Deceptive measures of progress in the NHS long-term plan for cancer: case-based vs. population-based measures.
The NHS Long Term Plan for cancer aims to increase early-stage diagnoses from 50% to 75% and to have 55,000 more people each year survive their cancer for at least 5 years following diagnosis. The targets measures are flawed and could be met without improving outcomes that really matter to patients. The proportion of early-stage diagnoses could increase, while the number of patients presenting at a late-stage remains the same. More patients could survive their cancer for longer, but lead time and overdiagnosis bias make it impossible to know whether anyone had their life prolonged. The target measures should switch from biased case-based measures to unbiased population-based measures that reflect the key objectives in cancer care: reducing late-stage incidence and mortality.


A photoelectrochemical aptasensor for the sensitive detection of streptomycin based on a TiO2/BiOI/BiOBr heterostructure.
In photoelectrochemical sensor (PEC sensor), sensitivity and selectivity are two essential factors which are determined by photosensitive of materials and identification of elements. Herein, a novel PEC aptamer sensor for streptomycin-specific detection was developed, with which the visible-light-active TiO2/BiOI/BiOBr heterostructure and aptamers were employed as photoactive material and bio-identification elements, separately. The combination of an appropriate amount of TiO2 with BiOI/BiOBr enhanced the photocurrent response, and thus is beneficial to the construction of PEC sensors. In addition, the one-pot synthesis of TiO2/BiOI/BiOBr has the advantage of being environmentally-friendly. Under optimized conditions, the photocurrent response of aptamer/TiO2/BiOI/BiOBr/ITO is linear with SRT concentration from 0.05 to 150 nM, and the detection limit (S/N = 3) is as low as 0.04 nM. This novel PEC sensing strategy provided an ultra-sensitive sensor with high selectivity and stability for SRT detection.


A single pulse of agrin triggers a pathway that acts to cluster acetylcholine receptors.
Agrin triggers signaling mechanisms of high temporal and spatial specificity to achieve phosphorylation, clustering, and stabilization of postsynaptic acetylcholine receptors (AChRs). Agrin transiently activates the kinase MuSK; MuSK activation has largely vanished when AChR clusters appear. Thus, a tyrosine kinase cascade acts downstream from MuSK, as illustrated by the agrin-evoked long-lasting activation of Src family kinases (SFKs) and their requirement for AChR cluster stabilization. We have investigated this cascade and report that pharmacological inhibition of SFKs reduces early but not later agrin-induced phosphorylation of MuSK and AChRs, while inhibition of Abl kinases reduces late phosphorylation. Interestingly, SFK inhibition applied selectively during agrin-induced AChR cluster formation caused rapid cluster dispersal later upon agrin withdrawal. We also report that a single 5-min agrin pulse, followed by extensive washing, triggered long-lasting MuSK and AChR phosphorylation and efficient AChR clustering. Following the pulse, MuSK phosphorylation increased and, beyond a certain level, caused maximal clustering. These data reveal novel temporal aspects of tyrosine kinase action in agrin signaling. First, during AChR cluster formation, SFKs initiate early phosphorylation and an AChR stabilization program that acts much later. Second, a kinase mechanism rapidly activated by agrin acts thereafter autonomously in agrin's absence to further increase MuSK phosphorylation and cluster AChRs.


Impact of a donor source on adult Philadelphia chromosome-negative acute lymphoblastic leukemia: a retrospective analysis from the Adult Acute Lymphoblastic Leukemia Working Group of the Japan Society for Hematopoietic Cell Transplantation.
We aimed to clarify the impact of the donor source of allogeneic stem cell transplantation (allo-SCT) on Philadelphia chromosome-negative acute lymphoblastic leukemia [Ph(-) ALL] with focus on cord blood (CB). We retrospectively analyzed data of 1726 patients who underwent myeloablative allo-SCT for adult Ph(-) ALL. The sources of the allo-SCT were related donors (RD; N = 684), unrelated donors (URD; N = 809), and CB (N = 233). Overall survival (OS) in patients after CB allo-SCT in first complete remission (CR1) was comparable with that after RD or URD allo-SCT (RD: 65%, URD: 64% and CB: 57% at 4 years, P = 0.11). CB was not a significant risk factor for relapse or non-relapse mortality as well as for OS in multivariate analyses. Similarly, the donor source was not a significant risk factor for OS in subsequent CR or non-CR (RD: 47%, URD: 39% and CB: 48% in subsequent CR, P = 0.33; RD: 15%, URD: 21% and CB: 18% in non-CR, P = 0.20 at 4 years). Allo-SCT using CB led to OS similar to those of RD or URD in any disease status. To avoid missing the appropriate timing, CB is a favorable alternative source for adult Ph(-) ALL patients without a suitable RD or URD.


Granular cell tumor of the tongue in a 6-year-old girl--a case report.
Granular cell tumor is a relatively uncommon benign hamartomatous lesion occurring in almost any part of the body. The tongue and the buccal mucosa are common intra oral sites. Granular cell lesions may be found in other diverse sites such as the jaw, skin, gastro intestinal tract and respiratory tract. The histogenesis of the lesion still remains unknown. However, histochemical and ultra structural studies propose the origin of the lesion from schwann cells, striated muscle, mesenchymal cells, histiocytes and epithelial cells. The tumor generally occurs in middle or older aged adults. The lesion is typically seen as an uninflammed asymptomatic mass measuring about two cms in diameter with a yellowish surface coloration. As most of the granular cell tumors are benign, surgical excision of the lesion is the treatment of choice. We describe a case of granular cell tumor of the tongue in a 6 year old girl along with a brief review of literature on granular cell tumors.


The dual role of human dignity in bioethics.
This paper argues that some of the misunderstandings surrounding the meaning and function of the concept of human dignity in bioethics arise from a lack of distinction between two different roles that this notion plays: one as an overarching policy principle, and the other as a moral standard of patient care. While the former is a very general concept which fulfils a foundational and a guiding role of the normative framework governing biomedical issues, the latter reflects a much more concrete and context-specific understanding of the patient as a "person". The importance of dignity as a policy principle will be described by appealing to the distinction between principles and rules as developed by some legal philosophers. The value of dignity as a standard of patient care will be illustrated with the help of concrete examples and by drawing on the taxonomies of dignity proposed by Jonathan Mann and other scholars. The overall scope of the article is to highlight this double and complementary role of human dignity in bioethics.


A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability.
The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally.


Androgen regulation of multidrug resistance-associated protein 4 (MRP4/ABCC4) in prostate cancer.
MRP4/ABCC4 is an ATP-binding cassette transporter expressed in normal prostate. This study aimed to define the pattern of MRP4/ABCC4 expression in normal and malignant prostate tissue and the relationship of MRP4/ABCC4 expression and function in response to androgen signaling. Eighty-four radical prostatectomy specimens from patients with localized prostate cancer (PC) (22 neoadjuvant androgen ablation, AA, 62 no AA), 42 non-cancer and 16 advanced PCs were assessed for MRP4/ABCC4 mRNA/protein expression. The effect of DHT and bicalutamide on LNCaP cells was assessed by immunoblotting. HEK293 cells (+/-MRP4/ABCC4) were assessed for the ability to efflux androgens and anti-androgens. MRP4/ABCC4 mRNA/protein levels were higher in localized PC compared to non-cancer (P = 0.006). MRP4/ABCC4 levels were significantly decreased in PCs treated with AA compared to cancers exposed to normal testosterone levels (P < 0.0001). MRP4/ABCC4 expression in normal human tissues was limited to the prostate and the renal tubules. MRP4/ABCC4 protein levels increased in LNCaP cells after DHT which was partially blocked by bicalutamide. However, DHT did not alter the activation of the MRP4/ABCC4 promotor in luciferase reporter assays and testosterone, DHT, flutamide and hydroxy-flutamide were not substrates for MRP4/ABCC4. Elevated MRP4/ABCC4 expression is found in malignant compared to benign prostate tissue while lower MRP4/ABCC4 expression is seen after AA. Furthermore, MRP4/ABCC4 is upregulated by androgen and downregulated by anti-androgen treatment in vitro potentially through an indirect mode of action. These data strongly suggest that MRP4/ABCC4 is an androgen-regulated gene important in the progression to PC and may be a potential drug target.


Hypersensitivity to metallic implants: pathophysiologic and diagnostic considerations.
Metals are ubiquitous in the surrounding environment and constitute an important class of substances that can act as allergens. Consumer products such as earings, zip, jewellery, cosmetics, paints, dental and body implants as well as endovascular, intracardiac and orthopedic devices are made from metals. Metal anions eluted from various metals are common allergic sensitizers. In Europe nickel, chromium and cobalt induce allergic skin reactions in about 20%, 4%, and 7% while in the United States14%, 4%, and 9% respectively.


A giant protease with potential to substitute for some functions of the proteasome.
An alanyl-alanyl-phenylalanyl-7-amino-4-methylcoumarin-hydrolyzing protease particle copurifying with 26S proteasomes was isolated and identified as tripeptidyl peptidase II (TPPII), a cytosolic subtilisin-like peptidase of unknown function. The particle is larger than the 26S proteasome and has a rod-shaped, dynamic supramolecular structure. TPPII exhibits enhanced activity in proteasome inhibitor-adapted cells and degrades polypeptides by exo- as well as predominantly trypsin-like endoproteolytic cleavage. TPPII may thus participate in extralysosomal polypeptide degradation and may in part account for nonproteasomal epitope generation as postulated for certain major histocompatibility complex class I alleles. In addition, TPPII may be able to substitute for some metabolic functions of the proteasome.


Cytological effects of prefixation treatment.
The effects produced by prefixation treatments on cells in metaphase from 10-day mouse fetuses and from several embryonic stages of the frog were investigated. The technical value of some of these pretreatments is noted. Pretreatment with isotonic solutions (both ionic and non-ionic in the case of the mouse, ionic only in the frog) generally produced a similar effect, viz., chromosomal swelling with little effect on the spindle. A notable exception is provided by frog embryos preceding the neurula stage; spindle disorganization without chromosomal swelling was produced by pretreatment in isotonic modified Niu-Twitty solution, containing no divalent cations. Pretreatment with hypotonic solutions (both ionic and non-ionic in the case of the mouse, ionic only in the frog) generally produced several major effects, viz., despiralization of chromosomes, chromatid separation, and spindle disorganization. The conclusion is drawn from the mouse data that, in order to produce these effects, pretreating solutions must be of low osmotic pressure. Low ionic strength alone (e.g., isotonic sucrose solutions) is not sufficient. As differentiation of frog embryos progressed, pretreatments either of longer duration or with solutions of increasing degrees of hypotonicity were required to produce comparable intensities of the same effects. Many of the effects on metaphases produced by hypotonic pretreatment of frog embryos were reversible by subsequent exposure to isotonic solutions. The significance of results presented here is discussed briefly with respect to some current considerations of the macromolecular structure of chromosomes.


