An analysis of the Kozak consensus in retinal genes and its relevance to gene therapy.
The classic Kozak consensus is a critical genetic element included in gene therapy transgenes to encourage the translation of the therapeutic coding sequence. Despite optimizations of other transgene elements, the Kozak consensus has not yet been considered for potential tissue-specific sequence refinement. We screened the -9 to -1 region relative to the AUG start codon of retina-specific genes to identify whether a Kozak consensus that is different from the classic sequence may be more appropriate for inclusion in gene therapy transgenes that treat inherited retinal disease. Sequences for 135 genes known to cause nonsyndromic inherited retinal disease were extracted from the NCBI database, and the -9 to -1 nucleotides were compared. This panel was then refined to 75 genes with specific retinal functions, for which the -9 to -1 nucleotides were placed in front of a GFP transcript sequence and RNAfold predictions performed. These were compared with a GFP sequence with the classic Kozak consensus (GCCGCCACC), and sequences from retinal genes with minimum free energy (MFE) predictions greater than the reference sequence were selected to generate an optimized Kozak consensus sequence. The original Kozak consensus and the refined retina Kozak consensus were placed upstream of the Renilla luciferase coding sequence, which were used to transfect retinoblastoma cell lines Y-79 and WERI-RB-1 and HEK 293T/17 cells. The nucleotide frequencies of the original panel of genes were determined to be comparable to the classic Kozak consensus. RNAfold analysis of a GFP transcript with the classic Kozak sequence in the 5' untranslated region (UTR) generated an MFE prediction of -503.3 kcal/mol. RNAfold analysis was then performed with a GFP transcript containing each -9 to -1 Kozak sequence of 75 retinal genes. Thirty-eight of the 75 genes provided a greater MFE value than -503.3 kcal/mol and exhibited an absence of stable secondary structures before the AUG codon. The -9 to -1 nucleotide frequencies of these genes identified a Kozak consensus of ACCGAGACC, differing from the classic Kozak consensus at positions -9, -5, and -4. Applying this sequence to the GFP transcript increased the MFE prediction to -500.1 kcal/mol. The newly identified retina Kozak sequence was also applied to Renilla luciferase plus the REP1 and RPGR transcripts used in current clinical trials. In all examples, the predicted transcript MFE score increased when compared with the current transcript sequences containing classic Kozak consensus sequences. In vitro transfections identified a 7%-9% increase in Renilla activity when incorporating the optimized Kozak sequence. The Kozak consensus is a critical element of eukaryotic genes; therefore, it is a required feature of gene therapy transgenes. To date, the classic sequence of GCCRCC (-6 to -1) has typically been incorporated in gene therapy transgenes, but the analysis described here suggests that, for vectors targeting the retina, using a Kozak consensus derived from retinal genes can provide increased expression of the target product.


Clostridium difficile Infection Among US Emergency Department Patients With Diarrhea and No Vomiting.
The incidence of Clostridium difficile infection has increased and has been observed among persons from the community who have not been exposed to antibiotics or health care settings. Our aims are to determine prevalence of C difficile infection among emergency department (ED) patients with diarrhea and the prevalence among patients without traditional risk factors. We conducted a prospective observational study of patients aged 2 years or older with diarrhea (≥3 episodes/24 hours) and no vomiting in 10 US EDs (2010 to 2013). We confirmed C difficile infection by positive stool culture result and toxin assay. C difficile infection risk factors were antibiotic use or overnight health care stay in the previous 3 months or previous C difficile infection. We typed strains with pulsed-field gel electrophoresis. Of 422 participants, median age was 46 years (range 2 to 94 years), with median illness duration of 3.0 days and 43.4% having greater than or equal to 10 episodes of diarrhea during the previous 24 hours. At least one risk factor for C difficile infection was present in 40.8% of participants; 25.9% were receiving antibiotics, 26.9% had health care stay within the previous 3 months, and 3.3% had previous C difficile infection. Forty-three participants (10.2%) had C difficile infection; among these, 24 (55.8%) received antibiotics and 19 (44.2%) had health care exposure; 17 of 43 (39.5%) lacked any risk factor. Among participants without risk factors, C difficile infection prevalence was 6.9%. The most commonly identified North American pulsed-field gel electrophoresis (NAP) strains were NAP type 1 (23.3%) and NAP type 4 (16.3%). Among mostly adults presenting to US EDs with diarrhea and no vomiting, C difficile infection accounted for approximately 10%. More than one third of patients with C difficile infection lacked traditional risk factors for the disease. Among participants without traditional risk factors, prevalence of C difficile infection was approximately 7%.


Arterial substitutes in peripheral vascular surgery: a review.
A large proportion of general vascular surgery involves diagnosis and operative treatment of disorders of the arterial system. To a great extent, advances in the operative care of patients with arterial disease have been dependent on the development of clinically satisfactory arterial substitutes. Currently, based on extensive basic research and clinical experience, prosthetic grafts are preferred as substitutes for larger caliber arteries, while autogenous veins provide the best results as substitutes for smaller, lower flow vessels in the extremities. In this review, we examine the arterial substitutes that have been and are currently important in the clinical practice of general vascular surgery. From this material, it should be apparent to the reader that the perfect arterial substitute does not exist and all, when implanted in humans, are associated with well-recognized short and long term complications.


How imprinting centres work.
Imprinted genes tend to be clustered in the genome. Most of these clusters have been found to be under the control of discrete DNA elements called imprinting centres (ICs) which are normally differentially methylated in the germline. ICs can regulate imprinted expression and epigenetic marks at many genes in the region, even those which lie several megabases away. Some of the molecular and cellular mechanisms by which ICs control other genes and regulatory regions in the cluster are becoming clear. One involves the insulation of genes on one side of the IC from enhancers on the other, mediated by the insulator protein CTCF and higher-order chromatin interactions. Another mechanism may involve non-coding RNAs that originate from the IC, targeting histone modifications to the surrounding genes. Given that several imprinting clusters contain CTCF dependent insulators and/or non-coding RNAs, it is likely that one or both of these two mechanisms regulate imprinting at many loci. Both mechanisms involve a variety of epigenetic marks including DNA methylation and histone modifications but the hierarchy of and interactions between these modifications are not yet understood. The challenge now is to establish a chain of developmental events beginning with differential methylation of an IC in the germline and ending with imprinting of many genes, often in a lineage dependent manner.


Structures of recombinant native and E202Q mutant human acetylcholinesterase complexed with the snake-venom toxin fasciculin-II.
Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a 'three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 A resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 A resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.


Airway management after maxillectomy: routine tracheostomy is unnecessary.
There is a paucity of data to guide the optimal management of the airway in patients after maxillectomy. The decision on whether a concomitant tracheostomy is needed is often dictated by the surgeon's training and experience. We reviewed our experience with maxillectomy to assess the need for tracheostomy in postoperative airway management. Retrospective analysis at a university hospital. We identified 121 patients who underwent 130 maxillectomies between October 1990 and September 2001. Twenty-four of these were total (all six walls removed), 45 were subtotal (two or more walls removed), and 61 were limited (only one wall removed). Reconstruction ranged from none to microvascular free flap, with split-thickness skin graft being the most common reconstructive option. Only 10 tracheostomies (7.7%) were performed at the time of maxillectomy. These included four tracheostomies in patients who underwent bulky flap reconstruction, two tracheostomies in patients who underwent both flap reconstruction and mandibulectomy, one tracheostomy in a patient who underwent mandibulectomy, one tracheostomy in a patient with mucormycosis in anticipation of prolonged ventilatory support postoperatively, and two tracheostomies at the surgeons' discretion because of concern for upper airway edema. Among the 111 patients who underwent 120 maxillectomies without concomitant tracheostomy, 1 patient (0.9%), a 74 year-old man with oxygen-dependent chronic obstructive pulmonary disease, required repeat intubation on day 3 and again on day 10 after the surgery, because of respiratory failure; fiberoptic examination confirmed the absence of upper airway compromise. The routine performance of tracheostomy in patients undergoing maxillectomy is unnecessary. Selective use of tracheostomy may be indicated in situations in which mandibulectomy or bulky flap reconstruction is performed or a concern for postoperative oropharyngeal airway obstruction because of edema or packing exists.


Structure and dynamics of multiple cationic vectors-siRNA complexation by all-atomic molecular dynamics simulations.
Understanding the molecular mechanism of gene condensation is a key component to rationalizing gene delivery phenomena, including functional properties such as the stability of the gene-vector complex and the intracellular release of the gene. In this work, we adopt an atomistic molecular dynamics simulation approach to study the complexation of short strand duplex RNA with four cationic carrier systems of varying charge and surface topology at different charge ratios. At lower charge ratios, polymers bind quite effectively to siRNA, while at high charge ratios, the complexes are saturated and there are free polymers that are unable to associate with RNA. We also observed reduced fluctuations in RNA structures when complexed with multiple polymers in solution as compared to both free siRNA in water and the single polymer complexes. These novel simulations provide a much better understanding of key mechanistic aspects of gene-polycation complexation and thereby advance progress toward rational design of nonviral gene delivery systems.


Evaluation of the effects of erythropoietin and interleukin-6 in rats submitted to acute spinal cord injury.
To evaluate the effects of interleukin-6 (IL-6) and erythropoietin (EPO) in experimental acute spinal cord injury (SCI) in rats. Using standardized equipment, namely, a New York University (NYU) Impactor, a SCI was produced in 50 Wistar rats using a 10-g weight drop from a 12.5-mm height. The rats were divided into the following 5 groups of 10 animals each: "Group EPO", treated with erythropoietin only; "Group EPO + IL-6", treated with both substances; "Group IL-6", receiving IL-6 administration only; "Group Placebo", receiving a placebo solution; and "Group Sham", submitted to an incomplete procedure (only laminectomy, without SCI). All drugs and the placebo solution were administered intraperitoneally for three weeks. The animals were followed up for 42 days. Functional motor recovery was monitored by the Basso, Beattie, and Bresnahan (BBB) scale on days 2, 7, 14, 21, 28, 35 and 42. Motor-evoked potential tests were performed on the 42nd day. Histological analysis was performed after euthanasia. The group receiving EPO exhibited superior functional motor results on the BBB scale. IL-6 administration alone was not superior to the placebo treatment, and the IL-6 combination with EPO yielded worse results than did EPO alone. Using EPO after acute SCI in rats yielded benefits in functional recovery. The combination of EPO and IL-6 showed benefits, but with inferior results compared to those of isolated EPO; moreover, isolated use of IL-6 resulted in no benefit.


