Characterization of the specific interaction between sialoadhesin and sialylated Campylobacter jejuni lipooligosaccharides.
In Campylobacter jejuni-induced Guillain-Barré syndrome (GBS), molecular mimicry between C. jejuni lipooligosaccharide (LOS) and host gangliosides leads to the production of cross-reactive antibodies directed against the peripheral nerves of the host. Currently, the presence of surface exposed sialylated LOS in C. jejuni is the single known bacterial pathogenesis factor associated with the development of GBS. Using a unique, well-characterized strain collection, we demonstrate that GBS-associated C. jejuni strains bind preferentially to sialoadhesin (Sn, Siglec-1, or CD169), a sialic acid receptor found on a subset of macrophages. In addition, using a whole-cell enzyme-linked immunosorbent assay (ELISA), C. jejuni strains with sialylated LOS bound exclusively to soluble Sn. Mass spectrometry revealed that binding was sialic acid-linkage specific with a preference for alpha(2,3)-linked sialic acid attached to the terminal galactose of the LOS chain as seen in the gangliosides GD1a, GM1b, and GM3. This molecular interaction was also related to functional consequences as a GBS-associated C. jejuni strain that bound Sn in a whole-cell ELISA adhered to surface-expressed Sn of Sn-transfected CHO cells but was unable to adhere to wild-type CHO cells. Moreover, a sialic acid-negative mutant of the same C. jejuni strain was unable to bind Sn-transfected CHO cells. This is the first report of the preferential binding of GBS-associated C. jejuni strains to the Sn immune receptor (P = 0.014). Moreover, because this binding is dependent on sialylated LOS, the main pathogenic factor in GBS progression, the present findings bring us closer to unraveling the mechanisms that lead to formation of cross-reactive antibodies in GBS disease.


Simultaneous chromatic dispersion and PMD compensation by using coded-OFDM and girth-10 LDPC codes.
Low-density parity-check (LDPC)-coded orthogonal frequency division multiplexing (OFDM) is studied as an efficient coded modulation scheme suitable for simultaneous chromatic dispersion and polarization mode dispersion (PMD) compensation. We show that, for aggregate rate of 10 Gb/s, accumulated dispersion over 6500 km of SMF and differential group delay of 100 ps can be simultaneously compensated with penalty within 1.5 dB (with respect to the back-to-back configuration) when training sequence based channel estimation and girth-10 LDPC codes of rate 0.8 are employed.


Butyrate attenuates inflammation and lipolysis generated by the interaction of adipocytes and macrophages.
Paracrine interaction between macrophages and adipocytes in obese visceral fat tissues is thought to be a trigger of chronic inflammation. The immunomodulatory effect of the short chain fatty acid, butyric acid, has been demonstrated. We hypothesize that sodium butyrate (butyrate) attenuates inflammatory responses and lipolysis generated by the interaction of macrophages and adipocytes. Using contact or transwell co-culture methods with differentiated 3T3-L1 adipocytes and RAW264.7 macrophages, we investigated the effects of butyrate on the production of tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), and the release of free glycerol, free fatty acids (FFAs) into the medium. We also examined the activity of nuclear factor-kappaB (NF-κB) and the phosphorylation of mitogen-activated protein kinases (MAPKs) in co-cultured macrophages, as well as lipase activity and expression in co-cultured adipocytes. We found increased production of TNF-α, MCP-1, IL-6, and free glycerol, FFAs in the co-culture medium, and butyrate significantly reduced them. Butyrate inhibited the phosphorylation of MAPKs, the activity of NF-κB in co-cultured macrophages, and suppressed lipase activity in co-cultured adipocytes. Lipase inhibitors significantly attenuated the production of TNF-α, MCP-1 and IL-6 in the co-culture medium as effectively as butyrate. Butyrate suppressed the protein production of adipose triglyceride lipase, hormone sensitive lipase, and fatty acid-binding protein 4 in co-cultured adipocytes. Pertussis toxin, which is known to block GPR41 completely, inhibited the antilipolysis effect of butyrate. Butyrate suppresses inflammatory responses generated by the interaction of adipocytes and macrophages through reduced lipolysis and inhibition of inflammatory signaling.


A threshold-free summary index for quantifying the capacity of covariates to yield efficient treatment rules.
When data on treatment assignment, outcomes, and covariates from a randomized trial are available, a question of interest is to what extent covariates can be used to optimize treatment decisions. Statistical hypothesis testing of covariate-by-treatment interaction is ill-suited for this purpose. The application of decision theory results in treatment rules that compare the expected benefit of treatment given the patient's covariates against a treatment threshold. However, determining treatment threshold is often context-specific, and any given threshold might seem arbitrary when the overall capacity towards predicting treatment benefit is of concern. We propose the Concentration of Benefit index (Cb ), a threshold-free metric that quantifies the combined performance of covariates towards finding individuals who will benefit the most from treatment. The construct of the proposed index is comparing expected treatment outcomes with and without knowledge of covariates when one of a two randomly selected patients is to be treated. We show that the resulting index can also be expressed in terms of the integrated efficiency of individualized treatment decision over the entire range of treatment thresholds. We propose parametric and semiparametric estimators, the latter being suitable for out-of-sample validation and correction for optimism. We used data from a clinical trial to demonstrate the calculations in a step-by-step fashion. The proposed index has intuitive and theoretically sound interpretation and can be estimated with relative ease for a wide class of regression models. Beyond the conceptual developments, various aspects of estimation and inference for such a metric need to be pursued in future research. R code that implements the method for a variety of regression models is provided at (https://github.com/msadatsafavi/txBenefit).


Concerns about the widespread use of rodent models for human risk assessments of endocrine disruptors.
Fetal testis is a major target of endocrine disruptors (EDs). During the last 20 years, we have developed an organotypic culture system that maintains the function of the different fetal testis cell types and have used this approach as a toxicological test to evaluate the effects of various compounds on gametogenesis and steroidogenesis in rat, mouse and human testes. We named this test rat, mouse and human fetal testis assay. With this approach, we compared the effects of six potential EDs ((mono-(2-ethylhexyl) phthalate (MEHP), cadmium, depleted uranium, diethylstilboestrol (DES), bisphenol A (BPA) and metformin) and one signalling molecule (retinoic acid (RA)) on the function of rat, mouse and human fetal testis at a comparable developmental stage. We found that the response is similar in humans and rodents for only one third of our analyses. For instance, RA and MEHP have similar negative effects on gametogenesis in the three species. For another third of our analyses, the threshold efficient concentrations that disturb gametogenesis and/or steroidogenesis differ as a function of the species. For instance, BPA and metformin have similar negative effects on steroidogenesis in human and rodents, but at different threshold doses. For the last third of our analyses, the qualitative response is species specific. For instance, MEHP and DES affect steroidogenesis in rodents, but not in human fetal testis. These species differences raise concerns about the extrapolation of data obtained in rodents to human health risk assessment and highlight the need of rigorous comparisons of the effects in human and rodent models, when assessing ED risk.