Nucleotide-Free sB-Raf is Preferentially Bound by Hsp90 and Cdc37 In Vitro.
The molecular chaperone Hsp90 and its cofactor Cdc37 are required for the stability of protein kinases in the cellular environment. Upon pharmacological inhibition of Hsp90, the Hsp90-dependent kinases are degraded quickly by the proteasome. Clear physiological evidence for the formation of heterooligomeric complexes between the chaperone system and its kinase clients exist, but the mechanisms of client processing are still enigmatic. Here, we investigate the interaction of the chaperone system with a stabilized fragment of the Hsp90-dependent protein kinase B-Raf (sB-Raf). sB-Raf is aggregation prone at elevated temperatures. We find that nucleotide binding strongly stabilizes the folded state of sB-Raf and suppresses its aggregation. Also, Cdc37 and Hsp90 in combination can suppress sB-Raf aggregation while forming a ternary complex with the kinase. The presence of nucleotides leads to the dissociation of the kinase from the ternary chaperone complex, implying that the stabilization of the kinase by nucleotides reduces its affinity toward the chaperone machinery. Human Cdc37-Hsp90 complexes can bind to kinase, if the NM domain of the chaperone is present. Nematode Cdc37, which does not require the N-terminal Hsp90 domain for binding, can form a ternary complex with the MC construct of Hsp90, which lacks the aggregation propensity of sB-Raf. Like the full-length complex, this interaction is sensitive to ATP binding to sB-Raf. We thus find that the interaction between sB-Raf and the Hsp90 chaperone system is based on contacts with the M domain of Hsp90, which contributes in forming the ternary complex with CeCdc37 as long as the kinase is not stabilized by nucleotide.


Neighborhood socioeconomic disadvantage and body mass index among residentially stable mid-older aged adults: Findings from the HABITAT multilevel longitudinal study.
Despite a body of evidence on the relationship between neighborhood socioeconomic disadvantage and body mass index (BMI), few studies have examined this relationship over time among ageing populations. This study examined associations between level of neighborhood socioeconomic disadvantage and the rate of change in BMI over time. The sample included 11,035 participants aged between 40 and 65years at baseline from the HABITAT study, residing in 200 neighborhoods in Brisbane, Australia. Data were collected biennially over four waves from 2007 to 2013. Self-reported height and weight were used to calculate BMI, while neighborhood disadvantage was measured using a census-based composite index. All models were adjusted for age, education, occupation, and household income. Analyses were conducted using multilevel linear regression models. BMI increased over time at a rate of 0.08kg/m2 (95% CI 0.02, 0.13) and 0.17kg/m2 (95% CI 0.11, 0.29) per wave for men and women respectively. Both men and women residing in the most disadvantaged neighborhoods had a higher average BMI than their counterparts living in the least disadvantaged neighborhoods. There were no evident differences in the rate of BMI change over time by level of neighborhood disadvantage. The findings suggest that by mid-older age, the influence of neighborhood socioeconomic conditions over time on BMI may have already played out. Future research should endeavor to identify the genesis of neighborhood socioeconomic inequalities in BMI, the determinants of these inequalities, and then suitable approaches to intervening.


After-brush rinsing protocols, frequency of toothpaste use: fluoride and other active ingredients.
The intra-oral retention or substantivity of active ingredients in toothpastes is important for their effectiveness, and this is influenced by product-related and user-related factors. Product-related factors include the formulation and the compatibility of active and other agents in the toothpaste and the concentration of the active ingredient. User-related factors include biological aspects such as salivary flow and salivary clearance, and behavioural aspects, such as frequency and duration of brushing, amount of toothpaste used and post-brushing rinsing behaviour. To date, product-related factors have dominated the research agenda for toothpastes, but user-related factors have the potential to significantly enhance or reduce the effectiveness of toothpaste. In this chapter, we will focus on two of the user-related factors that have been most widely studied: (1) frequency of toothbrushing and (2) post-brushing rinsing behaviour. We will then provide an overview of how evidence on these two behaviours has been used to produce guidance both for the profession and for the public, and make suggestions for the future direction of research in this area.


A longitudinal study of urinary phthalate excretion in 58 full-term and 67 preterm infants from birth through 14 months.
Some phthalates have shown antiandrogenic effects in rat offspring. Premature infants may be exposed to high amounts of specific phthalates during hospitalization, and thus are potentially at risk. We evaluated longitudinal phthalate exposure and metabolism in full-term (FT) and preterm (PT) infants. Fifty-eight FT and 67 PT (gestational age, 24.7-36.6 weeks) infants were recruited at birth and followed until 14 months (nine times). Urinary concentrations of metabolites of diethyl phthalate (DEP), dibutyl phthalate isomers (DiBP and DnBP), butylbenzyl phthalate (BBzP), di(2-ethylhexyl) phthalate (DEHP), and diisononyl phthalate (DiNP) were measured in 894 samples. Daily intake and a hazard index for antiandrogenic effects were estimated, and excretion patterns of DEHP and DiNP metabolites were analyzed. Metabolites of BBzP, DiNP, and DEHP were 5-50 times higher at day 7 (D7) and month 1 (M1) in PT than in FT infants. Thereafter, metabolite concentrations were similar between the two groups. The estimated hazard index for combined DiBP, DnBP, BBzP, and DEHP exposures 7 days after birth exceeded the antiandrogenic threshold in > 80% of PT and > 30% of FT infants, and after M2, in 30% of all infants. The excretion pattern of DEHP and DiNP metabolites changed with age. Most PT infants and approximately one-third of healthy FT newborns were exposed to phthalates during early life at a potentially harmful level according to the European Food Safety Authority's recommended limits of daily exposure. Changes in the relative proportions of secondary phthalate metabolites over time were consistent with maturation of infant metabolic pathways during the first year of life. Further research is needed on the health effects of phthalate exposures and the influence of changes in metabolic capacity in neonates and infants.


Percutaneous endovascular treatment of infrainguinal PAOD: Results of the PSI register study in 74 German vascular centers.
The percutaneous infrainguinal stent (PSI) register study aimed to collate all percutaneous endovascular procedures for infrainguinal peripheral arterial occlusive disease (PAOD) conducted in 74 German vascular centers between September and November 2015 (3 months). In order to obtain representative results all consecutive treatment procedures had to be submitted by the participating trial centers. This was a prospective, nonrandomized multicenter study design. All patients suffering from intermittent claudication (IC, Fontaine stage II) or critical limb ischemia (CLI, Fontaine stages III and IV) were included. Trial centers with less than 5 cases reported within the 3‑month trial period or centers that could not ensure the submission of all treated patients were excluded. In the final assessment 2798 treated cases from 74 trial centers were reported of which 65 (87.8 %) centers were under the leadership of a vascular surgeon. Approximately 33 % of the interventions in centers under the leadership of vascular surgeons were conducted by radiologists. Risk factors, especially chronic renal disease, diabetes and cardiac risk factors were significantly different between patients with IC and CLI. Of the patients with Fontaine stage II PAOD 41.3 % had 3 patent crural vessels compared to only 10.8 % of patients with Fontaine stage IV. With respect to peri-interventional complications, percutaneous endovascular treatment of IC was a safe procedure with severe complications in less than 1 % and no fatalities. Only 4.5 % of the procedures were conducted under ambulatory conditions. In the supragenual region self-expanding bare metal stents, standard percutaneous transluminal angioplasty (PTA) and drug-coated balloons were the most frequently used procedures. For interventions below the knee, standard PTA was the most commonly employed treatment. The main aim of the PSI study was to obtain a realistic picture of percutaneous endovascular techniques used to treat suprapopliteal and infrapopliteal PAOD lesions and to describe the treatment procedures used by vascular specialists in Germany. To investigate the change in trends for treatment over time, this study has to be repeated in the future in order to test how quickly the results of randomized studies can be implemented in practice.


Impaired perception of surface tilt in progressive supranuclear palsy.
Progressive supranuclear palsy (PSP) is characterized by early postural instability and backward falls. The mechanisms underlying backward postural instability in PSP are not understood. The aim of this study was to test the hypothesis that postural instability in PSP is a result of dysfunction in the perception of postural verticality. We gathered posturography data on 12 subjects with PSP to compare with 12 subjects with idiopathic Parkinson's Disease (PD) and 12 healthy subjects. Objective tests of postural impairment included: dynamic sensory perception tests of gravity and of surface oscillations, postural responses to surface perturbations, the sensory organization test of postural sway under altered sensory conditions and limits of stability in stance. Perception of toes up (but not toes down) surface tilt was reduced in subjects with PSP compared to both control subjects (p≤0.001 standing, p≤0.007 seated) and subjects with PD (p≤0.03 standing, p≤0.04 seated). Subjects with PSP, PD and normal controls accurately perceived the direction of gravity when standing on a tilting surface. Unlike PD and control subjects, subjects with PSP exerted less postural corrective torque in response to toes up surface tilts. Difficulty perceiving backward tilt of the surface or body may account for backward falls and postural impairments in patients with PSP. These observations suggest that abnormal central integration of sensory inputs for perception of body and surface orientation contributes to the pathophysiology of postural instability in PSP.


Intense slow hypoxic pulmonary vasoconstriction in gas-filled and liquid-filled lungs: an in vivo study in the rabbit.
To examine the hypothesis that hypoxic pulmonary vasoconstriction may have a slower time course and greater intensity than is currently recognized, experiments were conducted in twelve anaesthetized rabbits subjected to unilateral lung hypoxia for 6 h. Endobronchial cannulation was used to maintain apnoea of one lung at constant airway pressure whilst inflating the lung with nitrogen or liquid. The second lung was ventilated with oxygen to maintain normocapnia and oxygen transfer. A pulmonary ventilated with oxygen to maintain normocapnia and oxygen transfer. A pulmonary artery catheter was introduced non-invasively. Pulmonary shunt was derived from mixed venous and arterial blood gas parameters. Pulmonary artery pressure was monitored continuously and cardiac output was estimated from oxygen uptake measurements before and after 6 h unilateral hypoxia. The experiments show that a rapid phase of hypoxic pulmonary vasoconstriction is followed by a slow phase which develops over hours. The slow phase is associated with a massive blood flow diversion from the hypoxic lung, such that pulmonary shunt after 6 h unilateral hypoxia is indistinguishable from baseline shunt during bilateral ventilation with oxygen. The response is reversible, but with a similarly slow time course. Results from nitrogen and liquid filling of the lung are similar. These findings are consistent with early experiments by Dirken and Heemstra in 1948 (Quart F Exp Physiol 34, 193-211), and challenge the prevailing notion that hypoxic pulmonary vasoconstriction is always a rapid and relatively weak physiological response to hypoxia.