Fluoroscopic video to identify aberrant lumbar motion.
A prospective, case-control design. To develop a kinematic model that characterizes frequently observed movement patterns in patients with low back pain (LBP). Understanding arthrokinematics of lumbar motion in those with LBP may provide further understanding of this condition. Digital fluoroscopic video (DFV) was used to quantify the magnitude and rate of attainment of sagittal plane intersegmental angular and linear displacement from 20 individuals with LBP and 20 healthy control subjects during lumbar flexion and extension. Three fellowship-trained spine surgeons subsequently qualitatively analyzed the DFVs to determine normality of movement. Final classification was based on agreement between their symptom and motion status (11 with LBP and aberrant motion and 14 healthy controls without aberrant motion). Independent t tests, receiver operator characteristic curves, and accuracy statistics were calculated to determine the most parsimonious set of kinematic variables able to distinguish patients with LBP. Eight kinematic variables had a positive likelihood ratio > or = 2.5 and entered the model. Six of the variables described a disruption in the rate of attainment of angular or linear displacement during midrange postures. When 4 or more of these variables were present, the positive likelihood ratio was 14.0 (confidence interval 3.2-78.5), resulting in accurately identifying 96% of participants. DFV was useful for discriminating between individuals with and without LBP based on kinematic parameters. Disruptions in how the motion occurred during midrange motions were more diagnostic for LBP than range of motion variables. Cross validation of the model is required.


Auxin-induced degradation dynamics set the pace for lateral root development.
Auxin elicits diverse cell behaviors through a simple nuclear signaling pathway initiated by degradation of Aux/IAA co-repressors. Our previous work revealed that members of the large Arabidopsis Aux/IAA family exhibit a range of degradation rates in synthetic contexts. However, it remained an unresolved issue whether differences in Aux/IAA turnover rates played a significant role in plant responses to auxin. Here, we use the well-established model of lateral root development to directly test the hypothesis that the rate of auxin-induced Aux/IAA turnover sets the pace for auxin-regulated developmental events. We did this by generating transgenic plants expressing degradation rate variants of IAA14, a crucial determinant of lateral root initiation. Progression through the well-established stages of lateral root development was strongly correlated with the engineered rates of IAA14 turnover, leading to the conclusion that Aux/IAAs are auxin-initiated timers that synchronize developmental transitions.


Does transcranial direct current stimulation to prefrontal cortex affect mood and emotional memory retrieval in healthy individuals?
Studies using transcranial direct current stimulation (tDCS) of prefrontal cortex to improve symptoms of depression have had mixed results. We examined whether using tDCS to change the balance of activity between left and right dorsolateral prefrontal cortex (DLPFC) can alter mood and memory retrieval of emotional material in healthy volunteers. Participants memorised emotional images, then tDCS was applied bilaterally to DLPFC while they performed a stimulus-response compatibility task. Participants were then presented with a set of images for memory retrieval. Questionnaires to examine mood and motivational state were administered at the beginning and end of each session. Exploratory data analyses showed that the polarity of tDCS to DLPFC influenced performance on a stimulus-response compatibility task and this effect was dependent on participants' prior motivational state. However, tDCS polarity had no effect on the speed or accuracy of memory retrieval of emotional images and did not influence positive or negative affect. These findings suggest that the balance of activity between left and right DLPFC does not play a critical role in the mood state of healthy individuals. We suggest that the efficacy of prefrontal tDCS depends on the initial activation state of neurons and future work should take this into account.


Vitronectin enhances internalization of crocidolite asbestos by rabbit pleural mesothelial cells via the integrin alpha v beta 5.
The mechanism by which pleural mesothelial cells, the likely progenitor cells of asbestos-induced mesothelioma, recognize and internalize crocidolite asbestos is unknown. Because incubation of asbestos fibers with serum increases their association with cells, we asked whether a protein coat on asbestos increased internalization of fibers via specific cellular receptors. Coating crocidolite with citronectin, but not with fibronectin or other proteins, increased fiber internalization by rabbit pleural mesothelial cells, as measured by a new technique using fluorescence confocal microscopy. Receptors for vitronectin, alpha v beta 3 and alpha v beta 5, were identified on mesothelial cells. Inhibiting vitronectin receptors by plating cells on a vitronectin substrate or incubating cells with excess soluble vitronectin reduced internalization of vitronectin-coated crocidolite. Inhibition of alpha v beta 5, but not alpha v beta 3, with blocking antibodies similarly reduced internalization. In addition, alpha v beta 5, but not alpha v beta 3, showed immunocytochemical colocalization with fibers. Of biologic relevance, coating crocidolite with serum also increased internalization via alpha v beta 5, an effect dependent on the vitronectin in serum. We conclude that pleural mesothelial cells recognize and internalize vitronectin- and serum-coated asbestos via the integrin alpha v beta 5. Since integrins initiate some of the same signaling pathways as does asbestos, our findings may provide insights into the mechanisms of asbestos-induced biologic effects.


A simple method of mouse lung intubation.
A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.


THE FORMATION OF MACROCYTES AND MICROCYTES FROM RED CORPUSCLES IN HANGING DROP PREPARATIONS.
IN HANGING DROP PREPARATIONS OF NORMAL BLOOD FROM VARIOUS SPECIES OF ANIMALS INCLUDING MAN, THE FOLLOWING PROCESSES WERE DIRECTLY OBSERVED FOR THE FIRST TIME: 1. The process of fusion between two red corpuscles so as to form a round or slightly oval macrocyte with normal hemoglobin content, a diameter of 10micro+/- and no delle. These macrocytes appear to be slightly thicker in the centre than at the periphery. No thickness measurements could be made. 2. The process of fusion between two microcytes each 5micro+/- in diameter, so as to form a red corpuscle of approximately normal size but with dense hemoglobin and no delle. 3. The process of fusion between a red corpuscle and a microcyte, forming a macrocyte slightly larger than a normal red corpuscle and exhibiting a normal hemoglobin tint but no delle. 4. The process of microcyte formation from red corpuscles was seen occurring in four different ways: (a) By the intermittent compression of a single red corpuscle by the pull of three or more fibrin threads attached to the corpuscle. (b) By the steady, continuous compression of a single red corpuscle by the pull of two polar fibrin threads, (c) By intermittent rises of intracorpuscular pressure in a red corpuscle due to trauma, (d) By avulsion and retraction or contraction of the hemoglobin stroma from the intact surface layer of a red corpuscle. Thickness measurements in single microcytes were made in rabbit blood. 5. The process of a typical microcyte with dense hemoglobin and no delle changing back to a fairly large red corpuscle with practically normal hemoglobin tint and with a delle. 6. Macrocytes were seen expelling their hemoglobin in a fraction of a second and turning into shadows which were often paler than the background, but without any decrease in the visible surface area or change in the contour. 7. Extensibility and retractibility (or perhaps relaxation and contraction) may be exhibited by the corpuscle as a whole, by local sections of both surface layer and stroma, or retractility (contractility (?)) may be shown by the hemoglobin stroma alone. On the basis of observational evidence it is inferred that: 1. The surface layer of a red corpuscle is probably a condensed stroma rather than an anatomically defined membrane. 2. A pigment, probably hemoglobin, is present in the surface layer. 3. The surface layer of red corpuscles becomes rigid before or during spontaneous hemolysis. 4. Hemoglobin exists in an undissolved form in the intact red corpuscle. 5. The transverse diameter of microcytes (5micro+/-) represents the maximal shortening of which this diameter is capable in normal red corpuscles of man, rabbit and guinea pig under the conditions studied. 6. Fusing red corpuscle are destroyed by repeated washings with Ringer solution. 7. In stagnating blood there is no increase in fusion forms, nor in the production of microcytes.


Light treatment for seasonal Winter depression in African-American vs Caucasian outpatients.
To compare adherence, response, and remission with light treatment in African-American and Caucasian patients with Seasonal Affective Disorder. Seventy-eight study participants, age range 18-64 (51 African-Americans and 27 Caucasians) recruited from the Greater Baltimore Metropolitan area, with diagnoses of recurrent mood disorder with seasonal pattern, and confirmed by a Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders-IV, were enrolled in an open label study of daily bright light treatment. The trial lasted 6 wk with flexible dosing of light starting with 10000 lux bright light for 60 min daily in the morning. At the end of six weeks there were 65 completers. Three patients had Bipolar II disorder and the remainder had Major depressive disorder. Outcome measures were remission (score ≤ 8) and response (50% reduction) in symptoms on the Structured Interview Guide for the Hamilton Rating Scale for Depression (SIGH-SAD) as well as symptomatic improvement on SIGH-SAD and Beck Depression Inventory-II. Adherence was measured using participant daily log. Participant groups were compared using t-tests, chi square, linear and logistic regressions. The study did not find any significant group difference between African-Americans and their Caucasian counterparts in adherence with light treatment as well as in symptomatic improvement. While symptomatic improvement and rate of treatment response were not different between the two groups, African-Americans, after adjustment for age, gender and adherence, achieved a significantly lower remission rate (African-Americans 46.3%; Caucasians 75%; P = 0.02). This is the first study of light treatment in African-Americans, continuing our previous work reporting a similar frequency but a lower awareness of SAD and its treatment in African-Americans. Similar rates of adherence, symptomatic improvement and treatment response suggest that light treatment is a feasible, acceptable, and beneficial treatment for SAD in African-American patients. These results should lead to intensifying education initiatives to increase awareness of SAD and its treatment in African-American communities to increased SAD treatment engagement. In African-American vs Caucasian SAD patients a remission gap was identified, as reported before with antidepressant medications for non-seasonal depression, demanding sustained efforts to investigate and then address its causes.