Orchidectomy affects the innervation of the rat thymus.
To assess a putative role of the neural pathways in transfer of information from the gonads to the thymus, adult AO rats were orchidectomized (ORX) or sham ORX (controls); sacrificed 1, 3, 7, or 15 days later and their thymi were analyzed for: (a) the concentrations of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) and distribution of monoamine-containing nerve profiles and (b) the acetylcholinesterase (AChE) activity and distribution of AChE-containing nerve profiles. Three days after the castration, an elevation in the level of both catecholamines, reflecting an increase in the overall intensity of nerve fibers autofluorescence, was found. Seven days post castration neither NA nor DA concentration differed from the appropriate control values, while 15 days after the surgery the concentration of NA was lower than that in the controls, most likely, due to diminished density of noradrenergic nerve profiles. In both the rats sacrificed 7 and 15 days after orchidectomy the concentration of 5-HT was reduced as result of a decrease in the density of 5-HT-containing autofluorescent cells. The activity of AChE was depressed one day after the surgery; then increased, so that 3 days post castration its value was higher than that in the sham ORX. After this increase, AChE activity decreased being, at postoperative day 7 and 15, lower than that in the controls. It seems that this decrease in AChE activity reflected, not only a reduction in the density of AChE-containing nerve fibers, but also a decrease in the density of AChE positive cells. Thus, the results indicate that orchidectomy can evoke changes in the T-cell maturation altering modulatory influences on this process coming via neural route, as well as those coming from the mast cells and AChE positive epithelial cells which constitute important component of the thymus microenvironment.


Technical performance reduces during the extra-time period of professional soccer match-play.
Despite the importance of extra-time in determining progression in specific soccer tournament matches, few studies have profiled the demands of 120-minutes of soccer match-play. With a specific focus on the extra-time period, and using a within-match approach, we examined the influence of prolonged durations of professional soccer match-play on markers of technical (i.e., skilled) performance. In 18 matches involving professional European teams played between 2010 and 2014, this retrospective study quantified the technical actions observed during eight 15-minute epochs (E1: 00∶00-14∶59 min, E2: 15∶00-29∶59 min, E3: 30∶00-44∶59 min, E4: 45∶00-59∶59 min, E5: 60∶00-74∶59 min, E6: 75∶00-89∶59 min, E7: 90∶00-104∶59 min, E8: 105∶00-119∶59 min). Analysis of players who completed the demands of the full 120 min of match-play revealed that the cumulative number of successful passes observed during E8 (61±23) was lower than E1-4 (E1: 88±23, P = 0.001; E2: 77±21, P = 0.005; E3: 79±18, P = 0.001; E4: 80±21, P = 0.001) and E7 (73±20, P = 0.002). Similarly, the total number of passes made in E8 (71±25) was reduced when compared to E1 (102±22, P = 0.001), E3 (91±19, P = 0.002), E4 (93±22, P≤0.0005) and E7 (84±20, P = 0.001). The cumulative number of successful dribbles reduced in E8 (9±4) when compared to E1 (14±4, P = 0.001) and E3 (12±4, P≤0.0005) and the total time the ball was in play was less in E8 (504±61 s) compared to E1 (598±70 s, P≤0.0005). These results demonstrate that match-specific factors reduced particular indices of technical performance in the second half of extra-time. Interventions that seek to maintain skilled performance throughout extra-time warrant further investigation.


High-throughput quantum cascade laser (QCL) spectral histopathology: a practical approach towards clinical translation.
Infrared microscopy has become one of the key techniques in the biomedical research field for interrogating tissue. In partnership with multivariate analysis and machine learning techniques, it has become widely accepted as a method that can distinguish between normal and cancerous tissue with both high sensitivity and high specificity. While spectral histopathology (SHP) is highly promising for improved clinical diagnosis, several practical barriers currently exist, which need to be addressed before successful implementation in the clinic. Sample throughput and speed of acquisition are key barriers and have been driven by the high volume of samples awaiting histopathological examination. FTIR chemical imaging utilising FPA technology is currently state-of-the-art for infrared chemical imaging, and recent advances in its technology have dramatically reduced acquisition times. Despite this, infrared microscopy measurements on a tissue microarray (TMA), often encompassing several million spectra, takes several hours to acquire. The problem lies with the vast quantities of data that FTIR collects; each pixel in a chemical image is derived from a full infrared spectrum, itself composed of thousands of individual data points. Furthermore, data management is quickly becoming a barrier to clinical translation and poses the question of how to store these incessantly growing data sets. Recently, doubts have been raised as to whether the full spectral range is actually required for accurate disease diagnosis using SHP. These studies suggest that once spectral biomarkers have been predetermined it may be possible to diagnose disease based on a limited number of discrete spectral features. In this current study, we explore the possibility of utilising discrete frequency chemical imaging for acquiring high-throughput, high-resolution chemical images. Utilising a quantum cascade laser imaging microscope with discrete frequency collection at key diagnostic wavelengths, we demonstrate that we can diagnose prostate cancer with high sensitivity and specificity. Finally we extend the study to a large patient dataset utilising tissue microarrays, and show that high sensitivity and specificity can be achieved using high-throughput, rapid data collection, thereby paving the way for practical implementation in the clinic.


Real-time dynamics of dopamine released from neuronal transplants in experimental Parkinson's disease.
Intrastriatal transplantation of foetal midbrain dopamine (DA) neurons ameliorates the fundamental symptoms of dopaminergic denervation in clinical and experimental parkinsonism despite providing only restricted reinnervation. To understand how DA function is restored by these grafts we used fast-scan cyclic voltammetry at a carbon-fiber microelectrode in vitro to monitor directly and in "real time" the dynamics of graft-derived DA. Simulations of Michaelis-Menten kinetics were used to model the experimental observations. We show that the concentration of DA released by a single depolarizing pulse is significantly lower in grafted than intact striata. On the other hand, the extracellular lifetime of DA in grafts is extended due to a marked reduction in the rate maximum (V(max)) for DA reuptake by the DA transporter. Moreover, variations in V(max) and release occur in parallel: where DA release is lowest, V(max) is lowest and vice versa. The consequences of these dynamics are twofold. First, during repeated depolarization at a physiological firing frequency, when net extracellular concentrations reflect DA release versus uptake, ambient levels of extracellular DA within the graft are restored to normal. Second, the protracted extracellular lifetime of DA will increase the number and extracellular sphere of its postsynaptic actions. This effect will be most prominent where DA availability (and thus V(max)) is most restricted. Thus, these data demonstrate that dopaminergic grafts restore striatal dopaminergic function with extracellular dynamics of DA that are different from those of intact striatum but which can normalize ambient DA levels and permit transmission over an extended sphere.