Anticipation of Appetitive Operant Action Induces Sustained Dopamine Release in the Nucleus Accumbens.
The mesolimbic dopamine system is implicated in signaling reward-related information as well as in actions that generate rewarding outcomes. These implications are commonly investigated in either pavlovian or operant reinforcement paradigms, where only the latter requires instrumental action. To parse contributions of reward- and action-related information to dopamine signals, we directly compared the two paradigms: male rats underwent either pavlovian or operant conditioning while dopamine release was measured in the nucleus accumbens, a brain region central for processing this information. Task conditions were identical with the exception of the operant-lever response requirement. Rats in both groups released the same quantity of dopamine at the onset of the reward-predictive cue. However, only the operant-conditioning group showed a subsequent, sustained plateau in dopamine concentration throughout the entire 5 s cue presentation (preceding the required action). This dopamine ramp was unaffected by probabilistic reward delivery, occurred exclusively before operant actions, and was not related to task performance or task acquisition as it persisted throughout the 2 week daily behavioral training. Instead, the ramp flexibly increased in duration with longer cue presentation, seemingly modulating the initial cue-onset-triggered dopamine release, that is, the reward prediction error (RPE) signal, as both signal amplitude and sustainment diminished when reward timing was made more predictable. Thus, our findings suggest that RPE and action components of dopamine release can be differentiated temporally into phasic and ramping/sustained signals, respectively, where the latter depends on the former and presumably reflects the anticipation or incentivization of appetitive action, conceptually akin to motivation.SIGNIFICANCE STATEMENT It is unclear whether the components of dopamine signals that are related to reward-associated information and reward-driven approach behavior can be separated. Most studies investigating the dopamine system use either pavlovian or operant conditioning, which both involve the delivery of reward and necessitate appetitive approach behavior. Thus, used exclusively, neither paradigm can disentangle the contributions of these components to dopamine release. However, by combining both paradigms in the same study, we find that anticipation of a reward-driven operant action induces a modulation of reward-prediction-associated dopamine release, producing so-called dopamine ramps. Therefore, our findings provide new insight into dopamine ramps and suggest that dopamine signals integrate reward and appetitive action in a temporally distinguishable, yet dependent, manner.


Changes in muscle coordination and power output during sprint cycling.
This study investigated the changes in muscle coordination associated to power output decrease during a 30-s isokinetic (120rpm) cycling sprint. Modifications in EMG amplitude and onset/offset were investigated from eight muscles [gluteus maximus (EMGGMAX), vastus lateralis and medialis obliquus (EMGVAS), medial and lateral gastrocnemius (EMGGAS), rectus femoris (EMGRF), biceps femoris and semitendinosus (EMGHAM)]. Changes in co-activation of four muscle pairs (CAIGMAX/GAS, CAIVAS/GAS, CAIVAS/HAM and CAIGMAX/RF) were also calculated. Substantial power reduction (60±6%) was accompanied by a decrease in EMG amplitude for all muscles other than HAM, with the greatest deficit identified for EMGRF (31±16%) and EMGGAS (20±14%). GASonset, HAMonset and GMAXonset shifted later in the pedalling cycle and the EMG offsets of all muscles (except GASoffset) shifted earlier as the sprint progressed (P<0.05). At the end of the sprint, CAIVAS/GAS and CAIGMAX/GAS were reduced by 48±10% and 43±12%, respectively. Our results show that substantial power reduction during fatiguing sprint cycling is accompanied by marked reductions in the EMG activity of bi-articular GAS and RF and co-activation level between GAS and main power producer muscles (GMAX and VAS). The observed changes in RF and GAS EMG activity are likely to result in a redistribution of the joint powers and alterations in the orientation of the pedal forces.


The Adenomatous Polyposis Coli-protein (APC) interacts with the protein tyrosine phosphatase PTP-BL via an alternatively spliced PDZ domain.
Mutations of the tumor suppressor protein APC (Adenomatous Polyposis Coli) are linked to familiar and sporadic human colon cancer. Here we describe a novel interaction between the APC protein and the protein tyrosine phosphatase PTP-BL carrying five PDZ protein-protein interaction domains. Exclusively, the second PDZ domain (PDZ2) of PTP-BL is binding to the extreme C-terminus of the APC protein, as determined by yeast two-hybrid studies. Using surface plasmon resonance analysis we established a dissociation constant (K(D)) of 8.1 x 10(-9) M. We find that a naturally occurring splice insertion of five amino acids (PDZ2b) abolishes its binding affinity to the APC protein. The in vivo interaction between PTP-BL and the APC protein was shown by coprecipitation experiments in transfected COS cells. Furthermore, in cultured epithelial Madine Carnine Kidney cells the subcellular colocalization was demonstrated for the nucleus and also for the tips of cellular extensions. The interaction of the APC protein with a protein tyrosine phosphatase may indirectly modulate the steady state levels of tyrosine phosphorylations of associated proteins, such as beta-catenin playing a major role in the regulation of cell division, migration and cell adhesion.


Statistical primer: sample size and power calculations-why, when and how?
When designing a clinical study, a fundamental aspect is the sample size. In this article, we describe the rationale for sample size calculations, when it should be calculated and describe the components necessary to calculate it. For simple studies, standard formulae can be used; however, for more advanced studies, it is generally necessary to use specialized statistical software programs and consult a biostatistician. Sample size calculations for non-randomized studies are also discussed and two clinical examples are used for illustration.


Inverted DNA repeats: a source of eukaryotic genomic instability.
While inverted DNA repeats are generally acknowledged to be an important source of genetic instability in prokaryotes, relatively little is known about their effects in eukaryotes. Using bacterial transposon Tn5 and its derivatives, we demonstrate that long inverted repeats also cause genetic instability leading to deletion in the yeast Saccharomyces cerevisiae. Furthermore, they induce homologous recombination. Replication plays a major role in the deletion formation. Deletions are stimulated by a mutation in the DNA polymerase delta gene (pol3). The majority of deletions result from imprecise excision between small (4- to 6-bp) repeats in a polar fashion, and they often generate quasipalindrome structures that subsequently may be highly unstable. Breakpoints are clustered near the ends of the long inverted repeats (< 150 bp). The repeats have both intra- and interchromosomal effects in that they also create hot spots for mitotic interchromosomal recombination. Intragenic recombination is 4 to 18 times more frequent for heteroalleles in which one of the two mutations is due to the insertion of a long inverted repeat, compared with other pairs of heteroalleles in which neither mutation has a long repeat. We propose that both deletion and recombination are the result of altered replication at the basal part of the stem formed by the inverted repeats.


Polarization-sensitive hyperspectral imaging in vivo: a multimode dermoscope for skin analysis.
Attempts to understand the changes in the structure and physiology of human skin abnormalities by non-invasive optical imaging are aided by spectroscopic methods that quantify, at the molecular level, variations in tissue oxygenation and melanin distribution. However, current commercial and research systems to map hemoglobin and melanin do not correlate well with pathology for pigmented lesions or darker skin. We developed a multimode dermoscope that combines polarization and hyperspectral imaging with an efficient analytical model to map the distribution of specific skin bio-molecules. This corrects for the melanin-hemoglobin misestimation common to other systems, without resorting to complex and computationally intensive tissue optical models. For this system's proof of concept, human skin measurements on melanocytic nevus, vitiligo, and venous occlusion conditions were performed in volunteers. The resulting molecular distribution maps matched physiological and anatomical expectations, confirming a technologic approach that can be applied to next generation dermoscopes and having biological plausibility that is likely to appeal to dermatologists.


Home-based cardiac rehabilitation for people with heart failure: A systematic review and meta-analysis.
To assess the effectiveness of home-based cardiac rehabilitation (CR) for heart failure compared to either usual medical care (i.e. no CR) or centre-based CR on mortality, morbidity, exercise capacity, health-related quality of life, drop out, adherence rates, and costs. Randomised controlled trials were initially identified from previous systematic reviews of CR. We undertook updated literature searches of MEDLINE, EMBASE, CINAHL, PsycINFO and Cochrane Library to December 2015. A total of 19 trials with median follow up of 3months were included - 17 comparisons of home-based CR to usual care (995 patients) and four comparing home and centre-based CR (295 patients). Compared to usual care, home-based CR improved VO2max (mean difference: 1.6ml/kg/min, 0.8 to 2.4) and total Minnesota Living with Quality of Life score (-3.3, -7.5 to 1.0), with no difference in mortality, hospitalisation or study drop out. Outcomes and costs were similar between home-based and centre-based CR with the exception of higher levels of trial completion in the home-based group (relative risk: 1.2, 1.0 to 1.3). Home-based CR results in short-term improvements in exercise capacity and health-related quality of life of heart failure patients compared to usual care. The magnitude of outcome improvement is similar to centre-based CR. Home-based CR appears to be safe with no evidence of increased risk of hospitalisation or death. These findings support the provision of home-based CR for heart failure as an evidence-based alternative to the traditional centre-based model of provision.


Scattering of light from metamaterial gratings with finite length.
Using an integral equation approach based on the Rayleigh hypothesis, we investigate the scattering of a plane wave at the rough surface of a metamaterial with a finite number of sinusoidal grooves. To show the adequacy of the model, we present results that are in agreement with the predictions of physical optics and that quantitatively reproduce the polarization and angular dependences predicted by the C-formalism for metamaterial gratings with an infinite number of grooves.