Wound-associated skin fibrosis: mechanisms and treatments based on modulating the inflammatory response.
Skin fibrosis, in its mildest form, may present only a minor aesthetic problem, but in the most severe cases it can lead to debilitating pathologies of the skin, for example keloid and hypertrophic scars, and systemic sclerosis. In recent years, extensive basic research aimed at understanding the molecular mechanisms underlying fibrosis has revealed an impressive but baffling number of genes, molecules, and cell types that may contribute to this problem. However, one recurring and consistent theme in these studies is that inflammatory cells and their secreted mediators appear to be leading culprits in activating dermal fibroblasts to become fibrotic. This review will first describe the histology of normal versus fibrotic skin, and will also describe the process of wound repair, a primary cause of skin fibrosis. We will then focus on what is currently known about the molecular mechanisms underlying skin fibrosis, with particular attention paid to how inflammation contributes. Finally, current treatment strategies and emerging therapeutic targets will be discussed.


Control of Listeria monocytogenes virulence by 5'-untranslated RNA.
The Gram-positive bacterium Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. Expression of five of these factors has previously been shown to be subjected to post-transcriptional regulation as a result of their long 5'-untranslated region (5'-UTR). We have investigated the presence of 5'-UTRs among the other known virulence genes and genes that encode putatively virulence-associated surface proteins. Our results strongly suggest that L. monocytogenes controls many of its virulence genes by a mechanism that involves the 5'-UTR. These findings further emphasize the importance of post-transcriptional control for L. monocytogenes virulence.


Placebo conditioning and placebo analgesia modulate a common brain network during pain anticipation and perception.
The neural mechanisms whereby placebo conditioning leads to placebo analgesia remain unclear. In this study we aimed to identify the brain structures activated during placebo conditioning and subsequent placebo analgesia. We induced placebo analgesia by associating a sham treatment with pain reduction and used fMRI to measure brain activity associated with three stages of the placebo response: before, during and after the sham treatment, while participants anticipated and experienced brief laser pain. In the control session participants were explicitly told that the treatment was inactive. The sham treatment group reported a significant reduction in pain rating (p=0.012). Anticipatory brain activity was modulated during placebo conditioning in a fronto-cingulate network involving the left dorsolateral prefrontal cortex (DLPFC), medial frontal cortex and the anterior mid-cingulate cortex (aMCC). Identical areas were modulated during anticipation in the placebo analgesia phase with the addition of the orbitofrontal cortex (OFC). However, during altered pain experience only aMCC, post-central gyrus and posterior cingulate demonstrated altered activity. The common frontal cortical areas modulated during anticipation in both the placebo conditioning and placebo analgesia phases have previously been implicated in placebo analgesia. Our results suggest that the main effect of placebo arises from the reduction of anticipation of pain during placebo conditioning that is subsequently maintained during placebo analgesia.


Quantitative assessment of cell population diversity in single-cell landscapes.
Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, there is a need for methods to quantitatively and statistically define proportional shifts in cell population structures across datasets, such as expansion or shrinkage or emergence or disappearance of cell populations. Here we present sc-UniFrac, a framework to statistically quantify compositional diversity in cell populations between single-cell transcriptome landscapes. sc-UniFrac enables sensitive and robust quantification in simulated and experimental datasets in terms of both population identity and quantity. We have demonstrated the utility of sc-UniFrac in multiple applications, including assessment of biological and technical replicates, classification of tissue phenotypes and regional specification, identification and definition of altered cell infiltrates in tumorigenesis, and benchmarking batch-correction tools. sc-UniFrac provides a framework for quantifying diversity or alterations in cell populations across conditions and has broad utility for gaining insight into tissue-level perturbations at the single-cell resolution.


Morphine actions on supraoptic oxytocin neurones in anaesthetized rats: tolerance after i.c.v. morphine infusion.
1. The effects of acute i.v. administration of morphine on putative oxytocin neurones of the supraoptic nucleus were studied in urethane-anaesthetized female rats which had been exposed to i.c.v. infusion of morphine (up to 50 micrograms h-1) or vehicle for 5 days. 2. In vehicle-infused rats, i.v. morphine inhibited the spontaneous activity of six out of seven putative oxytocin neurones. Increasing doses of morphine were given, from 1 microgram kg-1 to 5 mg kg-1. The median cumulative threshold dose to produce significant inhibition was 20 micrograms kg-1 (seven cells in six rats); six out of seven cells were inhibited at 161 micrograms kg-1. The highest doses tested inhibited by approximately 90% (excluding one unaffected cell). Inhibition was fully reversed by i.v. naloxone without overshoot, indicating a lack of acute dependence. 3. Injection of morphine i.c.v. inhibited firing at doses that were ineffective by i.v. injection and the effects of i.c.v. morphine were reversed by i.v. naloxone. 4. Acute morphine (500 micrograms kg-1 i.v.) reduced the plasma concentration of oxytocin, measured after 15 min by specific radioimmunoassay, by 34% (n = 14). 5. In lactating rats i.c.v. injection of morphine (1-2 micrograms) inhibited the activity of supraoptic neurones identified as oxytocinergic by their responses to suckling. 6. In seventeen rats infused with i.c.v. morphine the initial firing rate of twenty-eight spontaneously active, non-phasic neurones was significantly less, by 24%, than thirty-four similar cells in control rats, indicating incomplete tolerance to i.c.v. morphine. Morphine (up to 161 micrograms kg-1 given i.v.) inhibited none of nine active non-phasic neurones (P less than 0.01 compared to control rats), but at higher doses inhibited four of nine cells; the overall median threshold cumulative dose (1660 micrograms kg-1) was significantly greater than in vehicle-infused controls, indicating tolerance to i.v. morphine. In contrast with control rats, some cells (5/9) were modestly excited by low doses of morphine. Naloxone (5 mg kg-1 i.v.) produced withdrawal excitation: the firing rate of putative oxytocin neurones increased to approximately 260% of the pre-i.v. morphine value, indicating dependence in mechanisms regulating the firing rate of these neurones. 7. In morphine-infused rats, the basal firing rate of nineteen phasically active, putative vasopressin supraoptic neurones was not different in nineteen phasic cells in controls (6.4 +/- 0.7 vs. 4.2 +/- 0.6 Hz). 8. Thus morphine potently inhibits the firing of magnocellular oxytocin neurones in the female rat, inhibiting oxytocin secretion. Morphine tolerance and dependence develop during i.c.v. infusion of morphine for 5 days. Similar tolerance to and dependence upon endogenous opioids during pregnancy may be important in the preparation of oxytocin neurones for parturition.


Effect of supplemental energy source and frequency on growing calf performance.
Crossbred heifers (n = 120; 265 kg, SD = 37) were fed individually (84 d) to determine the effect of supplement type, concentration, and frequency on intake and performance and to estimate the energy value of dry distillers grains plus solubles (DDGS) in a high-forage diet. Treatments were arranged in a 3 x 2 x 2 factorial, with 3 supplements, 2 concentrations, and 2 frequencies of supplementation. Supplements including dry-rolled corn (DRC), DRC with corn gluten meal (DRC + CGM), and DDGS were fed at 0.21% (LOW) or 0.81% (HIGH) of BW daily and were provided daily (DAILY) or 3 times weekly (ALT). Heifers were fed to consume grass hay (8.7% CP) ad libitum. Individual DMI, diet composition, BW, and ADG were used to calculate energy values for DDGS and DRC. Supplement type, concentration, frequency, and interactions were tested using the MIXED procedure of SAS, with BW included as a covariate. Supplement x concentration interactions for gain (P = 0.01) and G:F (P < 0.01) were detected. At the LOW concentration, heifers supplemented with DDGS gained more and were more efficient (P </= 0.03) than those supplemented with DRC or DRC + CGM. No performance differences were observed (P >/= 0.22) between DDGS and DRC + CGM in HIGH treatments, although both improved (P </= 0.01) gain and G:F relative to DRC. Calculated TDN content of DDGS was 18 to 30% greater than DRC. Gain and G:F were improved (P < 0.01) in heifers fed HIGH vs. LOW. Total intake was greater (P < 0.01) for HIGH than LOW, but LOW heifers consumed more hay (P < 0.01) than HIGH. The DAILY heifers consumed more (P < 0.01) hay and total DM than the ALT heifers. The DAILY heifers gained more (P < 0.01) than ALT, but G:F was not affected (P = 0.85) by supplementation frequency. In a high-forage diet, DDGS has greater energy value than corn.


Attention deficits predict phenotypic outcomes in syndrome-specific and domain-specific ways.
Attentional difficulties, both at home and in the classroom, are reported across a number of neurodevelopmental disorders. However, exactly how attention influences early socio-cognitive learning remains unclear. We addressed this question both concurrently and longitudinally in a cross-syndrome design, with respect to the communicative domain of vocabulary and to the cognitive domain of early literacy, and then extended the analysis to social behavior. Participants were young children (aged 4-9 years at Time 1) with either Williams syndrome (WS, N = 26) or Down syndrome (DS, N = 26) and typically developing controls (N = 103). Children with WS displayed significantly greater attentional deficits (as indexed by teacher report of behavior typical of attention deficit hyperactivity disorder (ADHD) than children with DS, but both groups had greater attentional problems than the controls. Despite their attention differences, children with DS and those with WS were equivalent in their cognitive abilities of reading single words, both at Time 1 and 12 months later, at Time 2, although they differed in their early communicative abilities in terms of vocabulary. Greater ADHD-like behaviors predicted poorer subsequent literacy for children with DS, but not for children with WS, pointing to syndrome-specific attentional constraints on specific aspects of early development. Overall, our findings highlight the need to investigate more precisely whether and, if so, how, syndrome-specific profiles of behavioral difficulties constrain learning and socio-cognitive outcomes across different domains.