Dimensional changes of demineralized human dentine during preparation for scanning electron microscopy.
Dentine rods measuring approx. 0.7 x 0.7 x 5.0mm were prepared from the crowns of extracted human third molars. The specimens were demineralized in 0.5 M EDTA (pH 7.0) for 3 days and their volume measured with a digital micrometer under a dissecting microscope. The specimens were randomly assigned to experimental groups and were chemically dehydrated in acetone. Next they were dried using either hexamethyldisilazane, Peldri II, or critical-point drying techniques. The dimensions of the specimens were measured again after each step and the changes in volume were expressed as a percentage of the original demineralized volume. The effects of fixing the specimens in 10% buffered formalin before dehydration with acetone were also investigated for every drying procedure. Dehydration in acetone caused a small but significant reduction in the volume of demineralized formalin-fixed specimens but unfixed specimens did not change significantly. In general, all three drying procedures caused some shrinkage in demineralized dentine specimens. Unfixed specimens exhibited a volumetric shrinkage of approx. 15-20% after drying with any of the methods. Fixed specimens shrank more than unfixed specimens after drying (25-35%). Regardless of the drying technique, the specimens shrank a further 10-20% when measured in the vacuum chamber of the scanning electron microscope. Among the three drying techniques employed, hexamethyldisilazane seems to be a very useful alternative to critical-point drying for the preparation of dentine specimens for scanning electron microscopy.


Improvements in exercise performance with high-intensity interval training coincide with an increase in skeletal muscle mitochondrial content and function.
Six sessions of high-intensity interval training (HIT) are sufficient to improve exercise capacity. The mechanisms explaining such improvements are unclear. Accordingly, the aim of this study was to perform a comprehensive evaluation of physiologically relevant adaptations occurring after six sessions of HIT to determine the mechanisms explaining improvements in exercise performance. Sixteen untrained (43 ± 6 ml·kg(-1)·min(-1)) subjects completed six sessions of repeated (8-12) 60 s intervals of high-intensity cycling (100% peak power output elicited during incremental maximal exercise test) intermixed with 75 s of recovery cycling at a low intensity (30 W) over a 2-wk period. Potential training-induced alterations in skeletal muscle respiratory capacity, mitochondrial content, skeletal muscle oxygenation, cardiac capacity, blood volumes, and peripheral fatigue resistance were all assessed prior to and again following training. Maximal measures of oxygen uptake (Vo2peak; ∼8%; P = 0.026) and cycling time to complete a set amount of work (∼5%; P = 0.008) improved. Skeletal muscle respiratory capacities increased, most likely as a result of an expansion of skeletal muscle mitochondria (∼20%, P = 0.026), as assessed by cytochrome c oxidase activity. Skeletal muscle deoxygenation also increased while maximal cardiac output, total hemoglobin, plasma volume, total blood volume, and relative measures of peripheral fatigue resistance were all unaltered with training. These results suggest that increases in mitochondrial content following six HIT sessions may facilitate improvements in respiratory capacity and oxygen extraction, and ultimately are responsible for the improvements in maximal whole body exercise capacity and endurance performance in previously untrained individuals.


Computed tomography perfusion in evaluating the therapeutic effect of transarterial chemoembolization for hepatocellular carcinoma.
To prospectively assess the changes in parameters of computed tomography (CT) perfusion pre- and post-transarterial chemoembolization (TACE) of hepatocellular carcinoma (HCC) in different treatment response groups, and to correlate the changes with various responses of HCC to TACE. Thirty-nine HCC patients underwent CT perfusion examinations pre-(1 d before TACE) and post-treatment (4 wk after TACE). The response evaluation criteria for solid tumors (RECIST) were referred to when treatment responses were distributed. Wilcoxon-signed ranks test was used to compare the differences in CT perfusion parameters pre- and post-TACE for different response groups. Only one case had treatment response to CR and the CT perfusion maps of post-treatment lesion displayed complete absence of signals. In the PR treatment response group, hepatic artery perfusion (HAP), hepatic arterial fracture (HAF) and hepatic blood volume (HBV) of viable tumors post-TACE were reduced compared with pre-TACE (P = 0.001, 0.030 and 0.001, respectively). In the SD group, all CT perfusion parameters were not significantly different pre- and post-TACE. In the PD group, HAP, HAF, portal vein perfusion (PVP) and hepatic blood flow (HBF) of viable tumors post-TACE were significantly increased compared with pre-TACE (P = 0.005, 0.012, 0.035 and 0.005, respectively). Changes in CT perfusion parameters of viable tumors are correlated with different responses of HCC to TACE. Therefore, CT perfusion imaging is a feasible technique for monitoring response of HCC to TACE.


Three-dimensional coherent transfer function for reflection confocal microscopy in the presence of refractive-index mismatch.
The three-dimensional (3-D) coherent transfer function for reflection confocal microscopy of high-numerical-aperture objectives is derived and calculated in the presence of refractive-index mismatch when a laser beam is focused into a medium of refractive index different from its immersion medium. This aberrated coherent transfer function is then used to estimate the readout efficiency of 3-D data bits recorded in a thick medium. It is shown that the readout efficiency of confocal microscopy for 3-D bit data storage is decreased with the focal depth of an objective in a recording medium. However, a high readout efficiency can be maintained if the tube length of a reading objective is linearly altered to compensate for the spherical aberration caused by the refractive-index mismatch.


Fermentation-induced variation in heat and oxidative stress phenotypes of Lactococcus lactis MG1363 reveals transcriptome signatures for robustness.
Lactococcus lactis is industrially employed to manufacture various fermented dairy products. The most cost-effective method for the preservation of L. lactis starter cultures is spray drying, but during this process cultures encounter heat and oxidative stress, typically resulting in low survival rates. However, viability of starter cultures is essential for their adequate contribution to milk fermentation, supporting the ambition to better understand and improve their robustness phenotypes. This study describes a transcriptome-phenotype matching approach in which the starter L. lactis MG1363 was fermented under a variety of conditions that differed in the levels of oxygen and/or salt, as well as the fermentation pH and temperature. Samples derived from these fermentations in the exponential phase of bacterial growth were analyzed by full-genome transcriptomics and the assessment of heat and oxidative stress phenotypes. Variations in the fermentation conditions resulted in up to 1000-fold differences in survival during heat and oxidative stress. More specifically, aeration during fermentation induced protection against heat stress, whereas a relatively high fermentation temperature resulted in enhanced robustness towards oxidative stress. Concomitantly, oxygen levels and fermentation temperature induced differential expression of markedly more genes when compared with the other fermentation parameters. Correlation analysis of robustness phenotypes and gene expression levels revealed transcriptome signatures for oxidative and/or heat stress survival, including the metC-cysK operon involved in methionine and cysteine metabolism. To validate this transcriptome-phenotype association we grew L. lactis MG1363 in the absence of cysteine which led to enhanced robustness towards oxidative stress. Overall, we demonstrated the importance of careful selection of fermentation parameters prior to industrial processing of starter cultures. Furthermore, established stress genes as well as novel genes were associated with robustness towards heat and/or oxidative stress. Assessment of the expression levels of this group of genes could function as an indicator for enhanced selection of fermentation parameters resulting in improved robustness during spray drying. The increased robustness after growth without cysteine appeared to confirm the role of expression of the metC-cysK operon as an indicator of robustness and suggests that sulfur amino acid metabolism plays a pivotal role in oxidative stress survival.