Polyamine oxidase in human retroplacental serum inhibits the growth of Plasmodium falciparum.
Human retroplacental serum (RPS) containing polyamine oxidase inhibited the growth of the Camp strain of Plasmodium falciparum in vitro as assayed by the parasite's decreased incorporation of 3H-hypoxanthine. Inhibition was dose-dependent on the concentrations of serum polyamine oxidase and added polyamines. Almost complete inhibition was seen in 96-hr asynchronous cultures containing 10% RPS and in those containing 1.2% RPS plus 50 microM polyamine. Subtle morphologic changes in mature stages and decreased numbers of new rings were associated with inhibition seen in 19-hr synchronous cultures initiated at the trophozoite stage. These incubation times were longer than in previous reports showing inhibition of malaria parasites by bovine polyamine oxidase but not by human polyamine oxidase. Macrophages contain polyamine oxidase, the reaction products of which are known to be similar to those of RPS polyamine oxidase but different from those of bovine polyamine oxidase. It remains to be determined whether human polyamine oxidase, acting upon ubiquitous polyamines, contributes to host defenses against malaria.


Genetic diversity in European Pisum germplasm collections.
The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collections has been used to determine their relatedness and to test previous ideas about the genetic diversity of Pisum. Our characterisation of genetic diversity among 4,538 Pisum accessions held in 7 European Genebanks has identified sources of novel genetic variation, and both reinforces and refines previous interpretations of the overall structure of genetic diversity in Pisum. Molecular marker analysis was based upon the presence/absence of polymorphism of retrotransposon insertions scored by a high-throughput microarray and SSAP approaches. We conclude that the diversity of Pisum constitutes a broad continuum, with graded differentiation into sub-populations which display various degrees of distinctness. The most distinct genetic groups correspond to the named taxa while the cultivars and landraces of Pisum sativum can be divided into two broad types, one of which is strongly enriched for modern cultivars. The addition of germplasm sets from six European Genebanks, chosen to represent high diversity, to a single collection previously studied with these markers resulted in modest additions to the overall diversity observed, suggesting that the great majority of the total genetic diversity collected for the Pisum genus has now been described. Two interesting sources of novel genetic variation have been identified. Finally, we have proposed reference sets of core accessions with a range of sample sizes to represent Pisum diversity for the future study and exploitation by researchers and breeders.


Probabilistic modeling of anatomical variability using a low dimensional parameterization of diffeomorphisms.
We present an efficient probabilistic model of anatomical variability in a linear space of initial velocities of diffeomorphic transformations and demonstrate its benefits in clinical studies of brain anatomy. To overcome the computational challenges of the high dimensional deformation-based descriptors, we develop a latent variable model for principal geodesic analysis (PGA) based on a low dimensional shape descriptor that effectively captures the intrinsic variability in a population. We define a novel shape prior that explicitly represents principal modes as a multivariate complex Gaussian distribution on the initial velocities in a bandlimited space. We demonstrate the performance of our model on a set of 3D brain MRI scans from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database. Our model yields a more compact representation of group variation at substantially lower computational cost than the state-of-the-art method such as tangent space PCA (TPCA) and probabilistic principal geodesic analysis (PPGA) that operate in the high dimensional image space.


Microplastics alter composition of fungal communities in aquatic ecosystems.
Despite increasing concerns about microplastic (MP) pollution in aquatic ecosystems, there is insufficient knowledge on how MP affect fungal communities. In this study, we explored the diversity and community composition of fungi attached to polyethylene (PE) and polystyrene (PS) particles incubated in different aquatic systems in north-east Germany: the Baltic Sea, the River Warnow and a wastewater treatment plant. Based on next generation 18S rRNA gene sequencing, 347 different operational taxonomic units assigned to 81 fungal taxa were identified on PE and PS. The MP-associated communities were distinct from fungal communities in the surrounding water and on the natural substrate wood. They also differed significantly among sampling locations, pointing towards a substrate and location specific fungal colonization. Members of Chytridiomycota, Cryptomycota and Ascomycota dominated the fungal assemblages, suggesting that both parasitic and saprophytic fungi thrive in MP biofilms. Thus, considering the worldwide increasing accumulation of plastic particles as well as the substantial vector potential of MP, especially these fungal taxa might benefit from MP pollution in the aquatic environment with yet unknown impacts on their worldwide distribution, as well as biodiversity and food web dynamics at large.


Determinants of the efficacy of HIV latency-reversing agents and implications for drug and treatment design.
HIV eradication studies have focused on developing latency-reversing agents (LRAs). However, it is not understood how the rate of latent reservoir reduction is affected by different steps in the process of latency reversal. Furthermore, as current LRAs are host-directed, LRA treatment is likely to be intermittent to avoid host toxicities. Few careful studies of the serial effects of pulsatile LRA treatment have yet been done. This lack of clarity makes it difficult to evaluate the efficacy of candidate LRAs or predict long-term treatment outcomes. We constructed a mathematical model that describes the dynamics of latently infected cells under LRA treatment. Model analysis showed that, in addition to increasing the immune recognition and clearance of infected cells, the duration of HIV antigen expression (i.e., the period of vulnerability) plays an important role in determining the efficacy of LRAs, especially if effective clearance is achieved. Patients may benefit from pulsatile LRA exposures compared with continuous LRA exposures if the period of vulnerability is long and the clearance rate is high, both in the presence and absence of an LRA. Overall, the model framework serves as a useful tool to evaluate the efficacy and the rational design of LRAs and combination strategies.


The presence of heparan sulfate proteoglycans in the neuritic plaques and congophilic angiopathy in Alzheimer's disease.
Two immunocytochemical probes were used to specifically identify and localize heparan sulphate proteoglycans (HSPGs) in 17 cases of Alzheimer's disease (AD). A monoclonal (HK-102) and an affinity-purified polyclonal antibody, each recognizing specific domains on the protein core of a basement membrane-derived HSPG, localized HSPGs to the amyloid fibrils present in neuritic plaques (NPs) and congophilic angiopathy (CA) in the brains of Alzheimer's patients, with weak to no immunostaining in neurofibrillary tangles from the same tissues. HSPGs were also demonstrated in "primitive plaques," suggesting that their accumulation takes place during early stages of plaque development. Immunolocalization of HSPGs to subsets of astrocytes and neuronal cells, particularly those in close proximity to NPs and CA, suggested possible involvement of these two cell types in deposition of HS-PGs into the amyloidotic lesions. The current study not only identifies a new component (HSPGs) present in the amyloid deposits of NPs and CA but also suggests that astrocytes, neurons, or both may be involved in its deposition at these sites.


Relief diffracted elements recorded on absorbent photopolymers.
Relief surface changes provide interesting possibilities for storing diffractive optical elements on photopolymers and are an important source of information for characterizing and understanding the material behavior. In this paper we use a 3-dimensional model, based on direct parameter measurements, for predicting the relief structures generated on without-coverplate photopolymers. We have analyzed different spatial frequency and recording intensity distributions such as binary and blazed periodic patterns. This model was successfully applied to different photopolymers with different values of monomer diffusion.


Biochar and manure affect calcareous soil and corn silage nutrient concentrations and uptake.
Carbon-rich biochar derived from the pyrolysis of biomass can sequester atmospheric CO, mitigate climate change, and potentially increase crop productivity. However, research is needed to confirm the suitability and sustainability of biochar application to different soils. To an irrigated calcareous soil, we applied stockpiled dairy manure (42 Mg ha dry wt) and hardwood-derived biochar (22.4 Mg ha), singly and in combination with manure, along with a control, yielding four treatments. Nitrogen fertilizer was applied when needed (based on preseason soil test N and crop requirements) in all plots and years, with N mineralized from added manure included in this determination. Available soil nutrients (NH-N; NO-N; Olsen P; and diethylenetriaminepentaacetic acid-extractable K, Mg, Na, Cu, Mn, Zn, and Fe), total C (TC), total N (TN), total organic C (TOC), and pH were evaluated annually, and silage corn nutrient concentration, yield, and uptake were measured over two growing seasons. Biochar treatment resulted in a 1.5-fold increase in available soil Mn and a 1.4-fold increase in TC and TOC, whereas manure produced a 1.2- to 1.7-fold increase in available nutrients (except Fe), compared with controls. In 2009 biochar increased corn silage B concentration but produced no yield increase; in 2010 biochar decreased corn silage TN (33%), S (7%) concentrations, and yield (36%) relative to controls. Manure produced a 1.3-fold increase in corn silage Cu, Mn, S, Mg, K, and TN concentrations and yield compared with the control in 2010. The combined biochar-manure effects were not synergistic except in the case of available soil Mn. In these calcareous soils, biochar did not alter pH or availability of P and cations, as is typically observed for acidic soils. If the second year results are representative, they suggest that biochar applications to calcareous soils may lead to reduced N availability, requiring additional soil N inputs to maintain yield targets.


Impact of Cone Beam Computed Tomography Dose in Pre-Surgical Implant Analysis.
Cone-Beam Computed Tomography (CBCT) produces vital information required for the accurate and prudent placement of dental implants. Lack of standardization between CBCT machines may result in unsafe patient exposure to harmful radiation; higher doses are not necessarily associated with improved image quality. The study aimed to assess the influence of low- and high-dose milliamperage settings on CBCT images for objective and subjective implant planning. Two dry skulls (4 hemi-maxillary segments of the maxilla and 4 hemi-maxillary segments of the mandible) were scanned under low (2 mA) and high (6.3 mA) dosage settings using a CBCT (Carestream CS 9300). Cross-sectional slices of both image qualities were evaluated by five expert clinicians, for image quality for implant planning and objective bone measurements. There were no significant differences in bone measurements taken on high or low dose images (p > 0.05). In qualitative image assessments, assessment and image quality for almost all observers were independent of each other. For planning posterior mandibular implant placement, increased dosage improved concordance and kappa values between low and high dose images. Reduction in milliamperage did not affect diagnostic image quality for objective bone measurements and produced sufficient intra-rater reliability for qualitative assessment; therefore dose reduction can be achieved without compromising diagnostic decision- making.


