair (NER). We hypothesized that NER normally functions to destabilize expanded TNRs. To test this hypothesis, we bred an Xpa null allele, which eliminates NER, into the TNR mouse model for spinocerebellar ataxia type 1 (SCA1), which carries an expanded CAG repeat tract at the endogenous mouse Sca1 locus. We find that Xpa deficiency does not substantially affect TNR instability in either the male or female germline; however, it dramatically reduces CAG repeat instability in neuronal tissues-striatum, hippocampus and cerebral cortex-but does not alter CAG instability in kidney or liver. The tissue-specific effect of Xpa deficiency represents a novel finding; it suggests that tissue-to-tissue variation in CAG repeat instability arises, in part, by different underlying mechanisms. These results validate our original findings in cultured human cells and suggest that transcription may induce NER-dependent TNR instability in neuronal tissues in humans.


Synthesis and characterization of sugar-bearing chitosan derivatives: aqueous solubility and biodegradability.
The extended use of chitosan in biomedical fields has been limited by its insoluble nature in a biological solution. To endow the water solubility in a broad range of pH, chitosan derivatives were prepared by the covalent attachment of a hydrophilic sugar moiety, gluconic acid, through the formation of an amide bond. These sugar-bearing chitosans (SBCs) were further modified by the N-acetylation in an alcoholic aqueous solution. Thereafter, the effect of the gluconyl group and the degree of N-acetylation (DA) on the water solubility at different pHs and on the biodegradability of chitosan were investigated. The SBCs showed the water solubility in a broader range of pH than chitosan, whereas they were still insoluble at neutral and alkali pH. The N-acetylation of SBCs significantly affected the water solubility, for example, the SBCs with the DA, ranging from 29% to 63%, were soluble in the whole range of pH. This might result from the improved hydrophilicity by the gluconyl group, accompanied by the role of the N-acetyl group that disturbed the hydrogen bonding between amino groups of chitosan. From the biodegradation tests, determined by the decrease in the viscosity of a polymer solution exposed to lysozyme, it was evident that the gluconyl group attached to chitosan improved the biodegradability. Thus, it was possible to control the biodegradability of chitosan by adjusting the amounts of gluconyl and N-acetyl groups in the chitosan backbone. The N-acetylated SBCs, soluble in the broad range of pH, might be useful for various biomedical applications.


Transmission characteristics of the 2009 H1N1 influenza pandemic: comparison of 8 Southern hemisphere countries.
While in Northern hemisphere countries, the pandemic H1N1 virus (H1N1pdm) was introduced outside of the typical influenza season, Southern hemisphere countries experienced a single wave of transmission during their 2009 winter season. This provides a unique opportunity to compare the spread of a single virus in different countries and study the factors influencing its transmission. Here, we estimate and compare transmission characteristics of H1N1pdm for eight Southern hemisphere countries/states: Argentina, Australia, Bolivia, Brazil, Chile, New Zealand, South Africa and Victoria (Australia). Weekly incidence of cases and age-distribution of cumulative cases were extracted from public reports of countries' surveillance systems. Estimates of the reproduction numbers, R(0), empirically derived from the country-epidemics' early exponential phase, were positively associated with the proportion of children in the populations (p = 0.004). To explore the role of demography in explaining differences in transmission intensity, we then fitted a dynamic age-structured model of influenza transmission to available incidence data for each country independently, and for all the countries simultaneously. Posterior median estimates of R₀ ranged 1.2-1.8 for the country-specific fits, and 1.29-1.47 for the global fits. Corresponding estimates for overall attack-rate were in the range 20-50%. All model fits indicated a significant decrease in susceptibility to infection with age. These results confirm the transmissibility of the 2009 H1N1 pandemic virus was relatively low compared with past pandemics. The pattern of age-dependent susceptibility found confirms that older populations had substantial--though partial--pre-existing immunity, presumably due to exposure to heterologous influenza strains. Our analysis indicates that between-country-differences in transmission were at least partly due to differences in population demography.


Neutralization of interleukin-18 ameliorates ischemia/reperfusion-induced myocardial injury.
Ischemia/reperfusion (I/R) injury is characterized by the induction of oxidative stress and proinflammatory cytokine expression. Recently demonstrating that oxidative stress and TNF-alpha each stimulate interleukin (IL)-18 expression in cardiomyocytes, we hypothesized that I/R also induces IL-18 expression and thus exacerbates inflammation and tissue damage. Neutralization of IL-18 signaling should therefore diminish tissue injury following I/R. I/R studies were performed using a chronically instrumented closed chest mouse model. Male C57BL/6 mice underwent 30 min of ischemia by LAD coronary artery ligation followed by various periods of reperfusion. Sham-operated or ischemia-only mice served as controls. A subset of animals was treated with IL-18-neutralizing antibodies 1 h prior to LAD ligation. Ischemic LV tissue was used for analysis. Our results demonstrate that, compared with sham operation and ischemia alone, I/R significantly increased (i) oxidative stress (increased MDA/4-HNE levels), (ii) neutrophil infiltration (increased MPO activity), (iii) NF-kappaB DNA binding activity (p50, p65), and (iv) increased expression of IL-18Rbeta, but not IL-18Ralpha or IL-18BP transcripts. Administration of IL-18-neutralizing antibodies significantly reduced I/R injury measured by reduced infarct size (versus control IgG). In isolated adult mouse cardiomyocytes, simulated ischemia/reperfusion enhanced oxidative stress and biologically active IL-18 expression via IKK-dependent NF-kappaB activation. These results indicate that IL-18 plays a critical role in I/R injury and thus represents a promising therapeutic target.


The rat testicular artery: a model of spontaneous aneurysmal-like structure formation.
The straight spermatic and highly convoluted testicular arteries were studied by light microscopy in adult and aging normotensive (NT) and spontaneously hypertensive (SHR) rats. In younger rats, on the internal part of bends of the testicular artery, areas lacking the media similar to classical cerebral arterial medial defects were observed. At the same location, in other bends, structurally defective areas (SDA) constituted by or including medial defects but also lacking the internal elastic lamina and which in some cases evaginated, were present. Structurally defective areas were less numerous in SHR than in NT rats at 6 months, suggesting that intrinsic differences may exist between rat strains. In contrast, in old rats, the number of SDA was higher in hypertensive than in normotensive rats, supporting the role of hemodynamics in SDA formation. With age, SDA enlarged in both rat strains, and most of them became structurally similar to aneurysms, ie, lacking the internal elastic lamina and medial cells and with a dilated lumen, supporting the view that medial defects are sites of aneurysmal structure development. In hypertensive rats, fibrin and lipid deposits occurred within these aneurysmal-like structures. In the straight part of the spermatic artery no such structural modifications occurred, suggesting that either hemodynamics and/or structural development, both dependent on arterial geometry, are determinant in SDA formation. The results are discussed in view of the use of the rat testicular artery as a possible model of the formation of spontaneous aneurysmal-like structures relevant to cerebral aneurysms.


Visual cortex operations and their implementation using the incoherent optical neuron model.
The incoherent optical neuron model uses two different device responses, an inhibitory response and a nonlinear output response, to realize a complete neuron unit that has both inhibitory and excitatory inputs. We describe its use to implement a model of simple cells of the visual cortex. Such simple cells perform the operations of edge detection, orientation selection, and in the case of moving objects, direction and speed selection. Experiments are described that utilize two Hughes liquid-crystal light valves to perform the functions of input transduction and optical neuron unit-array implementation. A multiplexed dichromated gelatin hologram serves as a holographic optical element that forms space-invariant (but otherwise arbitrary) point-spread functions for the network interconnections. Changing the holographic interconnection pattern permits implementation of different simple cells performing, e.g., transient response, edge detection, orientation preference, and direction and speed preference. Experimental results of these operations are presented.


Prematurity due to maternal brucella infection and review of the literature.
Brucellosis is a zoonosis caused by ingestion of unpasteurized milk or other dairy products from infected animals or through close contact with their secretions. Human-to-human transmission, which is rare, has been reported in association with blood transfusion, bone marrow transplantation, transplacental or perinatal exposure, and breastfeeding. In the neonatal period, congenital brucellosis, which is an extremely rare condition, can lead to serious clinical consequences with a high morbidity and mortality rate. The case presented here is a premature baby born at 25 weeks' gestation to a mother suffering from severe oligohydramnios, premature rupture of membranes and untreated acute brucellosis. The baby had severe respiratory distress and radiological findings compatible with pulmonary infection. The blood cultures of the baby and the mother were positive for Brucella melitensis, supporting the diagnosis of brucellosis with presumed transplacental transmission.


An efficient parallel algorithm for accelerating computational protein design.
Structure-based computational protein design (SCPR) is an important topic in protein engineering. Under the assumption of a rigid backbone and a finite set of discrete conformations of side-chains, various methods have been proposed to address this problem. A popular method is to combine the dead-end elimination (DEE) and A* tree search algorithms, which provably finds the global minimum energy conformation (GMEC) solution. In this article, we improve the efficiency of computing A* heuristic functions for protein design and propose a variant of A* algorithm in which the search process can be performed on a single GPU in a massively parallel fashion. In addition, we make some efforts to address the memory exceeding problem in A* search. As a result, our enhancements can achieve a significant speedup of the A*-based protein design algorithm by four orders of magnitude on large-scale test data through pre-computation and parallelization, while still maintaining an acceptable memory overhead. We also show that our parallel A* search algorithm could be successfully combined with iMinDEE, a state-of-the-art DEE criterion, for rotamer pruning to further improve SCPR with the consideration of continuous side-chain flexibility. Our software is available and distributed open-source under the GNU Lesser General License Version 2.1 (GNU, February 1999). The source code can be downloaded from http://www.cs.duke.edu/donaldlab/osprey.php or http://iiis.tsinghua.edu.cn/∼compbio/software.html.