A primary health-care intervention on pre- and postnatal risk factor behavior to prevent childhood allergy. The Prevention of Allergy among Children in Trondheim (PACT) study.
This study aimed to evaluate the impact of a primary prevention intervention program on risk behavior for allergic diseases among children up to 2 years of age. The setting was in ordinary pre- and postnatal primary health care in Trondheim, Norway. The Prevention of Allergy among Children in Trondheim, Norway (PACT) study invited all pregnant women and parents to children up to 2 years of age in the community to participate in a non-randomized, controlled, multiple life-style intervention study. Interventional topics was increased dietary intake of cod liver oil and oily fish for women during pregnancy and for infants during the first 2 years of life, reduced parental smoking and reduced indoor dampness. A control cohort was established prior to the intervention cohort with "follow up as usual". Questionnaires were completed in pregnancy, 6 weeks after birth and at 1 and 2 years of age. Trends in exposure and behavior are described. Intake of oily fish and cod liver oil increased statistically significantly among women and infants in the intervention cohort compared to the control cohort. There was a low postnatal smoking prevalence in both cohorts, with a trend towards a decreasing smoking prevalence in the control cohort. There was no change in indoor dampness or in behavior related to non- intervened life-style factors. The dietary intervention seemed to be successful. The observed reduced smoking behavior could not be attributed to the intervention program, and the latter had no effect on indoor dampness. (Current Controlled Trials registration number: ISRCTN28090297).


Ultra-high-throughput DArTseq-based silicoDArT and SNP markers for genomic studies in macadamia.
Macadamia (Macadamia integrifolia, M. tetraphylla and hybrids) is an Australian native nut crop and has a significant economic value in the food industries worldwide. Long juvenility along with traditional breeding strategies impede quick genetic improvement of this crop. The existing cultivars constitute only second to fourth generation of the wild germplasm in the rainforest. The utilisation of molecular markers for genomic selection and genome-wide association studies may accelerate genetic gains. Identification of a robust, reproducible, and cost-effective marker system is instrumental in increasing the efficiency of genomic studies. This study is the first to report the potential of two ultra-high-throughput diversity array technology (DArT) markers (silicoDArT and SNP) in macadamia. Both markers were used to identify the genetic diversity and population structure in 80 macadamia cultivars. Parentage analysis of 25 scions in a rootstock trial was conducted to confirm plant identity where recorded identities did not corroborate with phenotypic field observations. A total of 22,280 silicoDArT and 7,332 SNP markers were reported, of which 11,526 silicoDArT and 3,956 SNP markers were used for analyses after screening with quality control parameters including >95% call rate, >95% reproducibility, and >0.05 one ratio. The average polymorphic information content (PIC) values of silicoDArT and SNP markers were 0.29 and 0.21, respectively. Genetic variance among the cultivars ranged from 0.003 to 0.738 in silicoDArT and 0.004 to 0.412 in SNP markers. Four distinct population groups were identified from SNP data analysis. Most of the accessions used in this study were descended from two or more populations. Cluster analysis clearly separated genotypes of distinct origins, such as the Hawaii Agricultural Experiment Station and Hidden Valley Plantation accessions. Two wild accessions of Macadamia jansenii and M. ternifolia were found to be distantly related to the cultivars. Wild germplasm individuals and their hybrids with cv. '660' formed separate clusters, suggesting that crossing between wild and cultivated genepools can extend genetic diversity. DArTseq-based SNP markers were successfully utilized to confirm the genetic identity of 25 scions in a rootstock trial. Our study suggests that DArT platforms are a robust system for the facilitation of genomic studies with regard to macadamia.


Lameness in dairy cattle: A debilitating disease or a disease of debilitated cattle? A cross-sectional study of lameness prevalence and thickness of the digital cushion.
Lameness is the most significant challenge for the dairy industry to overcome, given its obvious disruption of animal welfare and severe economic losses. Sole ulcers and white line abscesses are ubiquitous chronic diseases with the highest associated economic losses among all foot lesions. Their underlying causes are still not fully understood. An observational cross-sectional study was carried out to investigate the association between claw horn lesions and the thickness of the digital cushion.The thickness of the digital cushion was evaluated by ultrasonographic examination of the sole at the typical ulcer site. A total of 501 lactating Holstein dairy cows were enrolled in the study. The prevalence of sole ulcers was 4.2 and 27.8% for parity 1 and parity >1, respectively.The prevalence of white line disease was 1.0 and 6.5% for parity 1 and >1, respectively. The prevalence of lameness (visual locomotion score > or = 3) was 19.8 and 48.2% for parity 1 and >1, respectively. The prevalence of sole ulcers and white line diseases was significantly associated with thickness of the digital cushion; cows in the upper quartile of digital cushion thickness had an adjusted prevalence of lameness 15 percentage points lower than the lower quartile. Body condition scores were positively associated with digital cushion thickness.The mean gray value of the sonographic image of the digital cushion had a negative linear association with digital cushion thickness (R2 = 0.14), indicating that the composition of the digital cushion may have changed with its thickness. Furthermore, digital cushion thickness decreased steadily from the first month of lactation and reached a nadir 120 d after parturition.These results support the concept that sole ulcers and white line abscesses are related to contusions within the claw horn capsule and such contusions are a consequence of the lesser capacity of the digital cushion to dampen the pressure exerted by the third phalanx on the soft tissue beneath.


FBXL13 directs the proteolysis of CEP192 to regulate centrosome homeostasis and cell migration.
Aberrant centrosome organisation with ensuing alterations of microtubule nucleation capacity enables tumour cells to proliferate and invade despite increased genomic instability. CEP192 is a key factor in the initiation process of centrosome duplication and in the control of centrosome microtubule nucleation. However, regulatory means of CEP192 have remained unknown. Here, we report that FBXL13, a binding determinant of SCF (SKP1-CUL1-F-box)-family E3 ubiquitin ligases, is enriched at centrosomes and interacts with the centrosomal proteins Centrin-2, Centrin-3, CEP152 and CEP192. Among these, CEP192 is specifically targeted for proteasomal degradation by FBXL13. Accordingly, induced FBXL13 expression downregulates centrosomal γ-tubulin and disrupts centrosomal microtubule arrays. In addition, depletion of FBXL13 induces high levels of CEP192 and γ-tubulin at the centrosomes with the consequence of defects in cell motility. Together, we characterise FBXL13 as a novel regulator of microtubule nucleation activity and highlight a role in promoting cell motility with potential tumour-promoting implications.