Estrogen, estrogen plus progestin therapy, and risk of breast cancer.
Epidemiologic evidence relating use of postmenopausal hormones to risk of breast cancer by nature relies on trends in prescribing practices. Data on the adverse effect of combination estrogen plus progestin used for long durations has only become available over the past decade. Evidence is reviewed relating estrogen alone and estrogen plus progestin to increased risk of breast cancer. Whereas current evidence indicates that longer duration of use increases risk of invasive breast cancer regardless of formulation, the rate of increase in risk is greater for combination estrogen plus progestin therapy. Although data are limited, continuous combined therapy and sequential therapy seem to have comparable impact on breast cancer risk. Combination therapy is more strongly related to lobular breast cancer than is estrogen alone. Unresolved issues remain about dose of estrogen and progestin in relation to risk, and about identification of women for whom short-term use to relieve menopausal symptoms may be safe and effective.


Femoral shaft fracture after hip arthroplasty: a system for classification and treatment.
Twenty-one consecutive cases of femoral shaft fracture after hip arthroplasty treated at the University of Illinois affiliated hospitals were reviewed. Adequate follow-up and radiographs were available for 19 patients. The length of follow-up after fracture ranged from 2 to 13 years, with a mean of 3.1 years. The time from index procedure to fracture averaged 2.6 years, with a range of 10 days to 11 years. The primary femoral stem was cemented in 11 hips and cementless in 8 hips. Six patients were treated nonoperatively and 13 operatively. Three had fracture fixation with retention of a well-fixed prosthesis and 10 had prosthetic revision. Cortical allograft was used in 5 cases. Sixteen of the 19 patients returned to their prefracture level of function and ambulation. The factors important to treatment are fracture stability, implant stability, and adequacy of bone stock. A classification system based on these factors and recommendations for treatment are proposed.


Exploring DRD4 and its interaction with SLC6A3 as possible risk factors for adult ADHD: a meta-analysis in four European populations.
Attention-deficit hyperactivity disorder (ADHD) is a common behavioral disorder affecting about 4-8% of children. ADHD persists into adulthood in around 65% of cases, either as the full condition or in partial remission with persistence of symptoms. Pharmacological, animal and molecular genetic studies support a role for genes of the dopaminergic system in ADHD due to its essential role in motor control, cognition, emotion, and reward. Based on these data, we analyzed two functional polymorphisms within the DRD4 gene (120 bp duplication in the promoter and 48 bp VNTR in exon 3) in a clinical sample of 1,608 adult ADHD patients and 2,352 controls of Caucasian origin from four European countries that had been recruited in the context of the International Multicentre persistent ADHD CollaboraTion (IMpACT). Single-marker analysis of the two polymorphisms did not reveal association with ADHD. In contrast, multiple-marker meta-analysis showed a nominal association (P = 0.02) of the L-4R haplotype (dup120bp-48bpVNTR) with adulthood ADHD, especially with the combined clinical subtype. Since we previously described association between adulthood ADHD and the dopamine transporter SLC6A3 9R-6R haplotype (3'UTR VNTR-intron 8 VNTR) in the same dataset, we further tested for gene × gene interaction between DRD4 and SLC6A3. However, we detected no epistatic effects but our results rather suggest additive effects of the DRD4 risk haplotype and the SLC6A3 gene.


EGFR regulation of epidermal barrier function.
Keratinocyte terminal differentiation is the process that ultimately forms the epidermal barrier that is essential for mammalian survival. This process is controlled, in part, by signal transduction and gene expression mechanisms, and the epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Using microarray analysis of a confluent cell density-induced model of keratinocyte differentiation, we identified 2,676 genes that are regulated by epidermal growth factor (EGF), a ligand of the EGFR. We further discovered, and separately confirmed by functional assays, that EGFR activation abrogates all of the known essential processes of keratinocyte differentiation by 1) decreasing the expression of lipid matrix biosynthetic enzymes, 2) regulating numerous genes forming the cornified envelope, and 3) suppressing the expression of tight junction proteins. In organotypic cultures of skin, EGF acted to impair epidermal barrier integrity, as shown by increased transepidermal water loss. As defective epidermal differentiation and disruption of barrier function are primary features of many human skin diseases, we used bioinformatic analyses to identify genes that are known to be associated with skin diseases. Compared with non-EGF-regulated genes, EGF-regulated genes were significantly enriched for skin disease genes. These results provide a systems-level understanding of the actions of EGFR signaling to inhibit keratinocyte differentiation, providing new insight into the role of EGFR imbalance in skin pathogenesis.


The H3K4me3 histone demethylase Fbxl10 is a regulator of chemokine expression, cellular morphology, and the metabolome of fibroblasts.
Fbxl10 (Jhdm1b/Kdm2b) is a conserved and ubiquitously expressed member of the JHDM (JmjC domain-containing histone demethylase) family. Fbxl10 was implicated in the demethylation of H3K4me3 or H3K36me2 thereby removing active chromatin marks and inhibiting gene transcription. Apart from the JmjC domain, Fbxl10 consists of a CxxC domain, a PHD domain, and an Fbox domain. By purifying the JmjC and the PHD domain of Fbxl10 and using different approaches we were able to characterize the properties of these domains in vitro. Our results suggest that Fbxl10 is rather a H3K4me3 than a H3K36me2 histone demethylase. The PHD domain exerts a dual function in binding H3K4me3 and H3K36me2 and exhibiting E3 ubiquitin ligase activity. We generated mouse embryonic fibroblasts stably overexpressing Fbxl10. These cells reveal an increase in cell size but no changes in proliferation, mitosis, or apoptosis. Using a microarray approach we were able to identify potentially new target genes for Fbxl10 including chemokines, the noncoding RNA Xist, and proteins involved in metabolic processes. Additionally, we found that Fbxl10 is recruited to the promoters of Ccl7, Xist, Crabp2, and RipK3. Promoter occupancy by Fbxl10 was accompanied by reduced levels of H3K4me3 but unchanged levels of H3K36me2. Furthermore, knockdown of Fbxl10 using small interfering RNA approaches showed inverse regulation of Fbxl10 target genes. In summary, our data reveal a regulatory role of Fbxl10 in cell morphology, chemokine expression, and the metabolic control of fibroblasts.


Aging contributes to inflammation in upper extremity tendons and declines in forelimb agility in a rat model of upper extremity overuse.
We sought to determine if tendon inflammatory and histopathological responses increase in aged rats compared to young rats performing a voluntary upper extremity repetitive task, and if these changes are associated with motor declines. Ninety-six female Sprague-Dawley rats were used in the rat model of upper extremity overuse: 67 aged and 29 young adult rats. After a training period of 4 weeks, task rats performed a voluntary high repetition low force (HRLF) handle-pulling task for 2 hrs/day, 3 days/wk for up to 12 weeks. Upper extremity motor function was assessed, as were inflammatory and histomorphological changes in flexor digitorum and supraspinatus tendons. The percentage of successful reaches improved in young adult HRLF rats, but not in aged HRLF rats. Forelimb agility decreased transiently in young adult HRLF rats, but persistently in aged HRLF rats. HRLF task performance for 12 weeks lead to increased IL-1beta and IL-6 in flexor digitorum tendons of aged HRLF rats, compared to aged normal control (NC) as well as young adult HRLF rats. In contrast, TNF-alpha increased more in flexor digitorum tendons of young adult 12-week HRLF rats than in aged HRLF rats. Vascularity and collagen fibril organization were not affected by task performance in flexor digitorum tendons of either age group, although cellularity increased in both. By week 12 of HRLF task performance, vascularity and cellularity increased in the supraspinatus tendons of only aged rats. The increased cellularity was due to increased macrophages and connective tissue growth factor (CTGF)-immunoreactive fibroblasts in the peritendon. In conclusion, aged rat tendons were overall more affected by the HRLF task than young adult tendons, particularly supraspinatus tendons. Greater inflammatory changes in aged HRLF rat tendons were observed, increases associated temporally with decreased forelimb agility and lack of improvement in task success.


Quercetin, a potent suppressor of NF-κB and Smad activation in osteoblasts.
Osteoclasts, the bone resorbing cells of the body, form when osteoclast precursors are exposed to the key osteoclastogenic cytokine receptor activator of NF-κB ligand (RANKL), a process requiring induction of NF-κB signaling. Quercetin is a ubiquitous plant-derived flavonoid with well documented anti-inflammatory properties, in part, a consequence of its capacity to downmodulate the NF-κB signal transduction pathway. Consistent with this mechanism of action quercetin is reported to suppress osteoclastogenesis in vitro and prevent bone loss in ovariectomized mice in vivo. By contrast, the effect of quercetin on osteoblasts, the cells responsible for bone formation, is contradictory with conflicting reports of inhibition as well as stimulation. Given our previous reports that NF-κB antagonists promote osteoblast differentiation and activity, we compared the effects of quercetin on osteoclast and osteoblast differentiation and on NF-κB signal transduction in vitro. As expected, quercetin potently suppressed osteoclastogenesis and NF-κB activation induced by RANKL in osteoclast precursors. However, the same doses of quercetin had no effect on osteoblast mineralization, and failed to significantly alleviate the inhibitory effect of NF-κB-induced by TNFα, even though quercetin potently suppressed NF-κB activation in these cells. This apparent contradiction was explained by the fact that addition to its anti-NF-κB activity, quercetin also potently antagonized both TGFβ and BMP-2-induced Smad activation in osteoblast precursors. Taken together our data suggest that multiple competing actions of quercetin mediate both stimulatory and inhibitory actions on osteoblasts with the final physiological effect likely a function of the net balance between these stimulatory and inhibitory effects.