Diamine oxidase (histaminase). A circulating marker for rat intestinal mucosal maturation and integrity.
Diamine oxidase (histaminase) is an enzyme found in high concentrations in the intestinal mucosa of humans and other mammalian species. We investigated whether plasma and mucosal levels of diamine oxidase activity reflect both the maturational status of the mucosa during its development in the newborn rate and the degree of mucosal damage during its injury in the adult rat. Litter mates were reared under identical conditions and killed at different ages from day 0 to day 40 after birth. Diamine oxidase in the small intestine was low at birth, increased gradually with age, reached a peak at 22 d, and then remained at normal adult levels, similar to the developmental patterns of maltase and sucrase. Plasma diamine oxidase rose in parallel with intestinal levels (n = 500, r = 0.84, P less than 0.001), reached a peak at 24 d, and then remained at normal adult levels. Diamine oxidase activity in 15 nonintestinal tissues was less than 5% of ileal mucosal activity, and no nonintestinal activities showed increase with age. Adult rat intestinal loops were perfused with hyperosmolar sodium sulfate solutions to produce selective damage to villus mucosa. With increasing mucosal damage, there was a progressive decrease in the enzyme activities studied; first, lactase levels fell, then maltase and sucrase, and finally mucosal and plasma diamine oxidase activity levels fell. The decrease in plasma diamine oxidase reflected the degree of mucosal damage (n = 29, P less than 0.04). Diamine oxidase activity is thus unique among intestinal mucosal enzymes studied to date in that circulating levels can serve as a marker of mucosal maturation and integrity.


Salt-load electrocochleography.
To introduce a new protocol for diagnostic electrocochleography using a pretest oral salt load to improve test sensitivity in patients with suspected inner ear fluid imbalance. A retrospective review of patients who reported vertigo that, by medical history, was suggestive of an inner ear fluid imbalance was preformed. The patients received a complete audiovestibular evaluation that included a baseline electrocochleogram. Despite the incapacitating nature of their vertigo, there were no symptoms or electrophysiologic abnormalities that would isolate an etiologic ear. After the baseline studies, the patients received 4 g of sodium chloride daily for 3 days before repeat electrocochleography. A control group of 13 healthy volunteers with normal baseline electrocochleography and pure tone audiometry was tested under similar conditions. This study was conducted at an ambulatory care clinic associated with a tertiary referral medical center. Electrocochleography was performed using alternating polarity clicks presented at a rate of 9.7/sec at 95 dB nHL by an extratympanic TIPtrode electrode and recorded with a Nicolet Spirit (Nicolet Instrument Corp., Madison, WI, U.S.A.). Responses were averaged for 1000 sweeps using a 10-msec time base with bandpass filtering from 5 to 1500 Hz. A summating potential/action potential (SP/AP) ratio of 0.37 was considered the upper limit of normal. Enhancement in the SP/AP ratio from a normal baseline value to > 0.37 after oral salt loading was indicative of a positive test. None of the ears from control subjects had a positive salt load electrocochleogram, and one or both ears in 38% of the patients in the study group with normal baseline SP/AP ratios and symptoms of inner ear fluid imbalance converted to abnormal. The mean SP/AP ratio of the control group for the conditions before and after salt-load was not statistically different (p = 0.48), although the difference in the mean SP/AP ratio in the study group after salt loading was statistically significant (p = 1.329 x 10(-5)). A group of patients who reported vertigo with no localizing abnormalities had a statistically significant increase in the mean SP/AP ratio after ingestion of a large quantity of sodium chloride. A modest percentage had elevation of the SP/AP ratio above the upper limit of normal for our audiovestibular lab. The localization of a "salt-senstitive" ear could assist the clinician in the management of these difficult problems with long-term medical therapy or surgical treatment when alternative measures fail.


Cytomegalovirus vaccine strain towne-derived dense bodies induce broad cellular immune responses and neutralizing antibodies that prevent infection of fibroblasts and epithelial cells.
Human cytomegalovirus (HCMV), a betaherpesvirus, can cause severe disease in immunosuppressed patients and following congenital infection. A vaccine that induces both humoral and cellular immunity may be required to prevent congenital infection. Dense bodies (DBs) are complex, noninfectious particles produced by HCMV-infected cells and may represent a vaccine option. As knowledge of the antigenicity and immunogenicity of DB is incomplete, we explored characterization methods and defined DB production methods, followed by systematic evaluation of neutralization and cell-mediated immune responses to the DB material in BALB/c mice. DBs purified from Towne-infected cultures treated with the viral terminase inhibitor 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) were characterized by nanoparticle tracking analysis (NTA), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), immunoblotting, quantitative enzyme-linked immunosorbent assay, and other methods. The humoral and cellular immune responses to DBs were compared to the immunogenicity of glycoprotein B (gB) administered with the adjuvant AddaVax (gB/AddaVax). DBs induced neutralizing antibodies that prevented viral infection of cultured fibroblasts and epithelial cells and robust cell-mediated immune responses to multiple viral proteins, including pp65, gB, and UL48. In contrast, gB/AddaVax failed to induce neutralizing antibodies that prevented infection of epithelial cells, highlighting a critical difference in the humoral responses induced by these vaccine candidates. Our data advance the potential for the DB vaccine approach, demonstrate important immunogenicity properties, and strongly support the further evaluation of DBs as a CMV vaccine candidate.


Sleep quality after initial chemotherapy for breast cancer.
The goal of this study is to characterize sleep quality and quantity prior to and in the first three nights after initial chemotherapy for breast cancer. This study makes use of secondary analysis of data from two separate randomized clinical trials (RCT) of behavioral interventions to improve fatigue and sleep. Patients came from two comprehensive cancer centers, three clinical cancer centers, and 10 community clinics in five states. Participants were women with stage I-IIIA breast cancer treated with anthracycline and/or cyclophosphamide-based regimens. Baseline data from each RCT were used in the analysis. Sixty-five percent of women self-reported poor sleep in the month preceding chemotherapy using the Pittsburgh Sleep Quality Index (PSQI) score >5. Three nights of actigraphy data indicated a wide range of sleep experience with an average of 10 awakenings and time (minutes) awake after sleep onset (WASO-M) averaging 61 min per night. The first night's sleep was the worst. There was no statistically significant relationship between self-reported poor sleep and sleep measures obtained by actigraphy. Women with poor sleep at baseline (global PSQI >5) had significantly lower (p < 0.001) physical (PCS) and mental (MCS) health status. However, neither the PCS nor MCS was associated with any of the average actigraphy sleep parameters or night 1 parameters in the aggregated sample. Increasing age was also associated with poorer sleep. A high percent of women with breast cancer begin chemotherapy with disturbed sleep and the initial nights after chemotherapy are characterized by sleep fragmentation that disrupts sleep maintenance. Interventions should focus on strategies to decrease the number and duration of night awakenings. Further research is needed to identify predictors of poor sleep during this time.


Basic cell physiological activities (cell adhesion, chemotaxis and proliferation) induced by selegiline and its derivatives in Mono Mac 6 human monocytes.
Selegiline (R-deprenyl), a monoamine oxidase-B (MAO-B) inhibitor, has complex pharmacological effect that contributes to treatment of neurodegenerative diseases such as Parkinson's and presumably Alzheimer's disease and might work as an inhibitor of tumor growth. In respect of tumorigenesis and metastasis formation, the controlled modifications of adhesion and migration have high therapeutic significance. In the present study, our purpose was to investigate cell physiological responses (adhesion, chemotaxis and proliferation) induced by selegiline, its metabolites and synthetic derivatives and to find some correlations between the molecular structure and the reported antitumor behavior of the derivatives. Our results demonstrated that both R- and S-deprenyls have the potency to elicit increased adhesion and a chemorepellent activity in monocyte model (Mono Mac 6 cell line derived from monoblastic leukemia); however, only the R-enantiomer proved to be cytotoxic. Among the metabolites R-amphetamine has retained the adhesion inducer and the chemorepellent effect of the parent drug on the most significant level. In contrast, a reversed chemotactic effect and an improved cytotoxic character were detected in the presence of fluoro group (p-fluoro-S-deprenyl). In summary, the adhesion inducer activity, chemorepellent and advantageous cytotoxic effects of selegiline and some derivatives indicate that these drug molecules might have inhibitory effects in metastasis formation in primary tumors.


Does the cerebellum contribute to human navigation by processing sequential information?
Several authors have proposed that the cerebellum has an important role in functions of higher order as a general mode of sequence detection, independently from the nature of the information. The aim of this study was to verify whether the cerebellum mediates the processing of navigational sequential information and to determine whether it is influenced by the modality of the stimuli presentation. We tested 12 cerebellar patients and 12 healthy age-matched participants in 2 comparable navigational tasks (Walking Corsi Test and the Magic Carpet) requiring to memorizing a sequence of spatial locations. The 2 tasks differ each other for the modality of stimuli presentation: in the Walking Corsi Test the sequence is shown by an examiner that walks on the carpet, whereas in the Magic Carpet it is shown by a computer that lights up the tiles in the sequence. We hypothesize that different mental processes are implicated between the Walking Corsi Test and the Magic Carpet. Indeed, whereas watching the examiner, who performs the sequence on the carpet, allows the patient to simulate the action mentally in the Walking Corsi Test, such simulation cannot be triggered in the Magic Carpet. Our results showed that cerebellar patients obtained scores significantly lower than control participants only in the Magic Carpet. We interpreted the patients' performance as a specific deficit in detecting and ordering single independent stimuli as a sequence, when the maintenance of stimulus-response associations is more demanding. (PsycINFO Database Record


Enhancing signal detection and completely eliminating scattering using quasi-phase-cycling in 2D IR experiments.
We demonstrate how quasi-phase-cycling achieved by sub-cycle delay modulation can be used to replace optical chopping in a box-CARS 2D IR experiment in order to enhance the signal size, and, at the same time, completely eliminate any scattering contamination. Two optical devices are described that can be used for this purpose, a wobbling Brewster window and a photoelastic modulator. They are simple to construct, easy to incorporate into any existing 2D IR setup, and have attractive features such as a high optical throughput and a fast modulation frequency needed to phase cycle on a shot-to-shot basis.