Craniocervical instability associated with rheumatoid arthritis: a case report and brief review.
Rheumatoid arthritis (RA) is an autoimmune disease that affects the synovial tissue which lines joints and tendons. The craniocervical junction is made up exclusively of synovial joints and ligaments and especially vulnerable to the inflammatory process of RA. The chronic inflammation of RA leads to loss of ligamentous restriction and erosion of the bony structures and results in craniocervical instability (CCI). This is a case report of an 80-year-old woman who had been diagnosed with seropositive RA two decades ago presented with head dropping and losing balance while walking for several months. Radiographic images of the cervical spine showed RA-related features of instability in the form of atlantoaxial instability, cranial settling and subaxial subluxation. Since physical therapy and acupuncture previously failed to provide a substantial, long-lasting outcome, the patient sought chiropractic care for her condition. The chiropractic regimen consisted of upper thoracic spine mobilization/adjustment, electrical muscle stimulation of the cervical extensors, home exercises and neck bracing. She regained substantial neck muscle strength, gaze angle and walking balance following a 4-month chiropractic treatment, although cervical kyphosis persisted. The current study aims to provide basic knowledge of CCI associated with RA and ability to modify a treatment program to accommodate the needs of patients with coexisting red flags.


Management of nodal diffuse large B-cell lymphomas: practice guidelines from the Italian Society of Hematology, the Italian Society of Experimental Hematology and the Italian Group for Bone Marrow Transplantation.
The Italian Society of Hematology (SIE) and two affiliate societies (SIES and GITMO) commissioned a project to develop clinical practice guidelines for the treatment of nodal diffuse large B-cell non Hodgkin lymphomas (DLBCL). Key questions clinically relevant to the management of patients with nodal DLBCL were formulated by an Advisory Committee (AC) and approved by an Expert Panel (EP) composed of eight senior hematologists. After a comprehensive and systematic literature review, the EP formulated therapy recommendations and graded them according to the supporting evidence. An explicit approach to consensus methodologies was used for evidence interpretation and for producing recommendations in the absence of strong evidence. The EP formulated recommendations on which first-line therapy to choose in patients with nodal DLBCL. Patients of all ages, with stage I-II disease and no adverse prognostic factors should receive abbreviated chemotherapy with an anthracycline-containing regimen plus involved field radiotherapy (35-40 Gy). Patients with stage I-II disease and at least one adverse prognostic factor, or with stage III-IV disease, should receive frontline chemoimmunotherapy with CHOP, CHOP-like or third-generation chemotherapy plus rituximab. Recommendations on stem cell transplantation and on which therapy to adopt for refractory or relapsed patients were also formulated.


Phytocannabinoids promote viability and functional adipogenesis of bone marrow-derived mesenchymal stem cells through different molecular targets.
The cellular microenvironment plays a critical role in the maintenance of bone marrow-derived mesenchymal stem cells (BM-MSCs) and their subsequent cell lineage differentiation. Recent studies suggested that individuals with adipocyte-related metabolic disorders have altered function and adipogenic potential of adipose stem cell subpopulations, primarily BM-MSCs, increasing the risk of heart attack, stroke or diabetes. In this study, we explored the potential therapeutic effect of some of the most abundant non-euphoric compounds derived from the Cannabis sativa plant (or phytocannabinoids) including tetrahydrocannabivarin (THCV), cannabidiol (CBD), cannabigerol (CBG), cannabidiolic acid (CBDA) and cannabigerolic acid (CBGA), by analysing their pharmacological activity on viability of endogenous BM-MSCs as well as their ability to alter BM-MSC proliferation and differentiation into mature adipocytes. We provide evidence that CBD, CBDA, CBGA and THCV (5 µM) increase the number of viable BM-MSCs; whereas only CBG (5 µM) and CBD (5 µM) alone or in combination promote BM-MSCs maturation into adipocytes via distinct molecular mechanisms. These effects were revealed both in vitro and in vivo. In addition, phytocannabinoids prevented the insulin signalling impairment induced by palmitate in adipocytes differentiated from BM-MSCs. Our study highlights phytocannabinoids as a potential novel pharmacological tool to regain control of functional adipose tissue in unregulated energy homeostasis often occurring in metabolic disorders including type 2 diabetes mellitus (T2DM), aging and lipodystrophy.


Family-focused treatment for bipolar disorder in adults and youth.
Levels of familial expressed emotion during an acute episode are consistently associated with rates of recurrence among bipolar patients. This article briefly reviews the evidence for expressed emotion (EE) as a prognostic indicator and then illustrates family-focused treatment (FFT) with adults and adolescents suffering from bipolar disorder. FFT is a time-limited, modularized treatment consisting of psychoeducation, communication enhancement training, and problem-solving skills. Controlled trials indicate that FFT is an efficacious adjunct to pharmacotherapy for patients with bipolar disorder. We describe its recent application to early onset bipolar patients and include a clinical vignette.


A brother and sister with breast cancer, BRCA2 mutations and bilateral supernumerary nipples.
We describe a 54-year-old man with breast cancer and a BRCA2 mutation who was also found to have bilateral supernumerary nipples. His sister, also with a BRCA2 mutation, was diagnosed with breast cancer in her late forties; she also had bilateral supernumerary nipples. We address the significance of breast cancer arising in breast tissue underlying supernumerary nipples; the known association between supernumerary nipples and genitourinary malignancies/malformations and the possible link between BRCA2 and supernumerary nipple development. We believe that this is the first described case of the latter. We then outline an approach to further management for supernumerary nipple cases.


Systematic review with meta-analysis: anxiety and depression in children and adolescents with inflammatory bowel disease.
The co-existence of psychological problems and paediatric inflammatory bowel disease (IBD) is receiving increasing attention. Most studies investigated anxiety and depression, with prevalence rates varying from 0% to 50%. A systematic review is necessary to provide clear insight into the prevalence of anxiety and depression in paediatric IBD. To systematically evaluate available data on the prevalence of anxiety and depressive symptoms and disorders in paediatric IBD (aged 6-18 years). Comprehensive searches were performed in Embase, Medline Ovid, Web of Science, Cochrane, PubMed, PsychInfo Ovid, and Google scholar for studies published from 1994 to 2017. Pooled prevalence rates were calculated using inverse variance heterogeneity models. Meta-regression was used to study if disease type, disease activity and gender influence prevalence. Twenty-eight studies (N = 8107, mean age: 14.3) were identified. Pooled prevalence estimates were 16.4% (95% confidence interval [CI] 6.8%-27.3%) for anxiety symptoms and 4.2% (95% CI 3.6%-4.8%) for anxiety disorders. Pooled prevalence estimates were 15.0% (95% CI 6.4%-24.8%) for depressive symptoms and 3.4% (95% CI 0%-9.3%) for depressive disorders. Meta-regression showed no influence of disease type or gender on these prevalence rates, but studies with a higher percentage of active disease had a higher rate of depressive symptoms. The described pooled prevalence of anxiety and depressive symptoms is lower than in adult IBD. However, due to varying instruments/cut-offs for measuring symptoms and few studies investigating disorders, the results should be interpreted with caution. Cross-cultural use of the same instruments is needed to gain better insight into prevalence rates.