SEMAC-VAT and MSVAT-SPACE sequence strategies for metal artifact reduction in 1.5T magnetic resonance imaging.
To evaluate the ability of four magnetic resonance imaging (MRI) techniques to correct for metallic artifacts. These techniques consisted of 3 2D techniques and one 3D technique. In 2D imaging the techniques View Angle Tilting (VAT), Slice Encoding for Metal Artifact Correction (SEMAC) and a technique that employed a combination of the first two (SEMAC-VAT) were evaluated. In 3D imaging the technique Multiple Slab acquisition with VAT based on a SPACE sequence was evaluated (MSVAT-SPACE). Agarose phantoms and tissue phantoms with two commonly used metal implants (stainless steel and titanium) as well as two volunteers with metal implants were imaged at 1.5T. All phantoms and volunteers were imaged using VAT, SEMAC, SEMAC-VAT and MSVAT-SPACE techniques, as well as 2D and 3D conventional imaging techniques. Each technique was optimized for different image contrast mechanisms. Artifact reduction was quantitatively assessed in the agarose phantoms by volumetric measurement. Image quality was qualitatively assessed by blinded reads employing two readers. Each reader independently viewed the tissue phantom images and in vivo human images. Statistical analysis was performed using a Friedman test, Wilcoxon test and weighted Cohen's kappa test. T1-weighted, T2-weighted, PD-weighted and STIR image contrasts were successfully implemented with the evaluated artifact reduction sequences in both the phantom experiments and in vivo images. For all evaluated image contrasts and both metal implants, a reduction in the volume of metal artifacts was seen when compared with 2D conventional acquisitions. The 2D metal artifact volumes on average were reduced by 49% ± 16%, 56% ± 15% and 63% ± 15% for VAT, SEMAC and SEMAC-VAT acquisitions respectively. When Friedman and Wilcoxon tests were applied the difference in metal artifact volume was found to be statistically significant when VAT, SEMAC and SEMAC-VAT were compared with the 2D conventional techniques. In 3D imaging on average MSVAT-SPACE reduced metal artifact volume compared with the 3D conventional imaging technique by 72% ± 23% for all evaluated image contrasts and both metal implants. The metal artifact volume differences were statistically significant when MSVAT-SPACE was compared with the 3D conventional technique. The blinded reads demonstrated that SEMAC-VAT and MSVAT-SPACE had distinctly superior quality compared with conventional acquisitions. Quality was measured in terms of artifact size, distortions, image quality and visualization of bone marrow and soft tissues adjacent to metal implants. This was the case for both tissue phantom images and human images with good interobserver agreement. SEMAC-VAT (2D) and MSVAT-SPACE (3D) demonstrated a consistent, marked reduction of metal artifacts for different metal implants and offered flexible image contrasts (T1, T2, PD and STIR) with high image quality. These techniques likely will improve the evaluation of postoperative patients with metal implants.


A flow-free droplet-based device for high throughput polymorphic crystallization.
Crystallization is one of the most crucial steps in the process of pharmaceutical formulation. In recent years, emulsion-based platforms have been developed and broadly adopted to generate high quality products. However, these conventional approaches such as stirring are still limited in several aspects, e.g., unstable crystallization conditions and broad size distribution; besides, only simple crystal forms can be produced. In this paper, we present a new flow-free droplet-based formation process for producing highly controlled crystallization with two examples: (1) NaCl crystallization reveals the ability to package saturated solution into nanoliter droplets, and (2) glycine crystallization demonstrates the ability to produce polymorphic crystallization forms by controlling the droplet size and temperature. In our process, the saturated solution automatically fills the microwell array powered by degassed bulk PDMS. A critical oil covering step is then introduced to isolate the saturated solution and control the water dissolution rate. Utilizing surface tension, the solution is uniformly packaged in the form of thousands of isolating droplets at the bottom of each microwell of 50-300 μm diameter. After water dissolution, individual crystal structures are automatically formed inside the microwell array. This approach facilitates the study of different glycine growth processes: α-form generated inside the droplets and γ-form generated at the edge of the droplets. With precise temperature control over nanoliter-sized droplets, the growth of ellipsoidal crystalline agglomerates of glycine was achieved for the first time. Optical and SEM images illustrate that the ellipsoidal agglomerates consist of 2-5 μm glycine clusters with inner spiral structures of ~35 μm screw pitch. Lastly, the size distribution of spherical crystalline agglomerates (SAs) produced from microwells of different sizes was measured to have a coefficient variation (CV) of less than 5%, showing crystal sizes can be precisely controlled by microwell sizes with high uniformity. This new method can be used to reliably fabricate monodispersed crystals for pharmaceutical applications.


The great escape; the hallmarks of resistance to antiangiogenic therapy.
The concept of antiangiogenic therapy in cancer treatment has led to the approval of different agents, most of them targeting the well known vascular endothelial growth factor pathway. Despite promising results in preclinical studies, the efficacy of antiangiogenic therapy in the clinical setting remains limited. Recently, awareness has emerged on resistance to antiangiogenic therapies. It has become apparent that the intricate complex interplay between tumors and stromal cells, including endothelial cells and associated mural cells, allows for escape mechanisms to arise that counteract the effects of these targeted therapeutics. Here, we review and discuss known and novel mechanisms that contribute to resistance against antiangiogenic therapy and provide an outlook to possible improvements in therapeutic approaches.


Dynamic Features for Iris Recognition.
The human eye is sensitive to visible light. Increasing illumination on the eye causes the pupil of the eye to contract, while decreasing illumination causes the pupil to dilate. Visible light causes specular reflections inside the iris ring. On the other hand, the human retina is less sensitive to near infra-red (NIR) radiation in the wavelength range from 800 nm to 1400 nm, but iris detail can still be imaged with NIR illumination. In order to measure the dynamic movement of the human pupil and iris while keeping the light-induced reflexes from affecting the quality of the digitalized image, this paper describes a device based on the consensual reflex. This biological phenomenon contracts and dilates the two pupils synchronously when illuminating one of the eyes by visible light. In this paper, we propose to capture images of the pupil of one eye using NIR illumination while illuminating the other eye using a visible-light pulse. This new approach extracts iris features called "dynamic features (DFs)." This innovative methodology proposes the extraction of information about the way the human eye reacts to light, and to use such information for biometric recognition purposes. The results demonstrate that these features are discriminating features, and, even using the Euclidean distance measure, an average accuracy of recognition of 99.1% was obtained. The proposed methodology has the potential to be "fraud-proof," because these DFs can only be extracted from living irises.


CRISPR/Cas9-Mediated Trp53 and Brca2 Knockout to Generate Improved Murine Models of Ovarian High-Grade Serous Carcinoma.
There is a need for transplantable murine models of ovarian high-grade serous carcinoma (HGSC) with regard to mutations in the human disease to assist investigations of the relationships between tumor genotype, chemotherapy response, and immune microenvironment. In addressing this need, we performed whole-exome sequencing of ID8, the most widely used transplantable model of ovarian cancer, covering 194,000 exomes at a mean depth of 400× with 90% exons sequenced >50×. We found no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, and Rb1), and p53 remained transcriptionally active. Homologous recombination in ID8 remained intact in functional assays. Further, we found no mutations typical of clear cell carcinoma (Arid1a, Pik3ca), low-grade serous carcinoma (Braf), endometrioid (Ctnnb1), or mucinous (Kras) carcinomas. Using CRISPR/Cas9 gene editing, we modeled HGSC by generating novel ID8 derivatives that harbored single (Trp53-/-) or double (Trp53-/-;Brca2-/-) suppressor gene deletions. In these mutants, loss of p53 alone was sufficient to increase the growth rate of orthotopic tumors with significant effects observed on the immune microenvironment. Specifically, p53 loss increased expression of the myeloid attractant CCL2 and promoted the infiltration of immunosuppressive myeloid cell populations into primary tumors and their ascites. In Trp53-/-;Brca2-/- mutant cells, we documented a relative increase in sensitivity to the PARP inhibitor rucaparib and slower orthotopic tumor growth compared with Trp53-/- cells, with an appearance of intratumoral tertiary lymphoid structures rich in CD3+ T cells. This work validates new CRISPR-generated models of HGSC to investigate its biology and promote mechanism-based therapeutics discovery. Cancer Res; 76(20); 6118-29. ©2016 AACR.


Shotgun lipidomics of phosphoethanolamine-containing lipids in biological samples after one-step in situ derivatization.
This article presents a novel methodology for the analysis of ethanolamine glycerophospholipid (PE) and lysoPE molecular species directly from lipid extracts of biological samples. Through brief treatment of lipid extracts with fluorenylmethoxylcarbonyl (Fmoc) chloride, PE and lysoPE species were selectively derivatized to their corresponding carbamates. The reaction solution was infused directly into the ion source of an electrospray ionization mass spectrometer after appropriate dilution. The facile loss of the Fmoc moiety dramatically enhanced the analytic sensitivity and allowed the identification and quantitation of low-abundance molecular species. A detection limitation of attomoles (amoles) per microliter for PE and lysoPE analysis was readily achieved using this technique (at least a 100-fold improvement from our previous method) with a >15,000-fold dynamic range. Through intrasource separation and multidimensional mass spectrometry array analysis of derivatized species, marked improvements in signal-to-noise ratio, molecular species identification, and quantitation can be realized. The procedure is both simple and effective and can be extended to analyze many other lipid classes or other cellular metabolites by adjustments in specific derivatization conditions. Thus, through judicious derivatization, a new dimension exploiting specific functional reactivities in each lipid class can be used in conjunction with shotgun lipidomics to penetrate farther into the low-abundance regime of cellular lipidomes.


Hypercapnic cerebral vascular reactivity is decreased, in humans, during sleep compared with wakefulness.
During wakefulness, increases in the partial pressure of arterial CO(2) result in marked rises in cortical blood flow. However, during stage III-IV, non-rapid eye movement (NREM) sleep, and despite a relative state of hypercapnia, cortical blood flow is reduced compared with wakefulness. In the present study, we tested the hypothesis that, in normal subjects, hypercapnic cerebral vascular reactivity is decreased during stage III-IV NREM sleep compared with wakefulness. A 2-MHz pulsed Doppler ultrasound system was used to measure the left middle cerebral artery velocity (MCAV; cm/s) in 12 healthy individuals while awake and during stage III-IV NREM sleep. The end-tidal Pco(2) (Pet(CO(2))) was elevated during the awake and sleep states by regulating the inspired CO(2) load. The cerebral vascular reactivity to CO(2) was calculated from the relationship between Pet(CO(2)) and MCAV by using linear regression. From wakefulness to sleep, the Pet(CO(2)) increased by 3.4 Torr (P < 0.001) and the MCAV fell by 11.7% (P < 0.001). A marked decrease in cerebral vascular reactivity was noted in all subjects, with an average fall of 70.1% (P = 0.001). This decrease in hypercapnic cerebral vascular reactivity may, at least in part, explain the stage III-IV NREM sleep-related reduction in cortical blood flow.