Target Residence Time-Guided Optimization on TTK Kinase Results in Inhibitors with Potent Anti-Proliferative Activity.
The protein kinase threonine tyrosine kinase (TTK; also known as Mps1) is a critical component of the spindle assembly checkpoint and a promising drug target for the treatment of aggressive cancers, such as triple negative breast cancer. While the first TTK inhibitors have entered clinical trials, little is known about how the inhibition of TTK with small-molecule compounds affects cellular activity. We studied the selective TTK inhibitor NTRC 0066-0, which was developed in our own laboratory, together with 11 TTK inhibitors developed by other companies, including Mps-BAY2b, BAY 1161909, BAY 1217389 (Bayer), TC-Mps1-12 (Shionogi), and MPI-0479605 (Myrexis). Parallel testing shows that the cellular activity of these TTK inhibitors correlates with their binding affinity to TTK and, more strongly, with target residence time. TTK inhibitors are therefore an example where target residence time determines activity in in vitro cellular assays. X-ray structures and thermal stability experiments reveal that the most potent compounds induce a shift of the glycine-rich loop as a result of binding to the catalytic lysine at position 553. This "lysine trap" disrupts the catalytic machinery. Based on these insights, we developed TTK inhibitors, based on a (5,6-dihydro)pyrimido[4,5-e]indolizine scaffold, with longer target residence times, which further exploit an allosteric pocket surrounding Lys553. Their binding mode is new for kinase inhibitors and can be classified as hybrid Type I/Type III. These inhibitors have very potent anti-proliferative activity that rivals classic cytotoxic therapy. Our findings will open up new avenues for more applications for TTK inhibitors in cancer treatment.


How development and manufacturing will need to be structured--heads of development/manufacturing. May 20-21, 2014 Continuous Manufacturing Symposium.
Continuous manufacturing (CM) is a process technology that has been used in the chemical industry for large-scale mass production of chemicals in single-purpose plants with benefit for many years. Recent interest has been raised to expand CM into the low-volume, high-value pharmaceutical business with its unique requirements regarding readiness for human use and the required quality, supply chain, and liability constraints in this business context. Using a fairly abstract set of definitions, this paper derives technical consequences of CM in different scenarios along the development-launch-supply axis in different business models and how they compare to batch processes. Impact of CM on functions in development is discussed and several operational models suitable for originators and other business models are discussed and specific aspects of CM are deduced from CM's technical characteristics. Organizational structures of current operations typically can support CM implementations with just minor refinements if the CM technology is limited to single steps or small sequences (bin-to-bin approach) and if the appropriate technical skill set is available. In such cases, a small, dedicated group focused on CM is recommended. The manufacturing strategy, as centralized versus decentralized in light of CM processes, is discussed and the potential impact of significantly shortened supply lead times on the organization that runs these processes. The ultimate CM implementation may be seen by some as a totally integrated monolithic plant, one that unifies chemistry and pharmaceutical operations into one plant. The organization supporting this approach will have to reflect this change in scope and responsibility. The other extreme, admittedly futuristic at this point, would be a highly decentralized approach with multiple smaller hubs; this would require a new and different organizational structure. This processing approach would open up new opportunities for products that, because of stability constraints or individualization to patients, do not allow centralized manufacturing approaches at all. Again, the entire enterprise needs to be restructured accordingly. The situation of CM in an outsourced operation business model is discussed. Next steps for the industry are recommended. In summary, opportunistic implementation of isolated steps in existing portfolios can be implemented with minimal organizational changes; the availability of the appropriate skills is the determining factor. The implementation of more substantial sequences requires business processes that consider the portfolio, not just single products. Exploration and implementation of complete process chains with consequences for quality decisions do require appropriate organizational support.


Distinctive cardiac autonomic dysfunction following stress exposure in both sexes in an animal model of PTSD.
It is unclear whether the poor autonomic flexibility or dysregulation observed in patients with posttraumatic stress disorder (PTSD) represents a pre-trauma vulnerability factor or results from exposure to trauma. We used an animal model of PTSD to assess the association between the behavioral response to predator scent stress (PSS) and the cardiac autonomic modulation in male and female rats. The rats were surgically implanted with radiotelemetry devices to measure their electrocardiograms and locomotor activity (LMA). Following baseline telemetric monitoring, the animals were exposed to PSS or sham-PSS. Continuous telemetric monitoring (24h/day sampling) was performed over the course of 7days. The electrocardiographic recordings were analyzed using the time- and frequency-domain indexes of heart rate variability (HRV). The behavioral response patterns were assessed using the elevated plus maze and acoustic startle response paradigms for the retrospective classification of individuals according to the PTSD-related cut-off behavioral criteria. During resting conditions, the male rats had significantly higher heart rates (HR) and lower HRV parameters than the female rats during both the active and inactive phases of the daily cycle. Immediately after PSS exposure, both the female and male rats demonstrated a robust increase in HR and a marked drop in HRV parameters, with a shift of sympathovagal balance towards sympathetic predominance. In both sexes, autonomic system habituation and recovery were selectively inhibited in the rats whose behavior was extremely disrupted after exposure to PSS. However, in the female rats, exposure to the PSS produced fewer EBR rats, with a more rapid recovery curve than that of the male rats. PSS did not induce changes to the circadian rhythm of the LMA. According to our results, PTSD can be conceptualized as a disorder that is related to failure-of-recovery mechanisms that impede the restitution of physiological homeostasis.


Interaction between ciprofloxacin and thiopental in the central nervous system of the male rat.
The effect of intravenous ciprofloxacin (CPX) pretreatment on the kinetics and brain sensitivity for thiopental was studied in male rats using a previously developed electroencephalographic (EEG) threshold method. Thiopental was administered intravenously with constant infusion rate. Immediately after the appearance of the first burst suppression of 1 sec. or more (the "silent-second") in the EEG the infusion was stopped and the rats were killed by decapitation. The dose of thiopental needed to reach the criterion of silent-second was slightly reduced in ciprofloxacin pretreated rats when compared with saline pretreated controls. One rat that developed seizures after CPX pretreatment needed a considerably reduced dose of thiopental to induce the silent-second. The serum concentrations of thiopental were markedly reduced in the experimental group while no significant differences were found in the concentrations of thiopental in the different parts of the central nervous system (CNS), fat or muscle tissue. The kinetics of CPX were also affected. The experimental group (CPX + thiopental treated) had significant higher brain concentrations of CPX than the corresponding only CPX treated control group while no differences were found in the serum concentrations of CPX between the groups. As previously suggested, the distribution of thiopental in the CNS is not only dependent on its lipid solubility, but also as a weak organic acid, on the transport system for organic acids out of the CNS which both thiopental and ciprofloxacin seem to use and mutually compete for it.


Ablation of p21-activated kinase-1 in mice promotes isoproterenol-induced cardiac hypertrophy in association with activation of Erk1/2 and inhibition of protein phosphatase 2A.
Earlier investigations in our lab indicated an anti-adrenergic effect induced by activation of p21-activated kinase (Pak-1) and protein phosphatase 2A (PP2A). Our objective was to test the hypothesis that Pak-1/PP2A is a signaling cascade controlling stress-induced cardiac growth. We determined the effects of ablation of the Pak-1 gene on the response of the myocardium to chronic stress of isoproterenol (ISO) administration. Wild-type (WT) and Pak-1-knockout (Pak-1-KO) mice were randomized into six groups to receive either ISO, saline (CTRL), or ISO and FR180204, a selective inhibitor of Erk1/2. Echocardiography revealed that hearts of the Pak-1-KO/ISO group had increased LV fractional shortening, reduced LV chamber volume in diastole and systole, increased cardiac hypertrophy, and enhanced transmitral early filling deceleration time, compared to all other groups. The changes were associated with an increase in relative Erk1/2 activation in Pak-1-KO/ISO mice versus all other groups. ISO-induced cardiac hypertrophy and Erk1/2 activation in Pak-1-KO/ISO were attenuated when the selective Erk1/2 inhibitor FR180204 was administered. Immunoprecipitation showed an association between Pak-1, PP2A, and Erk1/2. Cardiac myocytes infected with an adenoviral vector expressing constitutively active Pak-1 showed a repression of Erk1/2 activation. p38 MAPK phosphorylation was decreased in Pak-1-KO/ISO and Pak-1-KO/CTRL mice compared to WT. Levels of phosphorylated PP2A were increased in ISO-treated Pak-1-KO mice, indicating reduced phosphatase activity. Maximum Ca(2+)-activated tension in detergent-extracted bundles of papillary fibers from ISO-treated Pak-1-KO mice was higher than in all other groups. Analysis of cTnI phosphorylation indicated that compared to WT, ISO-induced phosphorylation of cTnI was blunted in Pak-1-KO mice. Active Pak-1 is a natural inhibitor of Erk1/2 and a novel anti-hypertrophic signaling molecule upstream of PP2A.


Characterization of void space in polydisperse sphere packings: Applications to hard-sphere packings and to protein structure analysis.
The implementation of a method for the exact evaluation of the volume and surface area of cavities and free volumes in polydisperse sphere packings is described. The generalization of an algorithm for Voronoi tessellation by Tanemura et al. is presented, employing the radical plane construction, as a part of the method. We employ this method to calculate the equation of state for monodisperse and polydisperse hard-sphere fluids, crystals, and for the metastable amorphous branch up to random close packing or jamming densities. We compute the distribution of free volumes, and compare with previous results employing a heuristic definition of free volume. We show the efficacy of the method for analyzing protein structure, by computing various quantities such as the distribution of sizes of buried cavities and pockets, the scaling of solvent accessible area to the corresponding occupied volume, the composition of residues lining cavities, etc.