Correlation of chromosome 17p loss with clinical outcome in medulloblastoma.
Medulloblastomas are primitive neuroectodermal tumors that arise in the cerebella of children. Cytogenetic and loss of heterozygosity (LOH) studies have shown that deletions on the short arm of chromosome 17 occur in 25-50% of cases, suggesting that loss of a tumor suppressor gene located on 17p plays a role in the genesis or progression of medulloblastoma. We report here on an LOH analysis of 21 patients with medulloblastoma using markers for 15 polymorphic loci previously ordered on chromosome 17 by meiotic break point mapping. Although the frequency of LOH in our patients (48%) was consistent with previous reports, our maps showed much larger deletions, the smallest spanning a 38-cM genetic distance distal to marker UT49 (D17S731). Survival analysis did not show a significant association between LOH on 17p and survival, although the sample size was too small to rule out a difference of less than 10-fold. Clinical risk factors were better prognostic indicators than LOH, with significantly prolonged survival in patients free of craniospinal metastasis following gross total tumor resection.


Mobile Phone Addiction and Its Relationship to Sleep Quality and Academic Achievement of Medical Students at King Abdulaziz University, Jeddah, Saudi Arabia.
Adverse effects of Mobile Phone (MP) usage could lead to dependency problems, and medical students are not excluded from it. We aimed to determine the pattern of MP usage, and its relation to sleep quality and academic performance between medical students at King Abdulaziz University (KAU), Jeddah, Saudi Arabia. A cross-sectional study. A multistage stratified random sample was used for selection of 610 participants, during 2016-2017. A validated, anonymous data collection sheet was used. It inquired about the Grade Point Averages (GPA). It included the Problematic Mobile Phone Use Questionnaire (PMPU-Q) for assessing various aspects of cellphone addiction (dependency, financial problems, prohibited and dangerous use). The Pittsburgh Sleep Quality Index (PSQI) was also included. Descriptive and inferential statistics were done. A high frequency of MP usage prevailed among participants (73.4% used it >5 h/day). About two-thirds of participants had poor sleep quality. Females, owners of smartphone for >1 yr, and increasing time spent on MP were associated with MP dependency. Lower academic achievers had significantly worse MP scores on financial problems, dangerous use, and total PUMP. MP dependency was correlated with subjective sleep quality score, and sleep latency. Global PSQI scale was correlated with prohibited MP use. Lower achievers had significantly worse scores on MP financial problems, dangerous usage, and the total PMPU. MP dependency was correlated with poor subjective sleep quality, and sleep latency. Rationale MP usage is needed to decrease the dependency, improve sleep quality, and academic achievement of medical students.


Differential roles of MAPK kinases MKK3 and MKK6 in osteoclastogenesis and bone loss.
Bone mass is maintained by osteoclasts that resorb bone and osteoblasts that promote matrix deposition and mineralization. Bone homeostasis is altered in chronic inflammation as well as in post-menopausal loss of estrogen, which favors osteoclast activity that leads to osteoporosis. The MAPK p38α is a key regulator of bone loss and p38 inhibitors preserve bone mass by inhibiting osteoclastogenesis. p38 function is regulated by two upstream MAPK kinases, namely MKK3 and MKK6. The goal of this study was to assess the effect of MKK3- or MKK6-deficiency on osteoclastogenesis in vitro and on bone loss in ovariectomy-induced osteoporosis in mice. We demonstrated that MKK3 but not MKK6, regulates osteoclast differentiation from bone marrow cells in vitro. Expression of NFATc1, a master transcription factor in osteoclastogenesis, is decreased in cells lacking MKK3 but not MKK6. Expression of osteoclast-specific genes Cathepsin K, osteoclast-associated receptor and MMP9, was inhibited in MKK3-/- cells. The effect of MKK-deficiency on ovariectomy-induced bone loss was then evaluated in female WT, MKK3-/- and MKK6-/- mice by micro-CT analysis. Bone loss was partially inhibited in MKK3-/- as well as MKK6-/- mice, despite normal osteoclastogenesis in MKK6-/- cells. This correlated with the lower osteoclast numbers in the MKK-deficient ovariectomized mice. These studies suggest that MKK3 and MKK6 differentially regulate bone loss due to estrogen withdrawal. MKK3 directly mediates osteoclastogenesis while MKK6 likely contributes to pro-inflammatory cytokine production that promotes osteoclast formation.


A spatial gradient of tau protein phosphorylation in nascent axons.
Mechanisms underlying axonogenesis remain obscure. Although a large number of proteins eventually become polarized to the axonal domain, in no case does protein compartmentalization occur before or simultaneous with the earliest morphological expression of axonal properties. How then might initially unpolarized proteins, such as the microtubule-associated protein tau, play a role in the microdifferentiation of axons? We hypothesized that tau function could be locally regulated by phosphorylation during the period of axonogenesis. To test this hypothesis, we mapped relative levels of tau phosphorylation within developing cultured hippocampal neurons. This was accomplished using calibrated immunofluorescence ratio measurements employing phosphorylation state-dependent and state-independent antibodies. Tau in the nascent axon is more highly dephosphorylated at the site recognized by the tau-1 antibody than tau in the somatodendritic compartment. The change in phosphorylation state from soma to axon takes the form of a smooth proximo-distal gradient, with tau in the soma, immature dendrites and proximal axon approximately 80% phosphorylated at the tau-1 site, and that in the axonal growth cone only 20% phosphorylated. The existence of real spatial differences in tau phosphorylation state was confirmed by in situ phosphatase and kinase treatment. Pervanadate, a tyrosine phosphatase inhibitor, induced rapid tau dephosphorylation within live cells, effectively abolishing the phosphorylation gradient. Thus, the gradient is dynamic and potentially regulatable by upstream signals involving tyrosine phosphorylation. Phosphorylation gradients are likely to be present on many neuronal proteins in addition to tau, and their modulation by transmembrane signals could direct the establishment of polarity.


Proposal for a multiphase fall model based on real-world fall recordings with body-fixed sensors.
Falls are by far the leading cause of fractures and accidents in the home environment. The current Cochrane reviews and other systematic reviews report on more than 200 intervention studies about fall prevention. A recent meta-analysis has summarized the most important risk factors of accidental falls. However, falls and fall-related injuries remain a major challenge. One novel approach to recognize, analyze, and work better toward preventing falls could be the differentiation of the fall event into separate phases. This might aid in reconsidering ways to design preventive efforts and diagnostic approaches. From a conceptual point of view, falls can be separated into a pre-fall phase, a falling phase, an impact phase, a resting phase, and a recovery phase. Patient and external observers are often unable to give detailed comments concerning these phases. With new technological developments, it is now at least partly possible to examine the phases of falls separately and to generate new hypotheses.The article describes the practicality and the limitations of this approach using body-fixed sensor technology. The features of the different phases are outlined with selected real-world fall signals.