Improved engraftment with minimal graft-versus-host disease after major histocompatibility complex-mismatched cord blood transplantation with photochemically treated donor lymphocytes.
There is a significant risk of severe graft-versus-host disease (GVHD) and graft failure after unrelated umbilical cord blood transplantation (CBT) if donor-recipient pairs are mismatched at major histocompatibility complex (MHC) loci. To mitigate these risks after MHC-mismatched CBT, we infused psoralen-treated, photochemically inactivated, mature donor T-lymphocytes with MHC (H2-haplotype) mismatched murine donor fetal near-term peripheral blood (FNPB) cells after sublethal irradiation. We analyzed the rates of donor engraftment, GVHD and long-term survival in H2 haplotype disparate (C57BL/6 [H-2(b)/Thy1.1] → AKR [H-2(k)/Thy1.2]) recipient mice. We observed inconsistent donor engraftment after transplantation with cord blood alone, but superior engraftment and long-term survival after FNPB transplantation supplemented with psoralen-treated donor T-lymphocytes. Additionally, there was fatal GVHD after FNPB co-infusion with untreated donor T-lymphocytes, but minimal GVHD after FNPB supplemented with psoralen-treated donor T-lymphocytes transplantation. Donor MHC(high)/c-Kit(+)/lineage(-)/CD34(-) stem cells were noted in the recipient bone marrow compartment following co-infusion of photochemically inactivated T-cells with FNPB. Despite the non-myeloablative preparation before FNPB infusion, complete hematological recovery was delayed until 50-60 d after transplantation. We observed that co-transplantation of psoralen-treated donor T-lymphocytes with FNPB facilitated durable engraftment of donor hematopoietic stem cells in the marrow and splenic compartments with complete but delayed recovery of all hematopoietic lineages. This CBT model establishes the possibility of ensuring donor engraftment across a MHC barrier without severe GVHD.


Evidence for the presence of disease-perturbed networks in prostate cancer cells by genomic and proteomic analyses: a systems approach to disease.
Prostate cancer is initially responsive to androgen ablation therapy and progresses to androgen-unresponsive states that are refractory to treatment. The mechanism of this transition is unknown. A systems approach to disease begins with the quantitative delineation of the informational elements (mRNAs and proteins) in various disease states. We employed two recently developed high-throughput technologies, massively parallel signature sequencing (MPSS) and isotope-coded affinity tag, to gain a comprehensive picture of the changes in mRNA levels and more restricted analysis of protein levels, respectively, during the transition from androgen-dependent LNCaP (model for early-stage prostate cancer) to androgen-independent CL1 cells (model for late-stage prostate cancer). We sequenced >5 million MPSS signatures, obtained >142,000 tandem mass spectra, and built comprehensive MPSS and proteomic databases. The integrated mRNA and protein expression data revealed underlying functional differences between androgen-dependent and androgen-independent prostate cancer cells. The high sensitivity of MPSS enabled us to identify virtually all of the expressed transcripts and to quantify the changes in gene expression between these two cell states, including functionally important low-abundance mRNAs, such as those encoding transcription factors and signal transduction molecules. These data enable us to map the differences onto extant physiologic networks, creating perturbation networks that reflect prostate cancer progression. We found 37 BioCarta and 14 Kyoto Encyclopedia of Genes and Genomes pathways that are up-regulated and 23 BioCarta and 22 Kyoto Encyclopedia of Genes and Genomes pathways that are down-regulated in LNCaP cells versus CL1 cells. Our efforts represent a significant step toward a systems approach to understanding prostate cancer progression.


Stability and Roughness of Interfaces in Mechanically Regulated Tissues.
Cell division and death can be regulated by the mechanical forces within a tissue. We study the consequences for the stability and roughness of a propagating interface by analyzing a model of mechanically regulated tissue growth in the regime of small driving forces. For an interface driven by homeostatic pressure imbalance or leader-cell motility, long and intermediate-wavelength instabilities arise, depending, respectively, on an effective viscosity of cell number change, and on substrate friction. A further mechanism depends on the strength of directed motility forces acting in the bulk. We analyze the fluctuations of a stable interface subjected to cell-level stochasticity, and find that mechanical feedback can help preserve reproducibility at the tissue scale. Our results elucidate mechanisms that could be important for orderly interface motion in developing tissues.


The Concerns and Experience of Decision-Making Regarding Do-Not-Resuscitate Orders Among Caregivers in Hospice Palliative Care.
A do-not-resuscitate (DNR) order is an important end-of-life decision. In Taiwan, family caregivers are also involved in this decision-making process. This study aimed to explore the concerns and experiences regarding DNR decisions among caregivers in Taiwan. Qualitative study was conducted. Convenience sampling was used, and 26 caregivers were recruited whose patients had a DNR order and had received hospice care or hospice home care. Semi-structured interviews were used for data collection, including the previous experiences of DNR discussions with the patients and medical staff and their concerns and difficulties in decision-making. The data analysis was based on the principle of thematic analysis. Four themes were identified: (1) Patients: The caregivers respected the patients' willingness and did not want to make them feel like "giving up." (2) Caregivers' self: They did not want to intensify the patients' suffering but sometimes found it emotionally difficult to accept death. (3) Other family members: They were concerned about the other family members' opinions on DNR orders, their blame, and their views on filial impiety. (4) Medical staff: The information and suggestions from the medical staff were foundational to their decision-making. The caregivers needed the health care professionals' supports to deal with the concerns from patients and other family members as well as their emotional reactions.


Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.
The locus for familial cortical myoclonic tremor with epilepsy (FCMTE) has long been mapped to 8q24 in linkage studies, but the causative mutations remain unclear. Recently, expansions of intronic TTTCA and TTTTA repeat motifs within SAMD12 were found to be involved in the pathogenesis of FCMTE in Japanese pedigrees. We aim to identify the causative mutations of FCMTE in Chinese pedigrees. We performed genetic linkage analysis by microsatellite markers in a five-generation Chinese pedigree with 55 members. We also used array-comparative genomic hybridisation (CGH) and next-generation sequencing (NGS) technologies (whole-exome sequencing, capture region deep sequencing and whole-genome sequencing) to identify the causative mutations in the disease locus. Recently, we used low-coverage (~10×) long-read genome sequencing (LRS) on the PacBio Sequel and Oxford Nanopore platforms to identify the causative mutations, and used repeat-primed PCR for validation of the repeat expansions. Linkage analysis mapped the disease locus to 8q23.3-24.23. Array-CGH and NGS failed to identify causative mutations in this locus. LRS identified the intronic TTTCA and TTTTA repeat expansions in SAMD12 as the causative mutations, thus corroborating the recently published results in Japanese pedigrees. We identified the pentanucleotide repeat expansion in SAMD12 as the causative mutation in Chinese FCMTE pedigrees. Our study also suggested that LRS is an effective tool for molecular diagnosis of genetic disorders, especially for neurological diseases that cannot be positively diagnosed by conventional clinical microarray and NGS technologies.


One-year Results of a Factorial Randomized Trial of Aspirin versus Placebo and Clonidine versus Placebo in Patients Having Noncardiac Surgery.
The authors previously reported that perioperative aspirin and/or clonidine does not prevent a composite of death or myocardial infarction 30 days after noncardiac surgery. Moreover, aspirin increased the risk of major bleeding and clonidine caused hypotension and bradycardia. Whether these complications produce harm at 1 yr remains unknown. The authors randomized 10,010 patients with or at risk of atherosclerosis and scheduled for noncardiac surgery in a 1:1:1:1 ratio to clonidine/aspirin, clonidine/aspirin placebo, clonidine placebo/aspirin, or clonidine placebo/aspirin placebo. Patients started taking aspirin or placebo just before surgery; those not previously taking aspirin continued daily for 30 days, and those taking aspirin previously continued for 7 days. Patients were also randomly assigned to receive clonidine or placebo just before surgery, with the study drug continued for 72 h. Neither aspirin nor clonidine had a significant effect on the primary 1-yr outcome, a composite of death or nonfatal myocardial infarction, with a 1-yr hazard ratio for aspirin of 1.00 (95% CI, 0.89 to 1.12; P = 0.948; 586 patients [11.8%] vs. 589 patients [11.8%]) and a hazard ratio for clonidine of 1.07 (95% CI, 0.96 to 1.20; P = 0.218; 608 patients [12.1%] vs. 567 patients [11.3%]), with effect on death or nonfatal infarction. Reduction in death and nonfatal myocardial infarction from aspirin in patients who previously had percutaneous coronary intervention at 30 days persisted at 1 yr. Specifically, the hazard ratio was 0.58 (95% CI, 0.35 to 0.95) in those with previous percutaneous coronary intervention and 1.03 (95% CI, 0.91to 1.16) in those without (interaction P = 0.033). There was no significant effect of either drug on death, cardiovascular complications, cancer, or chronic incisional pain at 1 yr (all P > 0.1). Neither perioperative aspirin nor clonidine have significant long-term effects after noncardiac surgery. Perioperative aspirin in patients with previous percutaneous coronary intervention showed persistent benefit at 1 yr, a plausible sub-group effect.


Factor XIIIa mediated attachment of S. aureus fibronectin-binding protein A (FnbA) to fibrin: identification of Gln103 as a major cross-linking site.
In the present study we investigated the role of factor XIIIa reactive Gln and Lys sites of staphylococcal FnbA receptor in cross-linking reaction with alpha chains of fibrin. For this purpose we produced two recombinant FnbA mutants in which either a single Gln103 site (1Q FnbA) or all identified reactive Gln103, 105, 783, 830 and Lys157, 503, 620, 762 sites (4Q4K FnbA) were substituted with Ala residues. The results of FXIIIa-catalyzed incorporation of dansylcadaverine and dansylated peptide patterned on the NH2-terminal segment of fibronectin revealed that the reactivity of Gln substrate sites was drastically reduced in 1Q FnbA and 4Q4K FnbA mutants, while the reactivity of Lys substrate sites was only moderately decreased in 4Q4K FnbA. When it was tested in the FXIIIa-mediated fibrin cross-linking reaction, the 1Q FnbA mutant exhibited about 70-85% reduction in reactivity compared to that of the wild-type FnbA. These results demonstrate that FnbA participates in cross-linking to alpha chains of fibrin predominantly via its Gln103 reactive site. Several minor sites, including residues replaced in 4Q4K FnbA mutant, contributed to an additional 15-30% of the total fibrin cross-linking reactivity of FnbA. Comparison of amino acid sequences that follow the major reactive Gln site in FnbA and several known substrate proteins revealed that FXIIIa displays a preference for the glutamine residue in an xQAxBxPx sequence, where Q represents reactive glutamine, x is any amino acid residue, A is a polar residue, B is either valine or leucine, and P is proline.