Large-Scale Photonic Ising Machine by Spatial Light Modulation.
Quantum and classical physics can be used for mathematical computations that are hard to tackle by conventional electronics. Very recently, optical Ising machines have been demonstrated for computing the minima of spin Hamiltonians, paving the way to new ultrafast hardware for machine learning. However, the proposed systems are either tricky to scale or involve a limited number of spins. We design and experimentally demonstrate a large-scale optical Ising machine based on a simple setup with a spatial light modulator. By encoding the spin variables in a binary phase modulation of the field, we show that light propagation can be tailored to minimize an Ising Hamiltonian with spin couplings set by input amplitude modulation and a feedback scheme. We realize configurations with thousands of spins that settle in the ground state in a low-temperature ferromagneticlike phase with all-to-all and tunable pairwise interactions. Our results open the route to classical and quantum photonic Ising machines that exploit light spatial degrees of freedom for parallel processing of a vast number of spins with programmable couplings.


Functional chest pain: esophageal or overlapping functional disorder.
Whether patients with functional chest pain have an esophageal or overlapping functional disorder of the gut is unclear. We investigated the prevalence of functional gastrointestinal disorders in patients with functional chest pain. One hundred patients with functional chest pain and normal cardiac, endoscopic, and manometric studies were evaluated for esophageal hypersensitivity with a balloon distension test. Subsequently, a modified Rome II functional bowel disorder questionnaire was mailed to these subjects. Prevalence of irritable bowel syndrome (IBS) and other functional disorder were determined using the Rome II criteria. In addition, we assessed the prevalence of chest pain in 81 patients with functional constipation. There were 69 responders (54 women); 2 were excluded. Fifty-five patients (82%) fulfilled criteria for other functional disorders besides chest pain. Although there was an overlap, IBS (27%) and abdominal bloating (22%) were most common; dyspepsia (7%), dysphagia (7%), nonspecific bowel disorder (7%), constipation (4%), abdominal pain (3%), and diarrhea (1%) were less common. Among responders, 52 (78%) had esophageal hypersensitivity and 15 (22%) had normosensitivity, with similar prevalence of functional disorders. Thirty-two (39%) of the subjects with functional constipation reported chest pain occasionally, and 5 (6%) frequently. Approximately 80% of patients with functional chest pain exhibit features of other functional disorders including IBS suggesting an overlap. This association is independent of esophageal hypersensitivity. Recognition of this overlap may facilitate better management of these patients.


Complex patterns of sex-biased demography in canines.
The demographic history of dogs is complex, involving multiple bottlenecks, admixture events and artificial selection. However, existing genetic studies have not explored variance in the number of reproducing males and females, and whether it has changed across evolutionary time. While male-biased mating practices, such as male-biased migration and multiple paternity, have been observed in wolves, recent breeding practices could have led to female-biased mating patterns in breed dogs. For example, breed dogs are thought to have experienced a popular sire effect, where a small number of males father many offspring with a large number of females. Here we use genetic variation data to test how widespread sex-biased mating practices in canines are during different evolutionary time points. Using whole-genome sequence data from 33 dogs and wolves, we show that patterns of diversity on the X chromosome and autosomes are consistent with a higher number of reproducing males than females over ancient evolutionary history in both dogs and wolves, suggesting that mating practices did not change during early dog domestication. By contrast, since breed formation, we found evidence for a larger number of reproducing females than males in breed dogs, consistent with the popular sire effect. Our results confirm that canine demography has been complex, with opposing sex-biased processes occurring throughout their history. The signatures observed in genetic data are consistent with documented sex-biased mating practices in both the wild and domesticated populations, suggesting that these mating practices are pervasive.


Quantifying the mediating effects of smoking and occupational exposures in the relation between education and lung cancer: the ICARE study.
Smoking only partly explains the higher lung cancer incidence observed among socially deprived people. Occupational exposures may account for part of these inequalities, but this issue has been little investigated. We investigated the extent to which smoking and occupational exposures to asbestos, silica and diesel motor exhaust mediated the association between education and lung cancer incidence in men. We analyzed data from a large French population-based case-control study (1976 lung cancers, 2648 controls). Detailed information on lifelong tobacco consumption and occupational exposures to various carcinogens was collected. We conducted inverse probability-weighted marginal structural models. A strong association was observed between education and lung cancer. The indirect effect through smoking varied by educational level, with the strongest indirect effect observed for those with the lowest education (OR = 1.34 (1.14-1.57)). The indirect effect through occupational exposures was substantial among men with primary (OR = 1.22 (1.15-1.30) for asbestos and silica) or vocational secondary education (OR = 1.18 (1.12-1.25)). The contribution of smoking to educational differences in lung cancer incidence ranged from 22 % (10-34) for men with primary education to 31 % (-3 to 84) for men with a high school degree. The contribution of occupational exposures to asbestos and silica ranged from 15 % (10-20) for men with a high school degree to 20 % (13-28) for men with vocational secondary education. Our results highlight the urgent need for public health policies that aim at decreasing exposure to carcinogens at work, in addition to tobacco control policies, if we want to reduce socioeconomic inequalities in the cancer field.


Epididymal fat is necessary for spermatogenesis, but not testosterone production or copulatory behavior.
Surgical removal of the epididymal white adipose tissue (EWAT) depot (lipectomy; EWATx) in laboratory rats or mice decreases spermatogenesis, but this phenomenal finding has not been investigated in depth. Specifically, detailed histology, neuroendocrine profiles, copulatory behavior, lipectomy of other WAT depots, rescue by autologous EWAT transplants, or tests whether this EWATx effect is due to disruption of testes innervation occurring during EWATx have not been performed. Therefore, in the first study, we performed EWATx in male Syrian hamsters and attempted to rescue spermatogenesis by transplanting the removed EWAT to the animal's subcutaneous dorsum, removed comparable or larger amounts of non-gonadal WAT [inguinal WAT (IWAT)] and conducted mating behavior tests. In a second study we conducted detailed testicular histology and assayed serum LH, FSH, and testosterone (T). In a third study, we surgically denervated the testes without removing EWAT and compared testicular histology with that of EWATx or sham surgery. We found that EWATx, but not IWATx, virtually eliminated spermatogenesis producing a marked decrease in size of the seminiferous tubule cellular lining including the Sertoli cells and spermatogonia that could not be rescued by autologous EWAT transplant to the subcutaneous dorsum. EWATx did not change serum LH or T concentrations but approximately doubled serum FSH concentrations. EWATx did not alter copulatory behavior but resulted in aspermatic ejaculate. Selective surgical testicular denervation did not affect spermatogenesis. Collectively, these results suggest the presence of a local, but currently unidentified, growth and/or nutritive factor from EWAT that promotes spermatogenesis.


Usefulness of biomarkers for predicting long-term mortality in patients with diabetes mellitus and non-ST-elevation acute coronary syndromes (a GUSTO IV substudy).
The present study evaluated whether biomarkers of ischemia, inflammation, myocardial damage, and dysfunction are equally useful in patients who have diabetes mellitus (DM) for prediction of cardiac events in non-ST-elevation acute coronary syndrome (ACS). DM was present in 1,677 of 7,800 patients (21.5%) who had non-ST-elevation ACS and were included in the Fourth Global Utilization of Strategies To Open Occluded Arteries (GUSTO IV) trial. Creatinine, N-terminal pro-B-type natriuretic peptide (NT-pro-BNP), troponin T, C-reactive protein, and interleukin-6 were analyzed in serum samples that were obtained at a median of 9.5 hours from symptom onset. One-year mortality rates were 13.5% among patients who had DM (n = 227) and 6.9% among those who did not (n = 418, p < 0.001). The median level of NT-pro-BNP was 2 times as high in patients who had DM, whereas troponin T levels did not differ by DM status. Mortality increased with ascending quartiles of NT-pro-BNP, with 1-year mortality rates of 3.9% (n = 11) in the bottom quartile and 29% (n = 103) in the top quartile. In multivariable analyses, factors that were predictive of 1-year mortality in patients who did not have DM were also significant for those who did. Presence of ST depression > 0.5 mm had the highest odds ratio of 2.3 (95% confidence interval 1.2 to 4.6). NT-pro-BNP levels > 669 ng/L (odds ratio 2.0, 95% confidence interval 1.1 to 3.6) and interleukin-6 levels > 10 ng/L (odds ratio 1.9, 95% confidence interval 1.2 to 3.0) were significant biomarker predictors. In conclusion, DM confers a high long-term mortality in non-ST-elevation ACS. Despite a larger proportion of ST depression and increased levels of NT-pro-BNP and interleukin-6 at admission, these factors provide independent prognostic information that may improve risk stratification and guidance of treatment.


On-chip membrane protein cell-free expression enables development of a direct binding assay: A curious case of potassium channel KcsA-Kv1.3.
Despite the significant role integral membrane proteins (IMPs) play in the drug discovery process, it remains extremely challenging to express, purify, and in vitro stabilize them for detailed biophysical analyses. Cell-free transcription-translation systems have emerged as a promising alternative for producing complex proteins, but they are still not a viable option for expressing IMPs due to improper post-translational folding of these proteins. We have studied key factors influencing in vitro folding of cell-free-expressed IMPs, particularly oligomeric proteins (i.e., ion channels). Using a chimeric ion channel, KcsA-Kv1.3 (K-K), as a model IMP, we have investigated several physiochemical determinants including artificial bilayer environments (i.e., lipid, detergent) for K-K in vitro stabilization. We observed that fusion of a 'superfolder' green fluorescent protein (sfGFP) to K-K as a protein expression reporter not only improves the protein yield, but surprisingly facilitates the K-K tetramer formation, probably by enhancing the solubility of monomeric K-K. Additionally, anionic lipids (i.e., DMPG) were found to be essential for the correct folding of cell-free-expressed monomeric K-K into tetramer, underscoring the importance of lipid-protein interaction in maintaining structural-functional integrity of ion channels. We further developed methods to integrate cell-free-expressed IMPs directly onto a biosensor chip. We employed a solid-supported lipid bilayer onto the surface plasmon resonance (SPR) chip to insert nascent K-K in a membrane. In a different approach, an anti-GFP-functionalized surface was used to capture in situ expressed K-K via its sfGFP tag. Interestingly, only the K-K-functionalized capture surface prepared by the latter strategy was able to interact with K-K's small binding partners. This generalizable approach can be further extended to other membrane proteins for developing direct binding assays involving small ligands.