Quercetin prevent proteoglycan destruction by inhibits matrix metalloproteinase-9, matrix metalloproteinase-13, a disintegrin and metalloproteinase with thrombospondin motifs-5 expressions on osteoarthritis model rats.
Prior study has shown that Ageratum conyzoides L. extract that containing quercetin has been proved to prevent inflammation and proteoglycan degradation by inhibiting tumor necrosis factor-alpha and matrix metalloproteinase (MMP-9) expression. Target of osteoarthritis (OA) treatment was in the synovial joint that requiring a drug delivery system. The aim of this study was to prove the efficacy of quercetin-loaded lecithin-chitosan nanoparticles on the OA model rats by observed its effect on interleukin (IL-1) β, MMP-9, MMP-13, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-5) expressions. In this study, 70 white male Sprague Dawley rats were divided into 14 groups, 7 groups each for destabilization of medial meniscus (DMM) and monoiodoacetate (MIA)-induced OA. After 28 days from induction, SHAM and negative group received gel base topically; positive group received sodium diclofenac gel; three-dose group received each 0.84, 1.68, 3.36 mg/g quercetin-loaded nanoparticles gel; and A. conyzoides L. group received A. conyzoides L. extract gel. Each group gets treatment until day 70, and then, blood sample was collected for serum analysis; knee joint was isolated and subjected to histology samples treatment. Quercetin-loaded nanoparticle gel dose 1 (0.84 mg/g gel), dose 2 (1.68 mg/g gel), dose 3 (3.36 mg/g), and A. conyzoides L. extract gel could decreased the level of IL-1 β, MMP-9, MMP-13, ADAMTS-5, and increasing color intensity significantly on histopathological observations on DMM and MIA-induced OA.


Electronic Health Record Logs Indicate That Physicians Split Time Evenly Between Seeing Patients And Desktop Medicine.
Time spent by physicians is a key resource in health care delivery. This study used data captured by the access time stamp functionality of an electronic health record (EHR) to examine physician work effort. This is a potentially powerful, yet unobtrusive, way to study physicians' use of time. We used data on physicians' time allocation patterns captured by over thirty-one million EHR transactions in the period 2011-14 recorded by 471 primary care physicians, who collectively worked on 765,129 patients' EHRs. Our results suggest that the physicians logged an average of 3.08 hours on office visits and 3.17 hours on desktop medicine each day. Desktop medicine consists of activities such as communicating with patients through a secure patient portal, responding to patients' online requests for prescription refills or medical advice, ordering tests, sending staff messages, and reviewing test results. Over time, log records from physicians showed a decline in the time allocated to face-to-face visits, accompanied by an increase in time allocated to desktop medicine. Staffing and scheduling in the physician's office, as well as provider payment models for primary care practice, should account for these desktop medicine efforts.


Measurement of serum phosphate levels using a mobile sensor.
The measurement of serum phosphate concentration is crucial for patients with advanced chronic kidney disease (CKD) and those on maintenance dialysis, as abnormal phosphate levels may be associated with severe health risks. It is important to monitor serum phosphate levels on a regular basis in these patients; however, such measurements are generally limited to every 0.5-3 months, depending on the severity of CKD. This is due to the fact that serum phosphate measurements can only be performed at regular clinic visits, in addition to cost considerations. Here we present a portable and cost-effective point-of-care device capable of measuring serum phosphate levels using a single drop of blood (<60 μl). This is achieved by integrating a paper-based microfluidic platform with a custom-designed smartphone reader. This mobile sensor was tested on patients undergoing dialysis, where whole blood samples were acquired before starting the hemodialysis and during the three-hour treatment. This sampling during the hemodialysis, under patient consent, allowed us to test blood samples with a wide range of phosphate concentrations, and our results showed a strong correlation with the ground truth laboratory tests performed on the same patient samples (Pearson coefficient r = 0.95 and p < 0.001). Our 3D-printed smartphone attachment weighs about 400 g and costs less than 80 USD, whereas the material cost for the disposable test is <3.5 USD (under low volume manufacturing). This low-cost and easy-to-operate system can be used to measure serum phosphate levels at the point-of-care in about 45 min and can potentially be used on a daily basis by patients at home.


Improving living and dying for people with advanced dementia living in care homes: a realist review of Namaste Care and other multisensory interventions.
Seventy percent of people with advanced dementia live and die in care homes. Multisensory approaches, such as Namaste Care, have been developed to improve the quality of life and dying for people with advanced dementia but little is known about effectiveness or optimum delivery. The aim of this review was to develop an explanatory account of how the Namaste Care intervention might work, on what outcomes, and in what circumstances. This is a realist review involving scoping of the literature and stakeholder interviews to develop theoretical explanations of how interventions might work, systematic searches of the evidence to test and develop the theories, and their validation with a purposive sample of stakeholders. Twenty stakeholders - user/patient representatives, dementia care providers, care home staff, researchers -took part in interviews and/or workshops. We included 85 papers. Eight focused on Namaste Care and the remainder on other types of sensory interventions such as music therapy or massage. We identified three context-mechanism-outcome configurations which together provide an explanatory account of what needs to be in place for Namaste Care to work for people living with advanced dementia. This includes: providing structured access to social and physical stimulation, equipping care home staff to cope effectively with complex behaviours and variable responses, and providing a framework for person-centred care. A key overarching theme concerned the importance of activities that enabled the development of moments of connection for people with advanced dementia. This realist review provides a coherent account of how Namaste Care, and other multisensory interventions might work. It provides practitioners and researchers with a framework to judge the feasibility and likely success of Namaste Care in long term settings. Key for staff and residents is that the intervention triggers feelings of familiarity, reassurance, engagement and connection. This study is registered as PROSPERO CRD42016047512.


Development and validation of a kinematically-driven discrete element model of the patellofemoral joint.
Quantifying the complex loads at the patellofemoral joint (PFJ) is vital to understanding the development of PFJ pain and osteoarthritis. Discrete element analysis (DEA) is a computationally efficient method to estimate cartilage contact stresses with potential application at the PFJ to better understand PFJ mechanics. The current study validated a DEA modeling framework driven by PFJ kinematics to predict experimentally-measured PFJ contact stress distributions. Two cadaveric knee specimens underwent quadriceps muscle [215 N] and joint compression [350 N] forces at ten discrete knee positions representing PFJ positions during early gait while measured PFJ kinematics were used to drive specimen-specific DEA models. DEA-computed contact stress and area were compared to experimentally-measured data. There was good agreement between computed and measured mean and peak stress across the specimens and positions (r = 0.63-0.85). DEA-computed mean stress was within an average of 12% (range: 1-47%) of the experimentally-measured mean stress while DEA-computed peak stress was within an average of 22% (range: 1-40%). Stress magnitudes were within the ranges measured (0.17-1.26 MPa computationally vs 0.12-1.13 MPa experimentally). DEA-computed areas overestimated measured areas (average error = 60%; range: 4-117%) with magnitudes ranging from 139 to 307 mm2 computationally vs 74-194 mm2 experimentally. DEA estimates of the ratio of lateral to medial patellofemoral stress distribution predicted the experimental data well (mean error = 15%) with minimal measurement bias. These results indicate that kinematically-driven DEA models can provide good estimates of relative changes in PFJ contact stress.


Probing the mechanism of the Ugi four-component reaction with charge-tagged reagents by ESI-MS(/MS).
The mechanism of the Ugi four-component reaction has been investigated by electrospray ionization (tandem) mass spectrometry using charge-tagged reagents (a carboxylic acid or an amine) to favour detection. Key intermediates were transferred directly via ESI(+) from the reaction solution to the gas phase and characterized by MS measurements and MS/MS collision induced dissociation. The Mumm rearrangement (final step) was also investigated by both travelling wave ion mobility mass spectrometry and DFT calculations. The data seem to consolidate the amazingly selective mechanism of this intricate four-component reaction.