Additive activation of hepatic NF-kappaB by ethanol and hepatitis B protein X (HBX) or HCV core protein: involvement of TNF-alpha receptor 1-independent and -dependent mechanisms.
Alcohol consumption and viral hepatitis infection synergistically accelerate liver injury, but the underlying mechanism is not fully understood. Here we have examined the effects of ethanol on hepatitis B protein X (HBX)- or hepatitis C core protein (HCV core protein)-mediated activation of NF-kappaB, a critical signal in hepatic injury, regeneration, and tumor transformation. Acute ethanol or acetaldehyde exposure potentiates HBX or HCV core protein activation of NF-kappaB in primary mouse hepatocytes. Such potentiation can be abolished by blocking ethanol metabolism or overexpression of dominant negative NF-kappaB-inducing kinase (NIK), IkappaB kinase (IKK), or IkappaB. Moreover, pertussis toxin attenuates NF-kappaB activation induced by acetaldehyde but not by HBX or HCV core protein, whereas HBX or HCV core protein-mediated activation of NF-kappaB is abolished completely in tumor necrosis factor a receptor 1 (TNFR1) (-/-) hepatocytes. Finally, chronic ethanol consumption induces hepatic CYP2E1 protein expression and potentiates HBX or HCV core protein activation of NF-kappaB in the liver. These findings suggest that ethanol activates hepatic NF-kappaB via its metabolism and that HBX or HCV core protein activates hepatic NF-kappaB via TNFR1. With the essential role of TNFR1 in alcoholic liver injury, targeting TNFR1 by hepatitis viral proteins could contribute to cooperative effects of alcohol consumption and viral hepatitis on liver disease.


Interim 18F-FDG-PET/CT during chemo-radiotherapy in the management of oesophageal cancer patients. A systematic review.
Oesophageal cancer is an aggressive disease. The possibility to early stratify patients as responsive and non-responsive with a non-invasive method is extremely appealing. The uptake of Fluorodeoxyglucose (18F-FDG) in tumours, provided by positron emission tomography (PET) images, has been proved to be useful to assess the initial staging of the disease, recurrence, and response to chemotherapy and chemo-radiotherapy (CRT). In the last years, efforts have been focused on the possibility to use ad interim 18F-FDG-PET/CT (PETint) to evaluate response during radiation therapy. However, controversial findings have been reported, although some relevant results would support its use for individual therapeutic decision. The present review assembles the comprehensive literature of the last decade to evaluate whether and in which cases PETint may offer predictive potential in oesophageal cancer. All the analysed studies (13 studies, 697 patients) denoted PETint as a challenging examination for early assessment of outcomes during CRT. In particular, 8 studies advocated the predictivity of PETint, whilst 5 did not find any correlation between the interim variation of PET parameters and the pathological complete response and/or the clinical outcome. The reasons that possibly have caused contradictions among the studies demand further research with prospective and uniform protocols and methods of analysis to assess the predictive and prognostic value of PETint in oesophageal cancer.


Characterisation of urinary monocyte chemoattractant protein 1: Potential biomarker for patients with overactive bladder.
Objective: To investigate urinary monocyte chemoattractant protein 1 (MCP-1) as a potential marker for idiopathic overactive bladder (OAB). This is a quantitative measurement of urinary MCP-1 to establish baseline normal values that could help in future index studies. Normalised urinary MCP-1 levels are measured in female patients with OAB and aged-matched controls. Severity of OAB symptoms is correlated to normalised urinary MCP-1 levels. Patients and methods: Urinary MCP-1 levels were measured in 29 female patients with OAB and 10 normal female controls. The patients with OAB were either newly diagnosed or off any OAB oral therapy for at least 2 weeks. OAB symptoms were assessed using validated OAB questionnaires. Urinary MCP-1 levels were measured using enzyme-linked immunosorbent assay and normalised by urinary creatinine (Cr) levels. Results: The baseline urinary MCP-1 levels in female patients with OAB were significantly higher than those of the controls, at a mean of 210.25 vs 48.02 pg/mg Cr (P < 0.001). Patients who had severe OAB bother symptoms had higher levels of urinary MCP-1 (r = 0.03), also patients with OAB-wet had higher levels of urinary MCP-1, at a mean (SEM) of 209.25 (30.5) vs OAB-dry 185.25 (10) pg/mg Cr (P < 0.001). Conclusion: Urinary MCP-1 levels were higher in female patients with idiopathic OAB. The close association of urinary MCP-1 and OAB bother severity symptoms and OAB-wet suggest that inflammation plays a major role in the pathophysiological mechanisms underlying the sensitisation of bladder afferent nerves. Establishing urinary MCP-1 levels in patients with OAB hopefully will help future studies to confirm the correlation as a baseline and changes with treatments. Abbreviations: BMI: body mass index; Cr: creatinine; MCP-1: monocyte chemoattractant protein 1; OAB: overactive bladder; OAB-q: Overactive Bladder Questionnaire; PPBC: Patient Perception of Bladder Condition; UI: urinary incontinence.


Steroidal regulation of biologically active luteinizing hormone secretion in men and women.
The biological activity of circulating luteinizing hormone (LH) molecules can be assessed with high sensitivity, specificity, and precision by the in-vitro rat interstitial (Leydig)-cell testosterone (RICT) bioassay. Compared to immunoreactive estimates of the LH content of plasma, the living Leydig-cell bioassay integrates the functional in-vitro potency of all circulating LH isoforms, while simultaneously reflecting the effects of otherwise potentially confounding in-vivo antagonists of LH action on steroidogenesis. Here, we review the pathophysiological regulation by steroid hormones of bioactive LH secretion and clearance in men and women. Our investigations and those in the available literature indicate that: (i) exogenous (non-aromatizable) 5-alpha-dihydrotestosterone infusion suppresses LH bioactivity, whereas blockade of the endogenous androgen receptor with flutamide increases secretion of biopotent LH in men; (ii) endogenous androgen excess due to a masculinizing testosterone-secreting adrenal tumour in a post-menopausal woman reversibly suppressed plasma LH bioactivity markedly; (iii) exogenous oestradiol infusion will reduce whereas the antioestrogen, tamoxifen-HCl, can increase bioactive LH secretion in young men; (iv) the effect of anti-oestrogen is blunted in healthy older men; (v) endogenous hyperoestrogenism due to an adrenal oestrogen-secreting tumour in a middle-aged man with gynaecomastia profoundly but reversibly inhibited bioactive LH secretion; (vi) in post-menopausal women, exogenous oestrogen administration, whether oral (diethylstilbestrol), percutaneous (oestradiol), or intravaginal (oestradiol-impregnated silastic ring), exerts a temporally biphasic effect on basal bioactive LH secretion, i.e. acute suppression (within 24 h), subacute escape (at 5-10 days), and longer-term inhibition (30 days); (vii) during the normal menstrual cycle, bioactive LH secretion is pulsatile and both frequency and amplitude regulated; and (viii) oestrogen exposure can enhance gonadotrophin releasing hormone (GnRH)'s stimulatory action on biologically active LH secretion, resulting in so-called GnRH self-priming. Oestrogen's facilitative effects are achieved by a novel mechanism, in which oestrogen augments LH secretory burst mass and duration. Moreover, GnRH dose-LH secretory response studies show that oestrogen promotes an increase in GnRH efficacy (maximal effects) but not GnRH sensitivity (potency or half-maximally effective doses of GnRH). We conclude that steroid hormones are primary regulators of physiologically pulsatile bioactive LH secretion in healthy men and women. Moreover, steroids exert potent pathological effects on biologically active LH release in conditions of selective hyperandrogenism or hyperoestrogenism due to steroid-secreting adrenal or gonadal tumours.


Heterogeneous nuclear ribonucleoprotein K represses transcription from a cytosine/thymidine-rich element in the osteocalcin promoter.
HnRNP K (heterogeneous nuclear ribonucleoprotein K) was biochemically purified from a screen of proteins co-purifying with binding activity to the osteocalcin promoter. We identify hnRNP K as a novel repressor of osteocalcin gene transcription. Overexpression of hnRNP K lowers the expression of osteocalcin mRNA by 5-fold. Furthermore, luciferase reporter assays demonstrate that overexpression of hnRNP K represses osteocalcin transcription from a CT (cytosine/thymidine)-rich element in the proximal promoter. Electrophoretic mobility-shift analysis reveals that recombinant hnRNP K binds to the CT-rich element, but binds ss (single-stranded), rather than ds (double-stranded) oligonucleotide probes. Accordingly, hnRNP K antibody can supershift a binding activity present in nuclear extracts using ss sense, but not antisense or ds oligonucleotides corresponding to the CT-rich -95 to -47 osteocalcin promoter. Importantly, addition of recombinant hnRNP K to ROS 17/2.8 nuclear extract disrupts formation of a DNA-protein complex on ds CT element oligonucleotides. This action is mutually exclusive with hnRNP K's ability to bind ss DNA. These results demonstrate that hnRNPK, although co-purified with a dsDNA-binding activity, does not itself bind dsDNA. Rather, hnRNP K represses osteocalcin gene transcription by inhibiting the formation of a transcriptional complex on the CT element of the osteocalcin promoter.


Digestibility of amino acids and concentrations of metabolizable energy and net energy are greater in high-shear dry soybean expellers than in soybean meal when fed to growing pigs.
Soybean expellers may be produced by dry extrusion and mechanical oil pressing of soybeans, but there is limited information about the nutritional value of expellers produced via this procedure. Therefore, 2 experiments were conducted to test the hypothesis that standardized ileal digestibility (SID) of CP and amino acids (AA), apparent total tract digestibility (ATTD) of energy and total dietary fiber (TDF), and concentrations of DE, ME, and NE are greater in soybean expellers than in soybean meal (SBM) when fed to growing pigs. Pigs were the offspring of Line 359 boars mated to Camborough females (Pig Improvement Company, Hendersonville, TN). In experiment 1, nine growing barrows (initial BW: 55.98 kg ± 13.75 kg) with T-cannulas installed in the distal ileum were allotted to 1 of 3 diets using a triplicated 3 × 3 Latin square design with 3 periods. Two diets were formulated to contain 35% soybean expellers or 33% SBM as the sole source of AA. A N-free diet was used to determine basal endogenous losses of AA. Ileal digesta were collected on days 6 and 7 of each 7-d period. Results indicated that the SID of most indispensable and dispensable AA were greater (P < 0.05) or tended (P < 0.10) to be greater in soybean expellers than in SBM. In experiment 2, a corn-based diet and 2 diets based on corn and each of the 2 soybean products were formulated. Twenty-four growing barrows (initial BW: 44.88 kg ± 2.17 kg) were allotted to 1 of the 3 diets with 8 pigs per diet. Urine and fecal samples were collected for 4 d after 5 d of adaptation. Results indicated that the ATTD of energy and TDF was not different between soybean expellers and SBM, but the ATTD of TDF in the 2 soybean products was greater (P < 0.05) than in corn. Concentrations of DE and ME in soybean expellers were greater (P < 0.05) compared with corn or SBM. Soybean expellers had greater (P < 0.05) calculated NE compared with SBM, but there was no difference in NE between corn and soybean expellers. In conclusion, values for SID of most AA and DE, ME, and NE in soybean expellers were greater than in SBM.