Variability in metabolic rate, feed intake and fatness among selection and inbred lines of mice.
Mouse populations differing in metabolic rate have been developed through selection for high (MH) and low (ML) heat loss (HLOSS), along with randomly selected controls (MC). Objectives of this study were to (a) compare MH, ML and MC lines for HLOSS and correlated traits of feed intake, body composition and organ weights; (b) compare three widely used inbred mouse lines with MH, ML and MC for the same traits; and (c) investigate potential genotype by diet interaction resulting from feeding diets differing in fat percentage. Heat loss (kcal/day) of MH and ML mice differed by 37% of the mean and remained significant (33%) when HLOSS was expressed on a fat-free mass basis. MH mice consumed more energy than ML with a greater difference in mice fed high-fat compared with standard diets (27% vs 13.9%). Despite greater energy consumption, MH mice were leaner than ML with a difference in total body fat percentage of 40%. The greatest difference in HLOSS between selection and inbred lines was between MH and C57BL/6J (BL), which differed by 26.3%. MH and BL mice also differed in energy intake (15.5%). Body composition of BL mice was similar to MH when fed a standard diet, but similar to ML when fed a high-fat diet. Crosses between MH and ML or between MH and BL would be useful to investigate the genetic regulation of, and identify quantitative trait loci influencing HLOSS, energy intake and body composition. Feeding of a high-fat diet may allow diet-specific loci influencing body composition to be identified in MH and BL lines.


Obstetric and perinatal outcome of babies born from vitrified oocytes.
To assess outcomes after oocyte vitrification on obstetric and perinatal outcomes compared with those achieved with fresh oocytes. Retrospective cohort study. Private university-affiliated IVF center. Children born after use of vitrified oocytes (1,027 from 804 pregnancies) and fresh oocytes (1,224 from 996 pregnancies). Singleton and multiples pregnancies from own and donated ova were included. Oocyte vitrification by the Cryotop method. Pregnancy, delivery, and neonatal outcomes. Vitrification had no clinically relevant adverse effects on obstetric and perinatal outcomes after adjusting for potential confounders. No differences were found between the vitrified and fresh oocyte groups in the rate of obstetric problems (including diabetes, pregnancy-induced hypertension, preterm birth, anemia, and cholestasis), gestational age at delivery, birth weight, Apgar scores, birth defects, admission to neonatal intensive care unit (ICU), perinatal mortality, and puerperal problems. Only a greater number of invasive procedures (adjusted odds ratio 2.12; 95% confidence interval 1.41-3.20), and a reduced occurrence of urinary tract infection (adjusted odds ratio 0.51; 95% confidence interval 0.28-0.91), were observed in the vitrified oocytes group. Although our data, the largest series to date, suggest that oocyte vitrification does not increase adverse obstetric and perinatal outcomes in children conceived with vitrified oocytes, further studies with larger samples are required to reinforce our conclusions.


Ventilation above closing volume reduces pulmonary vascular resistance hysteresis.
The aim of this study was to determine the relationship of pulmonary vascular resistance (PVR) hysteresis and lung volume, with special attention to the effects of ventilation around closing volume (CV). Isolated, blood-perfused canine left lower lung lobes (LLL) were incrementally inflated and deflated. Airway and pulmonary artery pressures (PAP) were recorded after each stepwise volume change. Constant blood flow was provided (600 ml/min) and the pulmonary vein pressure (PVP) was held constant at 5 cm H2O. PAP changes, therefore, were a direct index of PVR changes. Group 1 lobes underwent a full inflation from complete collapse to total lobe capacity (TLC) followed by a full deflation. Group 2 lobes underwent two deflation/inflation cycles, after an initial full inflation. These cycles, both beginning at TLC, had deflation end above and below CV, respectively. Significant PVR hysteresis was noted when the first inflation and deflation were compared. The maximum difference in PAP on deflation was 3.3 cm H2O or 11%. The mean decrease was 2.7 cm H2O for 18 lobes (p < 0.0001). The PAPs on all subsequent inflations or deflations that began above CV remained 9% lower than the initial inflation (n = 9, p < 0.0001), but were not different from each other. However, the final inflation which began from below CV resulted in a 30% return of PVR hysteresis (mean increase in PAP of 0.8 cm H2O, n = 7, p < 0.004). We conclude that there is hysteresis in the PVR response during ventilation, with decreased PVR during deflation relative to the initial inflation, that this hysteresis is absent when lung volume is maintained greater than CV, and that hysteresis returns when inflation occurs after deflation below CV.


Fibrodysplasia (myositis) ossificans progressiva: clinicopathological features and natural history.
Patients with fibrodysplasia (myositis) ossificans progressiva (FOP) (n = 28) were studied for up to 24 years. All had characteristic short big toes potentially recognizable at birth; there were radiographic changes in the toes, thumbs, cervical spine and metaphyses of the long bones, including exostoses. Ossification in the large skeletal muscles began from birth to 16 years (mean age 4.6 years) initially in 25 patients in the neck and upper spinal muscles, and later around the hips, major joints and jaw. The rate and extent of disability was unrelated to the time of onset. There was no evidence that any form of treatment produced consistent benefit. Despite the unique combination of skeletal abnormalities and ectopic ossification, the first diagnosis in patients with FOP was often wrong and usually delayed after ectopic ossification began (mean 2.7 years, range 0-14). Except where presentation was unusual, such as progressive stiffness, this delay was mainly due to failure to recognize the significance of the abnormal toes. The most frequent erroneous histological diagnoses were soft tissue sarcoma or fibromatosis. This series emphasizes the usually incorrect initial diagnosis, the misinterpretation of the histology, the unpredictable prognosis and the failure of current treatment. Despite its extreme rarity, there is a need for wider knowledge of this condition both to avoid clinical errors and to stimulate research.


Characterization of a T5-like coliphage, SPC35, and differential development of resistance to SPC35 in Salmonella enterica serovar typhimurium and Escherichia coli.
The potential of bacteriophage as an alternative biocontrol agent has recently been revisited due to the widespread occurrence of antibiotic-resistant bacteria. We isolated a virulent bacteriophage, SPC35, that can infect both Salmonella enterica serovar Typhimurium and Escherichia coli. Morphological analysis by transmission electron microscopy and analysis of its 118,351-bp genome revealed that SPC35 is a T5 group phage belonging to the family Siphoviridae. BtuB, the outer membrane protein for vitamin B(12) uptake, was found to be a host receptor for SPC35. Interestingly, resistant mutants of both E. coli and S. Typhimurium developed faster than our expectation when the cultures were infected with SPC35. Investigation of the btuB gene revealed that it was disrupted by the IS2 insertion sequence element in most of the resistant E. coli isolates. In contrast, we could not detect any btuB gene mutations in the resistant S. Typhimurium isolates; these isolates easily regained sensitivity to SPC35 in its absence, suggesting phase-variable phage resistance/sensitivity. These results indicate that a cocktail of phages that target different receptors on the pathogen should be more effective for successful biocontrol.


Brief Report: Influence of Physical Activity on Sleep Quality in Children with Autism.
Sleep-related problems are often documented in children with Autism Spectrum Disorders (ASD). This study examined physical activity as a variable that might influence sleep quality in children with ASD. Ten children, ages 9-16 years, were asked to wear accelerometer devices for 7 days in order to track objective measures of activity and sleep quality. Parents of the children also completed the Child's Sleep Habits Questionnaire and maintained a daily sleep log while their child wore the device. This study demonstrated that though over half of the children were identified as having at least one sleep-related problem, their activity levels were significantly related to their sleep patterns. Specifically, the more physically active children had overall higher sleep quality.


Uncovering Membrane-Bound Models of Coagulation Factors by Combined Experimental and Computational Approaches.
In the life sciences, including hemostasis and thrombosis, methods of structural biology have become indispensable tools for shedding light on underlying mechanisms that govern complex biological processes. Advancements of the relatively young field of computational biology have matured to a point where it is increasingly recognized as trustworthy and useful, in part due to their high space-time resolution that is unparalleled by most experimental techniques to date. In concert with biochemical and biophysical approaches, computational studies have therefore proven time and again in recent years to be key assets in building or suggesting structural models for membrane-bound forms of coagulation factors and their supramolecular complexes on membrane surfaces where they are activated. Such endeavors and the proposed models arising from them are of fundamental importance in describing and understanding the molecular basis of hemostasis under both health and disease conditions. We summarize the body of work done in this important area of research to drive forward both experimental and computational studies toward new discoveries and potential future therapeutic strategies.


Stimulation of the Mu DNA strand cleavage and intramolecular strand transfer reactions by the Mu B protein is independent of stable binding of the Mu B protein to DNA.
Interactions between the Mu A and Mu B proteins are important in the early steps of the in vitro transposition of a mini-Mu plasmid. We have examined these interactions by assaying Mu B stimulation of Mu A-mediated strand cleavage and strand transfer reactions. We have previously shown that in the presence of ATP the Mu B protein can stimulate the Mu A-directed cleavage reaction of mini-Mu plasmids carrying a terminal base pair mutation (Surette, M.G., Harkness, T., and Chaconas, G. (1991) J. Biol. Chem. 266, 3118-3124). Here we demonstrate that in the absence of a non-Mu DNA target molecule the Mu B protein stimulates intramolecular integration of a mini-Mu in an ATP-dependent fashion. Furthermore, modification of the Mu B protein with N-ethylmaleimide severely compromises the ability of B to form a stable complex with DNA; however, the modified protein stimulates the strand cleavage and intramolecular strand transfer reactions as efficiently as the untreated protein. These results indicate that the Mu B protein is capable of stimulating the Mu A protein through direct interaction in the absence of stable Mu B-DNA complex formation. Our results increase the spectrum of Mu B protein activities and uncouple the stimulatory properties of the Mu B protein from stable DNA binding but not the ATP cofactor requirement.