Hypofractionated versus conventionally fractionated radiotherapy for patients with prostate cancer (HYPRO): acute toxicity results from a randomised non-inferiority phase 3 trial.
In 2007, we began the randomised phase 3 multicentre HYPRO trial to investigate the effect of hypofractionated radiotherapy compared with conventionally fractionated radiotherapy on relapse-free survival in patients with prostate cancer. Here, we examine whether patients experience differences in acute gastrointestinal and genitourinary adverse effects. In this randomised non-inferiority phase 3 trial, done in seven radiotherapy centres in the Netherlands, we enrolled intermediate-risk or high-risk patients aged between 44 and 85 years with histologically confirmed stage T1b-T4 NX-0MX-0 prostate cancer, a PSA concentration of 60 ng/mL or lower, and WHO performance status of 0-2. A web-based application was used to randomly assign (1:1) patients to receive either standard fractionation with 39 fractions of 2 Gy in 8 weeks (five fractions per week) or hypofractionation with 19 fractions of 3·4 Gy in 6·5 weeks (three fractions per week). Randomisation was done with minimisation procedure, stratified by treatment centre and risk group. The primary endpoint is 5-year relapse-free survival. Here we report data for the acute toxicity outcomes: the cumulative incidence of grade 2 or worse acute and late genitourinary and gastrointestinal toxicity. Non-inferiority of hypofractionation was tested separately for genitourinary and gastrointestinal acute toxic effects, with a null hypothesis that cumulative incidences of each type of adverse event were not more than 8% higher in the hypofractionation group than in the standard fractionation group. We scored acute genitourinary and gastrointestinal toxic effects according to RTOG-EORTC criteria from both case report forms and patients' self-assessment questionnaires, at baseline, twice during radiotherapy, and 3 months after completion of radiotherapy. Analyses were done in the intention-to-treat population. Patient recruitment has been completed. This study is registered with www.controlled-trials.com, number ISRCTN85138529. Between March 19, 2007, and Dec 3, 2010, 820 patients were randomly assigned to treatment with standard fractionation (n=410) or hypofractionation (n=410). 3 months after radiotherapy, 73 (22%) patients in the standard fractionation group and 75 (23%) patients in the hypofractionation group reported grade 2 or worse genitourinary toxicity; grade 2 or worse gastrointestinal toxicity was noted in 43 (13%) patients in the standard fractionation group and in 42 (13%) in the hypofractionation group. Grade 4 acute genitourinary toxicity was reported for two patients, one (<1%) in each group. No grade 4 acute gastrointestinal toxicities were observed. We noted no significant difference in cumulative incidence by 120 days after radiotherapy of grade 2 or worse acute genitourinary toxicity (57·8% [95% CI 52·9-62·7] in the standard fractionation group vs 60·5% (55·8-65·3) in the hypofractionation group; difference 2·7%, 90% CI -2·99 to 8·48; odds ratio [OR] 1·12, 95% CI 0·84-1·49; p=0·43). The cumulative incidence of grade 2 or worse acute gastrointestinal toxicity by 120 days after radiotherapy was higher in patients given hypofractionation (31·2% [95% CI 26·6-35·8] in the standard fractionation group vs 42·0% [37·2-46·9] in the hypofractionation group; difference 10·8%, 90% CI 5·25-16·43; OR 1·6; p=0·0015; non-inferiority not confirmed). Hypofractionated radiotherapy was not non-inferior to standard fractionated radiotherapy in terms of acute genitourinary and gastrointestinal toxicity for men with intermediate-risk and high-risk prostate cancer. In fact, the cumulative incidence of grade 2 or worse acute gastrointestinal toxicity was significantly higher in patients given hypofractionation than in those given standard fractionated radiotherapy. Patients remain in follow-up for efficacy endpoints. The Dutch Cancer Society.


General lack of global dosage compensation in ZZ/ZW systems? Broadening the perspective with RNA-seq.
Species with heteromorphic sex chromosomes face the challenge of large-scale imbalance in gene dose. Microarray-based studies in several independent male heterogametic XX/XY systems suggest that dosage compensation mechanisms are in place to mitigate the detrimental effects of gene dose differences. However, recent genomic research on female heterogametic ZZ/ZW systems has generated surprising results. In two bird species and one lepidopteran no evidence for a global dosage compensating mechanism has been found. The recent advent of massively parallel RNA sequencing now opens up the possibility to gauge the generality of this observation with a broader phylogenetic sampling. It further allows assessing the validity of microarray-based inference on dosage compensation with a novel technology. We here exemplify this approach using massively parallel sequencing on barcoded individuals of a bird species, the European crow (Corvus corone), where previously no genetic resources were available. Testing for Z-linkage with quantitative PCR (qPCR,) we first establish that orthology with distantly related species (chicken, zebra finch) can be used as a good predictor for chromosomal affiliation of a gene. We then use a digital measure of gene expression (RNA-seq) on brain transcriptome and confirm a global lack of dosage compensation on the Z chromosome. RNA-seq estimates of male-to-female (m:f) expression difference on the Z compare well to previous microarray-based estimates in birds and lepidopterans. The data further lends support that an up-regulation of female Z-linked genes conveys partial compensation and suggest a relationship between sex-bias and absolute expression level of a gene. Correlation of sex-biased gene expression on the Z chromosome across all three bird species further suggests that the degree of compensation has been partly conserved across 100 million years of avian evolution. This work demonstrates that the study of dosage compensation has become amenable to species where previously no genetic resources were available. Massively parallele transcriptome sequencing allows re-assessing the degree of dosage compensation with a novel tool in well-studies species and, in addition, gain valuable insights into the generality of mechanisms across independent taxonomic group for both the XX/XY and ZZ/ZW system.


Telomere tethering at the nuclear periphery is essential for efficient DNA double strand break repair in subtelomeric region.
In the yeast Saccharomyces cerevisiae that lacks lamins, the nuclear pore complex (NPC) has been proposed to serve a role in chromatin organization. Here, using fluorescence microscopy in living cells, we show that nuclear pore proteins of the Nup84 core complex, Nup84p, Nup145Cp, Nup120p, and Nup133p, serve to anchor telomere XI-L at the nuclear periphery. The integrity of this complex is shown to be required for repression of a URA3 gene inserted in the subtelomeric region of this chromosome end. Furthermore, altering the integrity of this complex decreases the efficiency of repair of a DNA double-strand break (DSB) only when it is generated in the subtelomeric region, even though the repair machinery is functional. These effects are specific to the Nup84 complex. Our observations thus confirm and extend the role played by the NPC, through the Nup84 complex, in the functional organization of chromatin. They also indicate that anchoring of telomeres is essential for efficient repair of DSBs occurring therein and is important for preserving genome integrity.


Insights into hepatopancreatic functions for nutrition metabolism and ovarian development in the crab Portunus trituberculatus: gene discovery in the comparative transcriptome of different hepatopancreas stages.
The crustacean hepatopancreas has different functions including absorption, storage of nutrients and vitellogenesis during growth, and ovarian development. However, genetic information on the biological functions of the crustacean hepatopancreas during such processes is limited. The swimming crab, Portunus trituberculatus, is a commercially important species for both aquaculture and fisheries in the Asia-Pacific region. This study compared the transcriptome in the hepatopancreas of female P. trituberculatus during the growth and ovarian maturation stages by 454 high-throughput pyrosequencing and bioinformatics. The goal was to discover genes in the hepatopancreas involved in food digestion, nutrition metabolism and ovarian development, and to identify patterns of gene expression during growth and ovarian maturation. Our transcriptome produced 303,450 reads with an average length of 351 bp, and the high quality reads were assembled into 21,635 contigs and 31,844 singlets. Based on BLASTP searches of the deduced protein sequences, there were 7,762 contigs and 4,098 singlets with functional annotation. Further analysis revealed 33,427 unigenes with ORFs, including 17,388 contigs and 16,039 singlets in the hepatopancreas, while only 7,954 unigenes (5,691 contigs and 2,263 singlets) with the predicted protein sequences were annotated with biological functions. The deduced protein sequences were assigned to 3,734 GO terms, 25 COG categories and 294 specific pathways. Furthermore, there were 14, 534, and 22 identified unigenes involved in food digestion, nutrition metabolism and ovarian development, respectively. 212 differentially expressed genes (DEGs) were found between the growth and endogenous stage of the hepatopancreas, while there were 382 DEGs between the endogenous and exogenous stage hepatopancreas. Our results not only enhance the understanding of crustacean hepatopancreatic functions during growth and ovarian development, but also represent a basis for further research on new genes and functional genomics of P. trituberculatus or closely related species.


Fine mapping seronegative and seropositive rheumatoid arthritis to shared and distinct HLA alleles by adjusting for the effects of heterogeneity.
Despite progress in defining human leukocyte antigen (HLA) alleles for anti-citrullinated-protein-autoantibody-positive (ACPA(+)) rheumatoid arthritis (RA), identifying HLA alleles for ACPA-negative (ACPA(-)) RA has been challenging because of clinical heterogeneity within clinical cohorts. We imputed 8,961 classical HLA alleles, amino acids, and SNPs from Immunochip data in a discovery set of 2,406 ACPA(-) RA case and 13,930 control individuals. We developed a statistical approach to identify and adjust for clinical heterogeneity within ACPA(-) RA and observed independent associations for serine and leucine at position 11 in HLA-DRβ1 (p = 1.4 × 10(-13), odds ratio [OR] = 1.30) and for aspartate at position 9 in HLA-B (p = 2.7 × 10(-12), OR = 1.39) within the peptide binding grooves. These amino acid positions induced associations at HLA-DRB1(∗)03 (encoding serine at 11) and HLA-B(∗)08 (encoding aspartate at 9). We validated these findings in an independent set of 427 ACPA(-) case subjects, carefully phenotyped with a highly sensitive ACPA assay, and 1,691 control subjects (HLA-DRβ1 Ser11+Leu11: p = 5.8 × 10(-4), OR = 1.28; HLA-B Asp9: p = 2.6 × 10(-3), OR = 1.34). Although both amino acid sites drove risk of ACPA(+) and ACPA(-) disease, the effects of individual residues at HLA-DRβ1 position 11 were distinct (p < 2.9 × 10(-107)). We also identified an association with ACPA(+) RA at HLA-A position 77 (p = 2.7 × 10(-8), OR = 0.85) in 7,279 ACPA(+) RA case and 15,870 control subjects. These results contribute to mounting evidence that ACPA(+) and ACPA(-) RA are genetically distinct and potentially have separate autoantigens contributing to pathogenesis. We expect that our approach might have broad applications in analyzing clinical conditions with heterogeneity at both major histocompatibility complex (MHC) and non-MHC regions.


Conservation and diversity of influenza A H1N1 HLA-restricted T cell epitope candidates for epitope-based vaccines.
The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated. HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54) peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-gamma ELISpot assay. The 54 peptides were compared to the 2007-2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes. Seventeen (17) T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus.