Accurate Detection of SARS-CoV-2 by Next-Generation Sequencing in Low Viral Load Specimens.
As new SARS-CoV-2 variants emerge, there is an urgent need to increase the efficiency and availability of viral genome sequencing, notably to detect the lineage in samples with a low viral load. SARS-CoV-2 genome next-generation sequencing (NGS) was performed retrospectively in a single center on 175 positive samples from individuals. An automated workflow used the Ion AmpliSeq SARS-CoV-2 Insight Research Assay on the Genexus Sequencer. All samples were collected in the metropolitan area of the city of Nice (France) over a period of 32 weeks (from 19 July 2021 to 11 February 2022). In total, 76% of cases were identified with a low viral load (Ct ≥ 32, and ≤200 copies/µL). The NGS analysis was successful in 91% of cases, among which 57% of cases harbored the Delta variant, and 34% the Omicron BA.1.1 variant. Only 9% of cases had unreadable sequences. There was no significant difference in the viral load in patients infected with the Omicron variant compared to the Delta variant (Ct values, p = 0.0507; copy number, p = 0.252). We show that the NGS analysis of the SARS-CoV-2 genome provides reliable detection of the Delta and Omicron SARS-CoV-2 variants in low viral load samples.


Dispersion-managed solitons at normal average dispersion.
We find that in a dispersion-managed fiber, in which the strength of the dispersion management is above some threshold, solitons can exist with normal average dispersion. When the normal average dispersion is below some limiting value there exist two soliton solutions with the same pulse duration and different pulse energies. When the normal average dispersion is above this limiting value, no soliton exists. Both higher-energy and lower-energy solitons are dynamically stable in the parameter range that we considered.


Ancient DNA from lake sediments: bridging the gap between paleoecology and genetics.
Quaternary plant ecology in much of the world has historically relied on morphological identification of macro- and microfossils from sediments of small freshwater lakes. Here, we report new protocols that reliably yield DNA sequence data from Holocene plant macrofossils and bulk lake sediment used to infer ecological change. This will allow changes in census populations, estimated from fossils and associated sediment, to be directly associated with population genetic changes. We successfully sequenced DNA from 64 samples (out of 126) comprised of bulk sediment and seeds, leaf fragments, budscales, and samaras extracted from Holocene lake sediments in the western Great Lakes region of North America. Overall, DNA yields were low. However, we were able to reliably amplify samples with as few as 10 copies of a short cpDNA fragment with little detectable PCR inhibition. Our success rate was highest for sediments < 2000 years old, but we were able to successfully amplify DNA from samples up to 4600 years old. DNA sequences matched the taxonomic identity of the macrofossil from which they were extracted 79% of the time. Exceptions suggest that DNA molecules from surrounding nearby sediments may permeate or adhere to macrofossils in sediments. An ability to extract ancient DNA from Holocene sediments potentially allows exciting new insights into the genetic consequences of long-term environmental change. The low DNA copy numbers we found in fossil material and the discovery of multiple sequence variants from single macrofossil extractions highlight the need for careful experimental and laboratory protocols. Further application of these protocols should lead to better understanding of the ecological and evolutionary consequences of environmental change.


An interneurone mediating motor programme switching in the ventilatory system of the crab.
The central pattern generator controlling ventilation in the crab can generate two distinct motor programmes, which determine the direction of water flow during irrigation of the gills. An interneurone has been identified that depolarizes when the ventilatory motor output switches from forward to reverse ventilation and remains depolarized for the duration of the reverse motor programme. Depolarization of this neurone by intracellular current injection causes a switch in the motor programme from forward to reverse ventilation, which persists for the duration of the current step. Hyperpolarization of this cell during reverse ventilation terminates the reverse motor programme. The possible role of this reversal switch interneurone is considered in the context of the observed changes in the activity of other ventilatory interneurones and motor neurones during reverse ventilation.


The quantum epoché.
The theme of phenomenology and quantum physics is here tackled by examining some basic interpretational issues in quantum physics. One key issue in quantum theory from the very beginning has been whether it is possible to provide a quantum ontology of particles in motion in the same way as in classical physics, or whether we are restricted to stay within a more limited view of quantum systems, in terms of complementary but mutually exclusive phenomena. In phenomenological terms we could describe the situation by saying that according to the usual interpretation of quantum theory (especially Niels Bohr's), quantum phenomena require a kind of epoché (i.e. a suspension of assumptions about reality at the quantum level). However, there are other interpretations (especially David Bohm's) that seem to re-establish the possibility of a mind-independent ontology at the quantum level. We will show that even such ontological interpretations contain novel, non-classical features, which require them to give a special role to "phenomena" or "appearances", a role not encountered in classical physics. We will conclude that while ontological interpretations of quantum theory are possible, quantum theory implies the need of a certain kind of epoché even for this type of interpretations. While different from the epoché connected to phenomenological description, the "quantum epoché" nevertheless points to a potentially interesting parallel between phenomenology and quantum philosophy.


No evidence for association between tau gene haplotypic variants and susceptibility to Creutzfeldt-Jakob disease.
A polymorphism at codon 129 of the prion protein gene (PRNP) is the only well-known genetic risk factor for Creutzfeldt-Jakob disease (CJD). However, there is increasing evidence that other loci outside the PRNP open reading frame might play a role in CJD aetiology as well. We studied tau protein gene (MAPT) haplotypic variations in a population of sporadic and variant CJD patients. We tested 6 MAPT haplotype tagging SNPs (htSNPs) in a Dutch population-based sample of sporadic CJD (sCJD) patients and a cognitively normal control group of similar age distribution. We genotyped the same polymorphisms in two other sample groups of sCJD cases from Italy and the UK. In addition, we compared MAPT haplotypes between sCJD and variant CJD (vCJD) patients. Single locus and haplotype analyses did not detect any significant difference between sCJD cases and controls. When we compared MAPT haplotypes between sCJD and variant CJD (vCJD) patients, we found that two of them were represented differently (H1f: 8% in sCJD versus 2% in vCJD; H1j:1% in sCJD versus 7% in vCJD). However, these two haplotypes were rare in both groups of patients, and taking the small sample sizes into account, we cannot exclude that the differences are due to chance. None of the p-values remained statistically significant after applying a multiple testing correction. Our study shows no evidence for an association between MAPT gene variations and sCJD, and some weak evidence for an association to vCJD.