A Qualitative Analysis of Offenders' Modus Operandi in Sexually Exploitative Interactions With Children Online.
Transcripts of chat logs of naturally occurring, sexually exploitative interactions between offenders and victims that took place via Internet communication platforms were analyzed. The aim of the study was to examine the modus operandi of offenders in such interactions, with particular focus on the specific strategies they use to engage victims, including discursive tactics. We also aimed to ascertain offenders' underlying motivation and function of engagement in online interactions with children. Five cases, comprising 29 transcripts, were analyzed using qualitative thematic analysis with a discursive focus. In addition to this, police reports were reviewed for descriptive and case-specific information. Offenders were men aged between 27 and 52 years ( M = 33.6, SD = 5.6), and the number of children they communicated with ranged from one to 12 ( M = 4.6, SD = 4.5). Victims were aged between 11 and 15 ( M = 13.00, SD = 1.2), and were both female and male. Three offenders committed online sexual offenses, and two offenders committed contact sexual offenses in addition to online sexual offenses. The analysis of transcripts revealed that interactions between offenders and victims were of a highly sexual nature, and that offenders used a range of manipulative strategies to engage victims and achieve their compliance. It appeared that offenders engaged in such interactions for the purpose of sexual arousal and gratification, as well as fantasy fulfillment.


Bulbar urethroplasty using the dorsal approach: current techniques.
The use of flaps or grafts is mandatory in patients with longer and complex strictures. In 1995-96 we described a new dorsal onlay graft urethroplasty. Over time, our original technique was better defined and changed. Now this procedure (also named Barbagli technique) has been greeted with a fair amount of enthusiasm in Europe and in the United States. The patient is placed in normal lithotomy position, and a midline perineo-scrotal incision is made. The bulbar urethra is then free from the bulbo-cavernous muscles, and is dissected from the corpora cavernosa. The urethra is completely mobilized from the corpora cavernosa, it is rotated 180 degrees, and is incised along its dorsal surface. The graft (preputial skin or buccal mucosa) or the flap is fixed and quilted to the tunica albuginea of the corporal bodies. The right mucosal margin of the opened urethra is sutured to the right side of the patch-graft. The urethra is rotated back into its original position. The left urethral margin is sutured to the left side of the patch graft and to the corporal bodies, and the grafted area is entirely covered by the urethral plate. The bulbo-cavernous muscles are approximated over the grafted area. A 16F silicone Foley catheter is left in place. Dorsal onlay graft urethroplasty is a versatile procedure that may be combined with various substitute materials like preputial skin, buccal mucosa grafts or pedicled flaps.


The risk for chronic kidney disease in metabolically healthy obese patients: A systematic review and meta-analysis.
Chronic kidney disease (CKD) is associated with obesity and metabolic syndrome. Nevertheless, the association of CKD with phenotype referred as metabolically healthy obese or overweight is unclear. In this this systematic review and meta-analysis, we investigate the relationships between obesity and CKD independent of metabolic syndrome by appraising published evidence in studies focusing on metabolically healthy obese people. We performed a literature search through three databases Embase (Elsevier), the Cochrane Central Register of Controlled Trials (Wiley) and PubMed/Medline Web of Science up to March 2022 with the following terms: "chronic kidney disease", "kidney function", "obesity", "metabolic syndrome", "metabolically healthy obesity", "metabolically healthy overweight". Metabolically unhealthy was defined an individual having at least 3 of the following: abdominal obesity, high blood pressure, hypertriglyceridemia, low HDL cholesterol and hyperglycaemia. We used Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) for reporting. Prospective, retrospective, randomized and nonrandomized studies fitting the search criteria were included in our results. Our final analysis included 16 studies with a total number of 4.965.285 participants. There is considerable heterogeneity in terms of study design, participant characteristics and number of participants across individual studies. In comparison to healthy normal weight patients, the risk was progressively higher in overweight (RR 1.29, 95% CI 1.27 to 1.32, p < 0.001) and obese patients (RR 1.47, 95% CI 1.31 to 1.65, p < 0.001). Metabolically healthy overweight and obese individuals have higher risk of CKD compared to individuals without weight excess.


Direct Monte Carlo computation of time-resolved fluorescence in heterogeneous turbid media.
We show that a multiexponential model for time-resolved fluorescence allows the use of an absorption-perturbation Monte Carlo (MC) approach based on stored photon path histories. This enables the rapid fitting of fluorescence yield, lifetimes, and background tissue absorptions in complex heterogeneous media within a few seconds, without the need for temporal convolutions or MC recalculation of photon path lengths. We validate this method using simulations with both a slab and a heterogeneous model of the mouse head.


Association of ischemic heart disease to global and regional longitudinal strain in asymptomatic aortic stenosis.
Longitudinal deformation has been shown to deteriorate with progressive aortic stenosis as well as ischemic heart disease. Despite that both conditions share risk factors and are often coexisting, studies have not assessed the influence on longitudinal deformation for both conditions simultaneously. Thus the purpose of this study was to evaluate the association between subclinical ischemic heart disease and global and regional longitudinal strain in asymptomatic patients with significant aortic stenosis. Prevalent patients with a diagnosis of aortic stenosis at six hospitals in the Greater Copenhagen area were screened for inclusion. A total of 104 asymptomatic patients with moderate-severe aortic stenosis (aortic valve area ≤1.5 cm(2)) fulfilled study criteria and underwent advanced echocardiographic analysis and coronary angiography by multi-detector computed tomography. Angiography revealed coronary stenosis >50% in 31% (n = 32). All regional longitudinal strain measures (apical, mid and basal longitudinal strain) were significant predictors of significant coronary stenosis (>70% stenosis), but only apical and mid longitudinal strain were significant predictors in multivariable analyses independent of aortic valve area, stroke volume index, pro-BNP, valvulo-arterial impedance, body mass index and heart rate. In linear regression models with both aortic valve area and significant coronary stenosis, apical (p < 0.001) and mid (p < 0.01) longitudinal strain were associated to significant coronary stenosis but not aortic valve area. Conversely, basal longitudinal strain was significantly associated to aortic valve area (p = 0.001), but not to significant coronary stenosis. Subclinical coronary artery disease is frequent in moderate and severe aortic stenosis, and should be suspected when regional longitudinal dysfunction is predominant in the apical and mid ventricular segments.


Assessment of methods for predicting the effects of PTEN and TPMT protein variants.
Thermodynamic stability is a fundamental property shared by all proteins. Changes in stability due to mutation are a widespread molecular mechanism in genetic diseases. Methods for the prediction of mutation-induced stability change have typically been developed and evaluated on incomplete and/or biased data sets. As part of the Critical Assessment of Genome Interpretation, we explored the utility of high-throughput variant stability profiling (VSP) assay data as an alternative for the assessment of computational methods and evaluated state-of-the-art predictors against over 7,000 nonsynonymous variants from two proteins. We found that predictions were modestly correlated with actual experimental values. Predictors fared better when evaluated as classifiers of extreme stability effects. While different methods emerging as top performers depending on the metric, it is nontrivial to draw conclusions on their adoption or improvement. Our analyses revealed that only 16% of all variants in VSP assays could be confidently defined as stability-affecting. Furthermore, it is unclear as to what extent VSP abundance scores were reasonable proxies for the stability-related quantities that participating methods were designed to predict. Overall, our observations underscore the need for clearly defined objectives when developing and using both computational and experimental methods in the context of measuring variant impact.


Disability pension despite vocational rehabilitation? A study from six social insurance offices of a county.
Many long-term sick-listed individuals move from vocational rehabilitation to pension, rather than reaching the goal of return to work. There is thus reason to consider whether rehabilitation resources are being used optimally. Individuals receiving disability pensions are consuming financial and personnel resources at the insurance offices and also consume a large amount of health care. The general objective of the study was to evaluate the proportion of individuals granted vocational rehabilitation but then obtaining temporary or permanent disability pensions. All persons receiving any kind of rehabilitation and attending one of six local national insurance offices in a county in Sweden in 1998 and 1999 were studied. A 2-year follow-up was carried out to assess changes in status among those who had received temporary disability pensions. Of all individuals receiving rehabilitation, 46.2% ended up with a disability pension allowance. In addition, a large portion of the temporary disability pensions was transformed to permanent disability pensions within 2 years. For clients with a temporary disability pension, the rate of resuming work was close to nil. Among rehabilitation measures, investigation showed the lowest figures of work resumption while job training showed the best outcome in this respect. The study concluded that a large portion of the financial and personnel resources allocated by the national insurance offices to rehabilitation resulted in disability pensions.


Vancomycin in neonatal sepsis: predictive performance of a Chinese neonatal population pharmacokinetic model and clinical efficacy evaluation.
In the neonatal population, individual calculation and adjustment of vancomycin (VCM) doses has been recommended based on population pharmacokinetics (PPK) methods. Our previous study established a Chinese neonatal VCM PPK model. The main goal of this study was to evaluate the predictive performance of this PPK model for VCM trough concentration. The data on neonatal severe infection patients treated with VCM were retrospectively collected. The predictive performance of this PPK model was expressed using mean prediction error (MPE), mean absolute prediction error (MAPE), sensitivity and specificity. Linear regression analysis was used to compare predicted and measured VCM concentrations. We drew the receiver operating characteristic (ROC) curve to evaluate the predictive efficacy of the ratio of area under the concentration-time curve over 24 hours to minimum inhibitory concentration (AUC0-24/MIC) and trough concentration for clinical efficacy. A total of 40 neonates with Gram-positive bacterial sepsis were included. After VCM treatment, 32 (80%) neonates were clinically cured. Eight cases were a clinical failure: the trough concentrations and AUC0-24 were lower than that of the clinical cure patients (8.70±4.30 vs 14.30±4.50 mg/L, p=0.003; 404.30±122.80 vs 515.40±131.70, p=0.037). The measured and predicted trough concentration were 11.16 (5.96, 16.53) mg/L and 10.13 (6.61, 15.73) mg/L, respectively. The MPE and MAPE were 4.62% and 13.26% (5.30%, 25.88%), respectively. The proportion of MAPE <30% in the adjusted regimen was higher than the initial regimen (89.66% vs 65.00%, p=0.039). Predictions of sensitivity and specificity by this PPK model were 88.24% and 94.29%, respectively. The coefficients of determination of linear regression analysis were 0.9171 and 0.9009 for the initial and adjusted regimen, respectively. The AUC0-24 was correlated with the trough concentration (r=0.587, p<0.001). The ROC curve indicated that the optimal cut-off points for predicting clinical efficacy were AUC0-24/MIC >425.47 and trough concentration >9.45 mg/L. This PPK model has good predictive performance in Chinese neonatal patients. Both AUC0-24/MIC and trough concentration can predict the clinical efficacy of antibacterial treatment.