Is the universal population Hepatitis C virus screening a cost-effective strategy? A systematic review of the economic evidence.
Efficient strategies are needed in order to achieve the objective of the WHO of eradicating Hepatitis C virus (HCV). Hepatitis C infection can be eliminated by a combination of direct acting antiviral (DAA). The problem is that many individuals remain undiagnosed. The objective is to conduct a systematic review of the evidence on economic evaluations that analyze the screening of HCV followed by treatment with DAAs. Eleven databases were performed in a 2015-2018-systematic review. Inclusion criteria were economic evaluations that included incremental cost-effectiveness ratio (ICER) in terms of cost per life year gained or quality-adjusted life year. A total of 843 references were screened. Sixteen papers/posters meet the inclusion criteria. Ten of them included a general population screening. Other populations included were baby-boomer, people who inject drugs, prisoners or immigrants. Comparator was "standard of care", other high-risk populations or no-screening. Most of the studies are based on Markov model simulations and they mostly adopted a healthcare payer´s perspective. ICER for general population screening plus treatment versus high-risk populations or versus routinely performed screening showed to be below the accepted willingness to pay thresholds in most studies and therefore screening plus DAAs strategy is highly cost-effective. This systematic review shows that screening programmes followed by DAAs treatment is cost-effective not only for high risk population but for general population too. Because today HCV can be easily cured and its long-term consequences avoided, a universal HCV screening plus DAAs therapies should be the recommended strategy to achieve the WHO objectives for HCV eradication by 2030.


Gold nanoclusters with bright near-infrared photoluminescence.
The increase in nonradiative pathways with decreasing emission energy reduces the luminescence quantum yield (QY) of near-infrared photoluminescent (NIR PL) metal nanoclusters. Efficient surface ligand chemistry can significantly improve the luminescence QY of NIR PL metal nanoclusters. In contrast to the widely reported but modestly effective thiolate ligand-to-metal core charge transfer, we show that metal-to-ligand charge transfer (MLCT) can be used to greatly enhance the luminescence QY of NIR PL gold nanoclusters (AuNCs). We synthesized water-soluble and colloidally stable NIR PL AuNCs with unprecedentedly high QY (∼25%) upon introduction of triphenylphosphonium moieties into the surface capping layer. By using a combination of spectroscopic and theoretical methods, we provide evidence for gold core-to-ligand charge transfer occurring in AuNCs. We envision that this work can stimulate the development of these unusually bright AuNCs for promising optoelectronic, bioimaging, and other applications.


The impact on work-related stress of mental health teams following team-based learning on clinical risk management.
Risk management is viewed as a systematic process based on multiprofessional and multi-agency decision-making. A learning pack was developed as part of a team-based learning project aiming to encourage and develop collaborative working practice. This brought different professionals and agencies working in mental health together to learn. There is little doubt that mental health practice is a source of stress for practitioners. Apart from the stress associated with managing 'risky' situations, risk management is also a relatively new concept. This can increase stress around ability to cope, both on an individual practitioner level and in teams. This article reports the impact that the learning pack had on team members' stress, specifically work-related stress. A range of scales were used to measure change in stress and results demonstrated reduced work-related pressure in a number of areas following the learning. The implications for team learning in relation to clinical risk management are discussed in light of the findings.


Cervical human papillomavirus and HIV infection in women of child-bearing age in Abidjan, Côte d'Ivoire, 2010.
We sought to document the association of Human immunodeficiency Virus (HIV) infection and immunodeficiency with oncogenic Human Papillomavirus (HPV) infection in women with no cervical neoplastic lesions identified through a cervical cancer screening programme in Côte d'Ivoire. A consecutive sample of women stratified on their HIV status and attending the national blood donor clinic or the closest HIV clinic was recruited during a cervical cancer screening programme based on the visual inspection. Diagnosis of HPV infection and genotype identification were based on the Linear Array; HPV test. A total of 445 (254 HIV-positive and 191 HIV-negative) women were included. The prevalence of oncogenic HPV infection was 53.9% (95% confidence interval (CI) 47.9-59.9) in HIV-positive women and 33.7% (95% CI 27.1-40.3) in HIV-negative women (odds ratio (OR)=2.3 (95% CI 1.5-3.3)). In multivariate analysis, HIV-positive women with a CD4 count <200 cells mm(3) or between 200 and 499 cells mm(3) were more likely to harbour an oncogenic HPV compared with women with a CD4 count ≥500 cells mm(3) with OR of 2.8 (95% CI 1.1-8.1) and 1.7 (95% CI 1.0-2.9), respectively. A high prevalence of oncogenic HPV was found in women with no cervical neoplastic lesions, especially in HIV-positive women. Despite antiretroviral use, immunodeficiency was a main determinant of the presence of oncogenic HPV.


Phase I study of depsipeptide in pediatric patients with refractory solid tumors: a Children's Oncology Group report.
To determine the maximum-tolerated dose (MTD), dose-limiting toxicities (DLT), pharmacokinetic profile, and pharmacodynamics of the histone deacetylase inhibitor, depsipeptide, in children with refractory or recurrent solid tumors. Depsipeptide was administered as a 4-hour infusion weekly for 3 consecutive weeks every 28 days at dose levels of 10 mg/m2, 13 mg/m2, 17 mg/m2, and 22 mg/m2. Pharmacokinetics and histone acetylation studies were performed in the first course. The levels of H3 histone and acetyl-H3 histone were evaluated in peripheral blood mononuclear cells (PBMC) using immunofluorescence techniques. There were 24 patients, and 18 who were assessable were enrolled. DLTs included reversible, asymptomatic T-wave inversions, without any associated changes in troponin levels or evidence of ventricular dysfunction, in the inferior leads in two patients at 22 mg/m2 and in the lateral leads in one patient at 13 mg/m2 (n = 1), and transient asymptomatic sick sinus syndrome and hypocalcemia in one patient at 17 mg/m2. At the MTD (17 mg/m2), the median depsipeptide clearance was 6.8 L/h/m(2) with an area under the plasma depsipeptide concentration-time curve from 0 to infinity of 2,414 ng/mL/h, similar to adults. Accumulation of acetylated H3 histones was seen in all patients in a dose independent manner, with maximal accumulation at a median of 4 hours, (range, 0 hours to 20 hours) after the end of the infusion. No objective tumor responses were observed. Depsipeptide is well tolerated in children with recurrent or refractory solid tumors when administered weekly for 3 consecutive weeks every 28 days and inhibits histone deacetylase activity in PBMC in a dose-independent manner. The recommended phase II dose in children with solid tumors is 17 mg/m2.


Encountering the Body at the Site of the Suicide: A Population-Based Survey in Sweden.
Encountering the body of a child who died by suicide at the site of death is believed to be especially harmful for bereaved parents. We investigated the association between encountering the body at the site of the suicide and psychological distress in 666 suicide-bereaved parents. Parents who had encountered their child's body at the site of the suicide (n = 147) did not have a higher risk of nightmares (relative risk [RR] 0.95, 95% confidence interval [CI] 0.67-1.35), intrusive memories (RR 0.97, 95% CI 0.84-1.13), avoidance of thoughts (RR 0.97, 95% CI 0.74-1.27), avoidance of places or things (RR 0.91, 95% CI 0.66-1.25), anxiety (RR 0.93, 95% CI 0.64-1.33), or depression (RR 0.94, 95% CI 0.63-1.42) compared with parents who had not encountered the body (n = 512). Our results suggest that losing a child by suicide is sufficiently disastrous by itself to elicit posttraumatic responses or psychiatric morbidity whether or not the parent has encountered the deceased child at the site of death.


First case of canine peritoneal larval cestodosis caused by Mesocestoides lineatus in Germany.
A 13-year-old Dalmatian dog was presented with a history of abdominal enlargement and reduced appetite for several months. Acute clinical signs were anorexia, vomiting and diarrhoea. During exploratory laparotomy, acute intestinal perforation due to a foreign body and peritonitis was diagnosed. In addition, the abdominal cavity was filled with multiple small (0.5 cm), white, cyst-like structures. Histopathology revealed typical cestode structures of the cyst walls but no protoscolices were found. PCR was performed with cestode specific primers of the mitochondrial 12S rDNA. The sequence showed a 99.75% identity with a Mesocestoides lineatus isolate published in the NCBI GenBank. This is the first case of canine peritoneal larval cestodosis (CPLC) in Germany and the first evidence of M. lineatus as causal agent for CPLC.


Azole resistance in Mycobacterium tuberculosis is mediated by the MmpS5-MmpL5 efflux system.
Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. Moreover, the recent isolation of M. tuberculosis strains resistant to both first- and second-line antitubercular drugs (XDR-TB) threatens to make the treatment of this disease extremely difficult and becoming a threat to public health worldwide. Recently, it has been shown that azoles are potent inhibitors of mycobacterial cell growth and have antitubercular activity in mice, thus favoring the hypothesis that these drugs may constitute a novel strategy against tuberculosis disease. To investigate the mechanisms of resistance to azoles in mycobacteria, we isolated and characterized several spontaneous azoles resistant mutants from M. tuberculosis and Mycobacterium bovis BCG. All the analyzed resistant mutants exhibited both increased econazole efflux and increased transcription of mmpS5-mmpL5 genes, encoding a hypothetical efflux system belonging to the resistance-nodulation-division (RND) family of transporters. We found that the up-regulation of mmpS5-mmpL5 genes was linked to mutations either in the Rv0678 gene, hypothesized to be involved in the transcriptional regulation of this efflux system, or in its putative promoter/operator region.


Rearrangements of DNA mediated by terminal transferase.
To assess the involvement of terminal transferase in generating immunoglobulin diversity, the mutagenic potential of this enzyme has been measured. The frequency of single base substitutions during copying of phi X174 DNA by DNA polymerase beta is increased by, at most, 3-fold upon the addition of terminal transferase. However, terminal transferase is highly mutagenic, either alone or with DNA polymerase beta, in a forward mutation assay using M13mp2 DNA. The frequency of complex mutants, as determined by DNA sequence, is increased by greater than 100-fold. These mutants involve the deletion of a variable number of bases initially present in the template sequence and the addition of a sequence of nucleotides rich in guanine residues. Analysis of these mutants suggests an antibody diversity model implicating terminal transferase in the imprecise linkage of variable, joining, and diversity segments during the formation of functional immunoglobulin genes.


Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus.
Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore beta-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors.