Obesity Paradox: Comparison of Heart Failure Patients With and Without Comorbid Diabetes.
Diabetes is a common comorbid condition in patients with heart failure and is strongly associated with poor outcomes. Patients with heart failure who have diabetes are more likely to be obese than are those without diabetes. Obesity is positively associated with survival in patients with heart failure, but how comorbid diabetes influences the relationship between obesity and favorable prognosis is unclear. To explore whether the relationship between body mass index and survival differs between patients with heart failure who do or do not have diabetes. The sample consisted of 560 ambulatory patients with heart failure (mean age, 66 years; mean body mass index, 32; diabetes, 41%). The association between body mass index and all-cause mortality was examined by using multivariate Cox proportional hazards regression after adjustments for covariates. In patients without diabetes, higher body mass index was associated with a lower risk for all-cause mortality after adjustments for covariates (hazard ratio, 0.952; 95% CI, 0.909-0.998). In patients with diabetes, body mass index was not predictive of all-cause death after adjustments for covariates. Obesity was a survival benefit in heart failure patients without comorbid diabetes but not in those with comorbid diabetes. The mechanisms underlying the difference in the relationship between obesity and survival due to the presence of diabetes in patients with heart failure need to be elucidated.


Extension of Lander-Waterman theory for sequencing filtered DNA libraries.
The degree to which conventional DNA sequencing techniques will be successful for highly repetitive genomes is unclear. Investigators are therefore considering various filtering methods to select against high-copy sequence in DNA clone libraries. The standard model for random sequencing, Lander-Waterman theory, does not account for two important issues in such libraries, discontinuities and position-based sampling biases (the so-called "edge effect"). We report an extension of the theory for analyzing such configurations. The edge effect cannot be neglected in most cases. Specifically, rates of coverage and gap reduction are appreciably lower than those for conventional libraries, as predicted by standard theory. Performance decreases as read length increases relative to island size. Although opposite of what happens in a conventional library, this apparent paradox is readily explained in terms of the edge effect. The model agrees well with prototype gene-tagging experiments for Zea mays and Sorghum bicolor. Moreover, the associated density function suggests well-defined probabilistic milestones for the number of reads necessary to capture a given fraction of the gene space. An exception for applying standard theory arises if sequence redundancy is less than about 1-fold. Here, evolution of the random quantities is independent of library gaps and edge effects. This observation effectively validates the practice of using standard theory to estimate the genic enrichment of a library based on light shotgun sequencing. Coverage performance using a filtered library is significantly lower than that for an equivalent-sized conventional library, suggesting that directed methods may be more critical for the former. The proposed model should be useful for analyzing future projects.


Pituitary-adrenal responsiveness to corticotropin-releasing hormone in patients receiving chronic, alternate day glucocorticoid therapy.
We examined the responsiveness of the pituitary-adrenal axis to ovine corticotropin-releasing hormone (oCRH) in 14 women with systemic lupus erythematosus receiving chronic, alternate day glucocorticoid therapy with prednisone. Testing was done twice and in a random order (at 2000 h) on the day when the steroid was taken (12 h after the last dose) and on the day when no glucocorticoid was administered (36 h after the last dose). Plasma ACTH and cortisol responses were markedly blunted on the day of treatment and mildly blunted on the day off treatment compared to those in normal subjects. Altered metabolic clearance of exogenous oCRF was not responsible for this difference, since the plasma disappearance curves of immunoreactive oCRH were similar on both days. The degree of suppression was dependent on the dose of prednisone, and the amount of cortisol secreted during the oCRH test was directly proportional to the logarithm of the concurrent plasma ACTH level. Thus, the cortisol response to ACTH was normal in all patients. These data suggest that the blunting of responsiveness to oCRH on both days of testing represents prednisolone suppression of the corticotroph cell. Despite this, the adrenal glands retain normal responsiveness to ACTH, suggesting that moderate decreases in daily ACTH secretion are compatible with sustaining normal adrenal function. Hence, the site of the mild suppression of the hypothalamic-pituitary-adrenal axis during chronic, alternate day treatment with glucocorticoids is central, whereas the adrenal glands appear to remain functionally unaffected.


Resolving Recent Plant Radiations: Power and Robustness of Genotyping-by-Sequencing.
Disentangling species boundaries and phylogenetic relationships within recent evolutionary radiations is a challenge due to the poor morphological differentiation and low genetic divergence between species, frequently accompanied by phenotypic convergence, interspecific gene flow and incomplete lineage sorting. Here we employed a genotyping-by-sequencing (GBS) approach, in combination with morphometric analyses, to investigate a small western Mediterranean clade in the flowering plant genus Linaria that radiated in the Quaternary. After confirming the morphological and genetic distinctness of eight species, we evaluated the relative performances of concatenation and coalescent methods to resolve phylogenetic relationships. Specifically, we focused on assessing the robustness of both approaches to variations in the parameter used to estimate sequence homology (clustering threshold). Concatenation analyses suffered from strong systematic bias, as revealed by the high statistical support for multiple alternative topologies depending on clustering threshold values. By contrast, topologies produced by two coalescent-based methods (NJ$_{\mathrm{st}}$, SVDquartets) were robust to variations in the clustering threshold. Reticulate evolution may partly explain incongruences between NJ$_{\mathrm{st}}$, SVDquartets and concatenated trees. Integration of morphometric and coalescent-based phylogenetic results revealed (i) extensive morphological divergence associated with recent splits between geographically close or sympatric sister species and (ii) morphological convergence in geographically disjunct species. These patterns are particularly true for floral traits related to pollinator specialization, including nectar spur length, tube width and corolla color, suggesting pollinator-driven diversification. Given its relatively simple and inexpensive implementation, GBS is a promising technique for the phylogenetic and systematic study of recent radiations, but care must be taken to evaluate the robustness of results to variation of data assembly parameters.


Performance of soccer passing skills under moderate and high-intensity localized muscle fatigue.
Despite the acknowledged importance of fatigue on performance, ecologically sound studies investigating fatigue and its effects on sport-specific skills are surprisingly limited. The aim of this study was to investigate the effects of moderate and high-intensity localized muscle fatigue on passing performance in soccer. Twenty physically active male college students (age, 22.9 +/- 5.3 years) participated in this study. Subjects performed the modified Loughborough Soccer Passing Test under the following three conditions: rest, moderate fatigue, and high-intensity fatigue. Fatigue intensity was established using a percentage of the maximal number of alternate split squats performed by the subject in 1 minute. Results revealed a significant difference between performance at rest and performance following high-intensity, localized muscle fatigue. The results suggest the need for trainers and coaches to incorporate high-intensity exercise drills into training plans to help players better cope with the demands of the game on the pitch.


Tudor-domain protein PHF20L1 reads lysine methylated retinoblastoma tumour suppressor protein.
The retinoblastoma tumour suppressor protein (pRb) classically functions to regulate early cell cycle progression where it acts to enforce a number of checkpoints in response to cellular stress and DNA damage. Methylation at lysine (K) 810, which occurs within a critical CDK phosphorylation site and antagonises a CDK-dependent phosphorylation event at the neighbouring S807 residue, acts to hold pRb in the hypo-phosphorylated growth-suppressing state. This is mediated in part by the recruitment of the reader protein 53BP1 to di-methylated K810, which allows pRb activity to be effectively integrated with the DNA damage response. Here, we report the surprising observation that an additional methylation-dependent interaction occurs at K810, but rather than the di-methyl mark, it is selective for the mono-methyl K810 mark. Binding of the mono-methyl PHF20L1 reader to methylated pRb occurs on E2F target genes, where it acts to mediate an additional level of control by recruiting the MOF acetyltransferase complex to E2F target genes. Significantly, we find that the interplay between PHF20L1 and mono-methyl pRb is important for maintaining the integrity of a pRb-dependent G1-S-phase checkpoint. Our results highlight the distinct roles that methyl-lysine readers have in regulating the biological activity of pRb.


Thrombosed giant aneurysm of the distal anterior cerebral artery treated with aneurysm resection and proximal pericallosal artery-callosomarginal artery end-to-end anastomosis: Case report and review of the literature.
Giant distal anterior cerebral artery (DACA) aneurysms are extremely rare, with only 32 cases reported in the literature. Most giant DACA aneurysms have features that make standard neck clipping difficult, and bypass surgery is sometimes required, although this surgery was performed in only three reported cases. This report presents the fourth case treated with bypass surgery. A 69-year-old female presented with an unruptured thrombosed giant DACA aneurysm. She underwent wrapping operation 7 years before, but radiological imaging revealed enlargement of the aneurysm at the left pericallosal artery (PerA)-callosomarginal artery (CMA) junction. Before operation, three different strategies were considered for bypass surgery in case the neck could not be clipped. Aneurysm resection and left proximal PerA-CMA end-to-end anastomosis were successfully performed under intraoperative digital subtraction angiography (DSA) and motor-evoked potential (MEP) monitoring. Most DACA aneurysms are located at the PerA-CMA junction. In some cases, adequate retrograde flow to the distal PerA from the posterior or middle cerebral artery can be expected, making distal PerA reconstruction unnecessary. Moreover, when the distal PerA is cut, proximal PerA-CMA end-to-end anastomosis can be easily performed because of reduced tension in both vessels. We therefore conclude that this strategy should be utilized for treating such patients. We also presented here the effectiveness of intraoperative modalities, such as intraoperative DSA and MEP monitoring, for performing a safe operation.