Tuning Hot Carrier Cooling Dynamics by Dielectric Confinement in Two-Dimensional Hybrid Perovskite Crystals.
Hot carrier (HC) cooling is a critical photophysical process that significantly influences the optoelectronic performance of hybrid perovskite-based devices. The hot carrier extraction at the device interface is very challenging because of its ultrashort lifetime. Here, ultrafast transient reflectance spectroscopy measurements and time-domain ab initio calculations show how the dielectric constant of the organic spacers can control and slow the HC cooling dynamics in single-crystal 2D Ruddlesden-Popper hybrid perovskites. We find that (EA)2PbI4 (EA = HOC2H4NH3+) that correspond to a high dielectric constant organic spacer has a longer HC cooling time compared to that of (AP)2PbI4 (AP = HOC3H6NH3+) and (PEA)2PbI4 (PEA = C6H5C2H4NH3+). The slow HC relaxation process in the former case can be ascribed to a stronger screening of the Coulomb interactions, a small nonradiative internal conversion within the conduction bands, as well as a weak electron-phonon coupling. Our findings provide a strategy to prolong the hot carrier cooling time in low-dimensional hybrid perovskite materials by using organic spacers with reduced dielectric confinement.


Sexual dimorphism in Australopithecus afarensis was similar to that of modern humans.
The substantial fossil record for Australopithecus afarensis includes both an adult partial skeleton [Afar Locality (A.L.) 288-1, "Lucy"] and a large simultaneous death assemblage (A.L. 333). Here we optimize data derived from both to more accurately estimate skeletal size dimorphism. Postcranial ratios derived from A.L. 288-1 enable a significant increase in sample size compared with previous studies. Extensive simulations using modern humans, chimpanzees, and gorillas confirm that this technique is accurate and that skeletal size dimorphism in A. afarensis was most similar to that of contemporary Homo sapiens. These data eliminate some apparent discrepancies between the canine and skeletal size dimorphism in hominoids, imply that the species was not characterized by substantial sexual bimaturation, and greatly increase the probability that the reproductive strategy of A. afarensis was principally monogamy.


Using microscopy to assess chorion structural integrity and parasitoid oviposition sites on stink bug (Hemiptera: Pentatomidae) eggs.
Previous efficacy studies found that many insecticides used by growers could be having an adverse effect on egg parasitoids (Telenomus podisi) developing in the eggs of the brown stink bug (Euschistus servus), while unhatched stink bugs experienced lower levels of mortality. One plausible explanation for this was that insecticides might enter parasitized eggs more readily via oviposition wounds. Parasitized E. servus eggs, as well as nonparasitized stink bug (Acrosternum hilare, E. servus, Murgantia histrionica, and Podisus maculiventris) eggs, were examined using electron microscopy. Egg response to perforation by a tungsten probe served as a control. Microscopy images depicted the chorion surface as characterized by a matrix of ridges and micropylar processes in a ring around the margin of the operculum. Observations of oviposition sites showed a "scab" formed where the ovipositor penetrated the chorion, and at sites penetrated by the probe. These formations appeared to be the result of fluids from inside the egg leaking out, drying, and hardening after oviposition or probe perforation, suggesting that the response was not due to substances secreted by the parasitoid. Further, no open wounds or holes were seen to increase the possibility of insecticides entering parasitized eggs.


[Neurons that encode sound direction].
In the auditory system, the inner ear breaks down complex signals into their spectral components, and encodes the amplitude and phase of each. In order to infer sound direction in space, a computation on each frequency component of the sound must be performed. Space specific neurons in the owl s inferior colliculus respond only to sounds coming from a particular direction and represent the results of this computation. The interaural time difference (ITD) and interaural level difference (ILD define the auditory space for the owl and are processed in separate neural pathways. The parallel pathways that process these cues merge in the external nucleus of the inferior colliculus where the space specific neurons are selective to combinations of ITD and ILD. How do inputs from the two sources interact to produce combination selectivity to ITD ILD pairs? A multiplication of postsynaptic potentials tuned to ITD and ILD can account for the subthreshold responses of these neurons to ITD ILD pairs. Examples of multiplication by neurons or neural circuits are scarce, but many computational models assume the existence of this basic operation. The owl s auditory system uses such operation to create a 2 dimensional map of auditory space. The map of space in the owl s auditory system shows important similarities with representations of space in the cerebral cortex and other sensory systems. In encoding space or other stimulus features, individual neurons appear to possess analogous functional properties related to the synthesis of high order receptive fields.


Lateral superior olive function in congenital deafness.
The development of cochlear implants for the treatment of patients with profound hearing loss has advanced considerably in the last few decades, particularly in the field of speech comprehension. However, attempts to provide not only sound decoding but also spatial hearing are limited by our understanding of circuit adaptations in the absence of auditory input. Here we investigate the lateral superior olive (LSO), a nucleus involved in interaural level difference (ILD) processing in the auditory brainstem using a mouse model of congenital deafness (the dn/dn mouse). An electrophysiological investigation of principal neurons of the LSO from the dn/dn mouse reveals a higher than normal proportion of single spiking (SS) neurons, and an increase in the hyperpolarisation-activated I(h) current. However, inhibitory glycinergic input to the LSO appears to develop normally both pre and postsynaptically in dn/dn mice despite the absence of auditory nerve activity. In combination with previous electrophysiological findings from the dn/dn mouse, we also compile a simple Hodgkin and Huxley circuit model in order to investigate possible computational deficits in ILD processing resulting from congenital hearing loss. We find that the predominance of SS neurons in the dn/dn LSO may compensate for upstream modifications and help to maintain a functioning ILD circuit in the dn/dn mouse. This could have clinical repercussions on the development of stimulation paradigms for spatial hearing with cochlear implants.


Cytoplasmic control of premature activation of a secreted protease zymogen: deletion of staphostatin B (SspC) in Staphylococcus aureus 8325-4 yields a profound pleiotropic phenotype.
The cytoplasmic protein SspC of Staphylococcus aureus, referred to as staphostatin B, is a very specific, tightly binding inhibitor of the secreted protease staphopain B (SspB). SspC is hypothesized to protect intracellular proteins against proteolytic damage by prematurely folded and activated staphopain B (M. Rzychon, A. Sabat, K. Kosowska, J. Potempa, and A. Dubin, Mol. Microbiol. 49:1051-1066, 2003). Here we provide evidence that elimination of intracellular staphopain B activity is indeed the function of SspC. An isogenic sspC mutant of S. aureus 8325-4 exhibits a wide range of striking pleiotropic alterations in phenotype, which distinguish it from the parent. These changes include a defect in growth, a less structured peptidoglycan layer within the cell envelope, severely decreased autolytic activity, resistance to lysis by S. aureus phages, extensively diminished sensitivity to lysis by lysostaphin, the ability to form a biofilm, and a total lack of extracellular proteins secreted into the growth media. The same phenotype was also engineered by introduction of sspB into an 8325-4 sspBC mutant. In contrast, sspC inactivation in the SH1000 strain did not yield any significant changes in the mutant phenotype, apparently due to strongly reduced expression of sspB in the sigma B-positive background. The exact pathway by which these diverse aberrations are exerted in 8325-4 is unknown, but it is apparent that a very small amount of staphopain B (less than 20 ng per 200 microg of cell proteins) is sufficient to bring about these widespread changes. It is proposed that the effects observed are modulated through the proteolytic degradation of several cytoplasmic proteins within cells lacking the inhibitor. Seemingly, some of these proteins may play a role in protein secretion; hence, their proteolytic inactivation by SspB has pleiotropic effects on the SspC-deficient mutant.


Functional evidence for multiple gamma-aminobutyric acidB receptor subtypes in the rat cerebral cortex.
The aim of this work was the identification of pharmacologically distinct subtypes of gamma-aminobutyric acidB (GABAB) receptors in the central nervous system. Inasmuch as GABAB receptors are often sited on axon terminals where they mediate inhibition of transmitter release, we chose as models the GABAB receptors mediating inhibition of release of 1) endogenous GABA; 2) endogenous glutamate; and 3) somatostatin-like immunoreactivity (SRIF-LI). The experimental set up consisted of rat cerebrocortical synaptosomes depolarized in superfusion with 12 or 15 mM KCl. Endogenous GABA and glutamate were measured by high-performance liquid chromatography and SRIF-LI by radioimmunoassay. The selective GABAB receptor agonist (-)-baclofen inhibited in a concentration-dependent manner the K(+)-evoked release of GABA, glutamate and SRIF-Ll with similar potencies and efficacies [EC50 values, 1.1-1.5 microM; maximal inhibition, 45-50% at about 10 microM (-)-baclofen]. The GABAB receptor antagonist phaclofen concentration-dependently reduced the effects of (-)-baclofen on the release of GABA and SRIF-Ll but not on the release of glutamate, where it was ineffective up to 1000 microM. The rank order of potency (Ki values are shown in parentheses) are: SRIF-Ll (7.8 microM); GABA (10.4 microM); and glutamate (greater than 115 microM). The novel GABAB receptor antagonist 3-aminopropyl(diethoxymethyl) phosphinic acid (CGP 35348) displayed a different pattern on the three release systems examined (Ki values are shown in parentheses): SRIF-Ll (0.38 microM); glutamate (0.48 microM); and endogenous GABA (greater than 115 microM).(ABSTRACT TRUNCATED AT 250 WORDS)


Analysis of cyclic dynamics for networks of linear threshold neurons.
The network of neurons with linear threshold (LT) transfer functions is a prominent model to emulate the behavior of cortical neurons. The analysis of dynamic properties for LT networks has attracted growing interest, such as multistability and boundedness. However, not much is known about how the connection strength and external inputs are related to oscillatory behaviors. Periodic oscillation is an important characteristic that relates to nondivergence, which shows that the network is still bounded although unstable modes exist. By concentrating on a general parameterized two-cell network, theoretical results for geometrical properties and existence of periodic orbits are presented. Although it is restricted to two-dimensional systems, the analysis can provide a useful contribution to analyze cyclic dynamics of some specific LT networks of high dimension. As an application, it is extended to an important class of biologically motivated networks of large scale: the winner-take-all model using local excitation and global inhibition.