Antibiotics for exacerbations of chronic obstructive pulmonary disease.
Many patients with an exacerbation of chronic obstructive pulmonary disease (COPD) are treated with antibiotics. However, the value of antibiotics remains uncertain, as systematic reviews and clinical trials have shown conflicting results. To assess effects of antibiotics on treatment failure as observed between seven days and one month after treatment initiation (primary outcome) for management of acute COPD exacerbations, as well as their effects on other patient-important outcomes (mortality, adverse events, length of hospital stay, time to next exacerbation). We searched the Cochrane Central Register of Controlled Trials (CENTRAL), in the Cochrane Library, MEDLINE, Embase, and other electronically available databases up to 26 September 2018. We sought to find randomised controlled trials (RCTs) including people with acute COPD exacerbations comparing antibiotic therapy and placebo and providing follow-up of at least seven days. Two review authors independently screened references and extracted data from trial reports. We kept the three groups of outpatients, inpatients, and patients admitted to the intensive care unit (ICU) separate for benefit outcomes and mortality because we considered them to be clinically too different to be summarised as a single group. We considered outpatients to have a mild to moderate exacerbation, inpatients to have a severe exacerbation, and ICU patients to have a very severe exacerbation. When authors of primary studies did not report outcomes or study details, we contacted them to request missing data. We calculated pooled risk ratios (RRs) for treatment failure, Peto odds ratios (ORs) for rare events (mortality and adverse events), and mean differences (MDs) for continuous outcomes using random-effects models. We used GRADE to assess the quality of the evidence. The primary outcome was treatment failure as observed between seven days and one month after treatment initiation. We included 19 trials with 2663 participants (11 with outpatients, seven with inpatients, and one with ICU patients).For outpatients (with mild to moderate exacerbations), evidence of low quality suggests that currently available antibiotics statistically significantly reduced the risk for treatment failure between seven days and one month after treatment initiation (RR 0.72, 95% confidence interval (CI) 0.56 to 0.94; I² = 31%; in absolute terms, reduction in treatment failures from 295 to 212 per 1000 treated participants, 95% CI 165 to 277). Studies providing older antibiotics not in use anymore yielded an RR of 0.69 (95% CI 0.53 to 0.90; I² = 31%). Evidence of low quality from one trial in outpatients suggested no effects of antibiotics on mortality (Peto OR 1.27, 95% CI 0.49 to 3.30). One trial reported no effects of antibiotics on re-exacerbations between two and six weeks after treatment initiation. Only one trial (N = 35) reported health-related quality of life but did not show a statistically significant difference between treatment and control groups.Evidence of moderate quality does not show that currently used antibiotics statistically significantly reduced the risk of treatment failure among inpatients with severe exacerbations (i.e. for inpatients excluding ICU patients) (RR 0.65, 95% CI 0.38 to 1.12; I² = 50%), but trial results remain uncertain. In turn, the effect was statistically significant when trials included older antibiotics no longer in clinical use (RR 0.76, 95% CI 0.58 to 1.00; I² = 39%). Evidence of moderate quality from two trials including inpatients shows no beneficial effects of antibiotics on mortality (Peto OR 2.48, 95% CI 0.94 to 6.55). Length of hospital stay (in days) was similar in antibiotic and placebo groups.The only trial with 93 patients admitted to the ICU showed a large and statistically significant effect on treatment failure (RR 0.19, 95% CI 0.08 to 0.45; moderate-quality evidence; in absolute terms, reduction in treatment failures from 565 to 107 per 1000 treated participants, 95% CI 45 to 254). Results of this trial show a statistically significant effect on mortality (Peto OR 0.21, 95% CI 0.06 to 0.72; moderate-quality evidence) and on length of hospital stay (MD -9.60 days, 95% CI -12.84 to -6.36; low-quality evidence).Evidence of moderate quality gathered from trials conducted in all settings shows no statistically significant effect on overall incidence of adverse events (Peto OR 1.20, 95% CI 0.89 to 1.63; moderate-quality evidence) nor on diarrhoea (Peto OR 1.68, 95% CI 0.92 to 3.07; moderate-quality evidence). Researchers have found that antibiotics have some effect on inpatients and outpatients, but these effects are small, and they are inconsistent for some outcomes (treatment failure) and absent for other outcomes (mortality, length of hospital stay). Analyses show a strong beneficial effect of antibiotics among ICU patients. Few data are available on the effects of antibiotics on health-related quality of life or on other patient-reported symptoms, and data show no statistically significant increase in the risk of adverse events with antibiotics compared to placebo. These inconsistent effects call for research into clinical signs and biomarkers that can help identify patients who would benefit from antibiotics, while sparing antibiotics for patients who are unlikely to experience benefit and for whom downsides of antibiotics (side effects, costs, and multi-resistance) should be avoided.


Video assisted right mini-thoracotomy for aortic root replacement.
Aortic root replacement is typically performed through a median sternotomy. The right anterior mini-thoracotomy approach has been shown to decrease hospital length of stay in aortic valve surgery when compared to sternotomy. This approach is rare in ascending aortic surgery due to technical challenges which include exposure and annular suture placement. Automated suturing technology is now available to facilitate the placement of annular sutures. The use of a camera greatly enhances visualization of the aortic root. A right anterior mini-thoracotomy is performed via a 5 cm incision in the right second intercostal space with a camera port placed lateral to the incision. Peripheral arterial and venous cannulation are performed. The aortic cross clamp is placed through a 5 mm incision in the third interspace anterior to mid axillary line. Histidine tryptophan ketoglutarate (HTK) cardioplegia is administered and deep hypothermic circulatory arrest is achieved followed by completion of the distal anastomosis with a 4-0 polypropylene running suture. After aortic leaflet removal, annular and prosthetic sutures are placed with shafted instruments or with automated suturing technology. Coronary button suturing and graft-to-graft anastomoses are performed with shafted instruments. Aortic root procedures can be performed safely through a right anterior mini-thoracotomy. The use of a camera and automated suturing technology may further facilitate this procedure, enabling more surgeons to offer this less invasive approach to patients.


Analysis of substrate specificity and cyclin Y binding of PCTAIRE-1 kinase.
PCTAIRE-1 (cyclin-dependent kinase [CDK] 16) is a highly conserved serine/threonine kinase that belongs to the CDK family of protein kinases. Little is known regarding PCTAIRE-1 regulation and function and no robust assay exists to assess PCTAIRE-1 activity mainly due to a lack of information regarding its preferred consensus motif and the lack of bona fide substrates. We used positional scanning peptide library technology and identified the substrate-specificity requirements of PCTAIRE-1 and subsequently elaborated a peptide substrate termed PCTAIRE-tide. Recombinant PCTAIRE-1 displayed vastly improved enzyme kinetics on PCTAIRE-tide compared to a widely used generic CDK substrate peptide. PCTAIRE-tide also greatly improved detection of endogenous PCTAIRE-1 activity. Similar to other CDKs, PCTAIRE-1 requires a proline residue immediately C-terminal to the phosphoacceptor site (+1) for optimal activity. PCTAIRE-1 has a unique preference for a basic residue at +4, but not at +3 position (a key characteristic for CDKs). We also demonstrate that PCTAIRE-1 binds to a novel cyclin family member, cyclin Y, which increased PCTAIRE-1 activity towards PCTAIRE-tide >100-fold. We hypothesised that cyclin Y binds and activates PCTAIRE-1 in a way similar to which cyclin A2 binds and activates CDK2. Point mutants of cyclin Y predicted to disrupt PCTAIRE-1-cyclin Y binding severely prevented complex formation and activation of PCTAIRE-1. We have identified PCTAIRE-tide as a powerful tool to study the regulation of PCTAIRE-1. Our understanding of the molecular interaction between PCTAIRE-1 and cyclin Y further facilitates future investigation of the functions of PCTAIRE-1 kinase.


Surgical management of airway obstruction in primary tuberculosis in children.
The role of surgery in the management of airway obstruction from lymphobronchial tuberculosis is discussed in the present article. Nine patients were operated on over a 4-year period and are currently presented. The age of the patients ranged between 5 and 28 months and 7 patients were male. Six patients required preoperative ventilation due to respiratory failure and all received standard posterolateral thoracotomies. Partial dissection and enucleation of bulky lymph nodes was performed in all but 1 patient. In that patient, the group of lymph nodes could be removed fully, including the sheath. All patients showed marked improvement and were weaned off the ventilator between 24 and 72 hours postoperatively. Long term follow-up was available in 7 patients and they are all doing well and are free of symptoms. Enucleation of mediastinal lymph nodes obstructing the airways in young patients with lymphobronchial tuberculosis is safe. It successfully relieves obstruction and is devoid of complication providing that incision, evacuation, and curettage of lymph nodes is performed avoiding overzealous dissection.


Triggering the apoptosis of targeted human renal cancer cells by the vibration of anisotropic magnetic particles attached to the cell membrane.
Cancer cells develop resistance to chemotherapy, and the side effects encountered seriously limit the effectiveness of treatments. For these reasons, the search for alternative therapies that target cancer cells without affecting healthy tissues is currently one of the most active areas of research on cancer. The present study focuses on a recently proposed approach for cancer cell destruction based on the targeted triggering of cancer cell spontaneous death through the mechanical vibration of anisotropic magnetic micro/nanoparticles attached to the cell membranes at low frequencies (∼20 Hz) and in weak magnetic fields (∼30 mT). The study was conducted in vitro, on human renal cancer cells with superparamagnetic-like particles. Three types of such particles made of NiFe or magnetite were prepared and characterized (either synthetic antiferromagnetic, vortex or polycrystalline with random grain anisotropy). The triggering of the apoptosis of these cancer cells was demonstrated with NiFe vortex particles and statistically characterized by flow-cytometry studies. The death pathway via apoptosis and not necrosis was identified by the clear observation of caspase activation.