Shifting care from hospital to community, a strategy to integrate care in Singapore: process evaluation of implementation fidelity.
Accessibility to efficient and person-centered healthcare delivery drives healthcare transformation in many countries. In Singapore, specialist outpatient clinics (SOCs) are commonly congested due to increasing demands for chronic care. To improve this situation, the National University Health System (NUHS) Regional Health System (RHS) started an integrated care initiative,the Right-Site Care (RSC) program in 2014. Through collaborations between SOCs at the National University Hospital and primary and community care (PCC) clinics in the western region of the county, the program was designed to facilitate timely discharge and appropriate transition of patients, who no longer required specialist care, to the community. The aim of this study was to evaluate the implementation fidelity of the NUHS RHS RSC program using the modified Conceptual Framework for Implementation Fidelity (CFIF), at three distinct levels; providers, organizational, and system levels to explain outcomes of the program and to inform further development of (similar) programs. A convergent parallel mixed methods study using the realist evaluation approach was used. Data were collected between 2016 and 2018 through non-participatory observations, reviews of medical records and program database, together with semi-structured interviews with healthcare providers. Triangulation of data streams was applied guided by the modified CFIF. Our findings showed four out of six program components were implemented with low level of fidelity, and 9112 suitable patients were referred to the program while 3032 (33.3%) declined to be enrolled. Moderating factors found to influence fidelity included: (i) complexity of program, (ii) evolving providers' responsiveness, (iii) facilitation through synergistic partnership, training of PCC providers by specialists and supportive structures: care coordinators, guiding protocols, shared electronic medical record and shared pharmacy, (iv) lack of organization reinforcement, and (v) mismatch between program goals, healthcare financing and providers' reimbursement. Functional integration alone is insufficient for a successful right-site care program implementation. Improvement in relationships between providers, organizations, and patients are also warranted for further development of the program.


Valuing life and evaluating suffering in infants with life-limiting illness.
In this paper, we explore three separate questions that are relevant to assessing the prudential value of life in infants with severe life-limiting illness. First, what is the value or disvalue of a short life? Is it in the interests of a child to save her life if she will nevertheless die in infancy or very early childhood? Second, how does profound cognitive impairment affect the balance of positives and negatives in a child's future life? Third, if the life of a child with life-limiting illness is prolonged, how much suffering will she experience and can any of it be alleviated? Is there a risk that negative experiences for such a child (suffering) will remain despite the provision of palliative care? We argue that both the subjective and objective components of well-being for children could be greatly reduced if they are anticipated to have a short life that is affected by profound cognitive impairment. This does not mean that their overall well-being will be negative, but rather that there may be a higher risk of negative overall well-being if they are expected to experience pain, discomfort, or distress. Furthermore, we point to some of the practical limitations of therapies aimed at relieving suffering, such that there is a risk that suffering will go partially or completely unrelieved. Taken together, these considerations imply that some life-prolonging treatments are not in the best interests of infants with severe life-limiting illness.


Optimising the timing of whooping cough immunisation in mums (OpTIMUM) through investigating pertussis vaccination in pregnancy: an open-label, equivalence, randomised controlled trial.
Pertussis vaccination in pregnancy is recommended in many countries to provide protection to young infants. The best timing for this vaccination is uncertain. In the UK, vaccination is recommended between 16 weeks and 32 weeks of gestation. In this trial we aimed to investigate the equivalence of three time periods for pertussis vaccination in pregnancy. In this open-label, equivalence, randomised controlled trial to investigate equivalence of different time windows for pertussis vaccination in pregnancy, participants were randomly assigned (1:1:1 ratio) to receive a pertussis-containing vaccine (Boostrix-inactivated poliovirus vaccine) in one of three gestational age groups, comprising group 1 (≤23 weeks + 6 days), group 2 (24-27 weeks + 6 days), and group 3 (28-31 weeks + 6 days) using a computer-generated randomisation list. The primary outcome was concentration of pertussis-specific antibodies in the infant born at term at birth. Maternal blood sampling was done before and 2 weeks after vaccination and at delivery, together with a cord sample, and an infant sample was collected at least 4 weeks after primary vaccination. Reactogenicity was assessed for 7 days after vaccination. This trial was registered with ClinicalTrials.gov (NCT03908164). Between May 7, 2019, and Feb 13, 2020, of 1010 women assessed for eligibility, 364 women were recruited and 351 received the intervention (120 in group 1, 119 in group 2, and 112 in group 3). Equivalence of time periods was demonstrated for anti-pertussis toxin and anti-pertactin IgG concentrations. The cord blood geometric mean concentrations of anti-filamentous haemagglutinin IgG were higher with increasing gestational age at vaccination, such that for infants in group 1 (≤23 weeks + 6 days), equivalence to group 3 (28-31 weeks + 6 days) was not shown. Reported rates of fever were similar between study groups. Pertussis vaccination at three different time intervals in pregnancy resulted in equivalent concentrations of IgG antibodies in infants against two of the three pertussis antigens assessed. Overall, these findings support recommendations to vaccinate any time between 16 weeks and 32 weeks of gestation. The Thrasher Research Fund and the National Immunisation Schedule Evaluation Consortium through the National Institute for Health and Care Research policy research programme.


Optogenetic inhibition of the caudal substantia nigra inflates behavioral responding to uncertain threat and safety.
Defensive responding is adaptive when it approximates current threat, but maladaptive when it exceeds current threat. Here we asked if the substantia nigra, a region consistently implicated in reward, is necessary to show appropriate levels of defensive responding in Pavlovian fear discrimination. Rats received bilateral transduction of the caudal substantia nigra with halorhodopsin or a control fluorophore, and bilateral ferrule implants. Rats then behaviorally discriminated cues predicting unique foot shock probabilities (danger, p =1; uncertainty, p =0.25; and safety, p =0). Green-light illumination (532 nm) during cue presentation inflated defensive responding of halorhodopsin rats - measured by suppression of reward seeking - to uncertainty and safety beyond control levels. Green-light illumination outside of cue presentation had no impact on halorhodopsin or control rat responding. The results reveal caudal substantia nigra cue activity is necessary to inhibit defensive responding to non-threatening and uncertain threat cues.


Arthrogenic quadriceps inhibition and rehabilitation of patients with extensive traumatic knee injuries.
1. The relationship between joint damage, quadriceps weakness and arthrogenic muscle inhibition was investigated in eight patients who had sustained extensive traumatic knee injury. Isometric and isokinetic quadriceps and hamstring voluntary strength, and quadriceps arthrogenic muscle inhibition during isometric contractions, were measured before and after 4 weeks (approximately 100 h) of intensive rehabilitation. 2. Compared with the uninjured leg, before rehabilitation the injured leg had larger amounts of quadriceps arthrogenic muscle inhibition (P < 0.025), quadriceps (P < 0.0001) and hamstring (P < 0.0001) weakness and severe functional joint instability. There was a negative correlation between the amount of arthrogenic muscle inhibition and quadriceps voluntary contraction force (P < 0.025). 3. After rehabilitation in the injured leg there were small hamstring strength increases (P < 0.05-0.025), but no overall significant quadricep strength increase. Arthrogenic muscle inhibition was statistically unchanged. Severe functional joint instability was still reported by all patients. 4. Previous studies have shown that minimal joint damage evokes relatively less arthrogenic muscle inhibition that does not impede rehabilitation. These data indicate that greater joint damage is associated with greater arthrogenic muscle inhibition, quadriceps weakness and joint instability. Furthermore, intensive rehabilitation had little affect on either quadriceps arthrogenic muscle inhibition or atrophy.


Regression trees for predicting mortality in patients with cardiovascular disease: what improvement is achieved by using ensemble-based methods?
In biomedical research, the logistic regression model is the most commonly used method for predicting the probability of a binary outcome. While many clinical researchers have expressed an enthusiasm for regression trees, this method may have limited accuracy for predicting health outcomes. We aimed to evaluate the improvement that is achieved by using ensemble-based methods, including bootstrap aggregation (bagging) of regression trees, random forests, and boosted regression trees. We analyzed 30-day mortality in two large cohorts of patients hospitalized with either acute myocardial infarction (N = 16,230) or congestive heart failure (N = 15,848) in two distinct eras (1999-2001 and 2004-2005). We found that both the in-sample and out-of-sample prediction of ensemble methods offered substantial improvement in predicting cardiovascular mortality compared to conventional regression trees. However, conventional logistic regression models that incorporated restricted cubic smoothing splines had even better performance. We conclude that ensemble methods from the data mining and machine learning literature increase the predictive performance of regression trees, but may not lead to clear advantages over conventional logistic regression models for predicting short-term mortality in population-based samples of subjects with cardiovascular disease.


Hospital restructuring: does it adversely affect care and outcomes?
The past decade has witnessed pronounced changes in the organization of U.S. hospitals, many the direct result of restructuring and reengineering initiative intended to decrease costs and increase productivity. Little is known about how these initiatives have affected clinical care and patient outcomes. Using data from a variety of sources, the authors describe initiatives that hospitals undertook over this period, indicate how staffing changed relative to the case-mix of patients receiving care, and examine changes in nursing practice environments over the period from 1996 to 1998. The authors found that apparent increases in nurse-to-patient ratios may be deceiving and that increases in patient acuity and nurses' responsibilities may have increased the workload of nurses in hospitals in ways, when coupled with a deteriorating practice environment, may adversely affect patient outcomes.