Iodinated contrast opacification gradients in normal coronary arteries imaged with prospectively ECG-gated single heart beat 320-detector row computed tomography.
To define and evaluate coronary contrast opacification gradients using prospectively ECG-gated single heart beat 320-detector row coronary angiography (CTA). Thirty-six patients with normal coronary arteries determined by 320 x 0.5-mm detector row coronary CTA were retrospectively evaluated with customized image postprocessing software to measure Hounsfield Units at 1-mm intervals orthogonal to the artery center line. Linear regression determined correlation between mean Hounsfield Units and distance from the coronary ostium (regression slope defined as the distance gradient G(d)), lumen cross-sectional area (G(a)), and lumen short-axis diameter (G(s)). For each gradient, differences between the 3 coronary arteries were analyzed with ANOVA. Linear regression determined correlations between measured gradients, heart rate, body mass index, and cardiac phase. To determine feasibility in lesions, all 3 gradients were evaluated in 22 consecutive patients with left anterior descending artery lesions > or =50% stenosis. For all 3 coronary arteries in all patients, the gradients G(a) and G(s) were significantly different from zero (P<0.0001), highly linear (Pearson r values, 0.77 to 0.84), and had no significant difference between the left anterior descending, left circumflex, and right coronary arteries (P>0.503). The distance gradient G(d) demonstrated nonlinearities in a small number of vessels and was significantly smaller in the right coronary artery when compared with the left coronary system (P<0.001). Gradient variations between cardiac phases, heart rates, body mass index, and readers were low. Gradients in patients with lesions were significantly different (P<0.021) than in patients considered normal by CTA. Measurement of contrast opacification gradients from temporally uniform coronary CTA demonstrates feasibility and reproducibility in patients with normal coronary arteries. For all patients, the gradients defined with respect to the coronary lumen cross-sectional area and short-axis diameters are highly linear, not significantly influenced by the coronary artery (left anterior descending artery versus left circumflex versus right coronary artery), and have only small variation with respect to patient parameters. Preliminary evaluation of gradients across coronary artery lesions is promising but requires additional study.


FGFR2 and other loci identified in genome-wide association studies are associated with breast cancer in African-American and younger women.
Twenty-nine single-nucleotide polymorphisms (SNPs) from previously published genome-wide association studies (GWAS) and multiple ancestry informative markers were genotyped in the Carolina Breast Cancer Study (CBCS) (742 African-American (AA) cases, 1230 White cases; 658 AA controls, 1118 White controls). In the entire study population, 9/10 SNPs in fibroblast growth factor receptor 2 (FGFR2) were significantly associated with breast cancer after adjusting for age, race and European ancestry [odds ratios (OR) range 1.17-1.81]. Associations were observed for SNPs in FGFR2, LSP1, H19, TLR1/TLR6 and RELN for AA; FGFR2, TNRC9, H19 and MAP3K1 for Whites; FGFR2, TNRC9, Msc5A1 and chromosome 8q for women > or =50 years old and FGFR2 and TNRC9 for women <50 years old. FGFR2 haplotypes based upon rs11200014, rs2981579, rs1219648 and rs2420946 were associated with increased risk of breast cancer, including the GTGT haplotype in AAs [OR = 1.27, 95% confidence interval (CI) 1.04-1.56] and younger women of either race [OR = 1.35, 95% CI 1.02-1.78) and the ATGT haplotype in Whites (OR = 1.30, 95% CI 1.15-1.46). Recent GWAS hits for breast cancer in Europeans and Whites (i.e. women of European descent) thus showed evidence of replication among AAs and Whites in the CBCS. Several new haplotypes were associated with breast cancer in AA and younger women, particularly the FGFR2 GTGT haplotype. These results highlight the need to conduct GWAS among younger women and in a variety of racial-ethnic populations.


Electrodiagnostic findings in COVID-19 patients: A single center experience.
Neurological manifestations in patients with coronavirus disease 2019 (COVID-19) have been reported from early features of anosmia and dysgeusia to widespread involvement of the central nervous system, peripheral nervous system, as well as the neuromuscular junction and muscle. Our study objective is to evaluate the electromyography and nerve conduction study (EMG/NCS) findings among COVID-19 patients and look for possible correlations. This is a hospital-based retrospective observational study. All COVID-19 patients between the period of 1st January 2020 to 31st December 2020 undergoing an EMG/NCS were included. Eighteen patients (12 male and 6 female) were included. Mean age was 55 ± 12 years. 11 patients required intubation for a mean period of 18.6 days (range: 3-37 days). Electrodiagnostic findings were consistent with a myopathy in a majority of these patients (82%). Five of them also had a concurrent axonal neuropathy. In the remaining patients who did not require intubation (n = 7), three patients had myopathic EMG changes and one had Guillain Barre syndrome. At this time, there are no neuromuscular-specific recommendations for patients who contract COVID-19. Only time and additional data will unveil the varying nature and potential neurological sequelae of COVID-19. Myopathic EMG changes are commonly seen in critically ill COVID-19 patients, especially with a prolonged hospital stay.


Assessing characteristics of RNA amplification methods for single cell RNA sequencing.
Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5-10 molecules. Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.


Modulation of angiogenesis during adipose tissue development in murine models of obesity.
Development of vasculature and mRNA expression of 17 pro- or antiangiogenic factors were studied during adipose tissue development in nutritionally induced or genetically determined murine obesity models. Subcutaneous (SC) and gonadal (GON) fat pads were harvested from male C57Bl/6 mice kept on standard chow [standard fat diet (SFD)] or on high-fat diet for 0-15 wk and from male ob/ob mice kept on SFD. Ob/ob mice and C57Bl/6 mice on high-fat diet had significantly larger SC and GON fat pads, accompanied by significantly higher blood content, increased total blood vessel volume, and higher number of proliferating cells. mRNA and protein levels of angiopoietin (Ang)-1 were down-regulated, whereas those of thrombospondin-1 were up-regulated in developing adipose tissue in both obesity models. Ang-1 mRNA levels correlated negatively with adipose tissue weight in the early phase of nutritionally induced obesity as well as in genetically determined obesity. Placental growth factor and Ang-2 expression were increased in SC adipose tissue of ob/ob mice, and thrombospondin-2 was increased in both their SC and GON fat pads. mRNA levels of vascular endothelial growth factor (VEGF)-A isoforms VEGF-B, VEGF-C, VEGF receptor-1, -2, and -3, and neuropilin-1 were not markedly modulated by obesity. This modulation of angiogenic factors during development of adipose tissue supports their important functional role in obesity.


Investigation of the pattern of hepatitis C virus sequence diversity in different geographical regions: implications for virus classification. The International HCV Collaborative Study Group.
Genotypes of hepatitis C virus (HCV) present within 104 samples from HCV-infected individuals from Africa, the Middle East, the Indian subcontinent and South-East Asia were identified by sequence comparisons in the core and NS-5 regions. Relatively short sequences (such as the 222 bp fragment of NS-5) provided effective discrimination of types, subtypes and isolates, and produced equivalent relationships between genotypes as were found upon comparison of longer sequences of NS-5, of the core region, and by comparison of the limited number of complete genomic sequences currently available. Measurement of evolutionary distances in the core and NS-5 regions allowed 79 of the 104 samples to be identified as examples of known genotypes, while 17 of the remainder could be provisionally classified as new subtypes of types 1 (Nigeria), 2 (Gambia), 3 (India, Pakistan and Bangladesh) and 4 (Middle East) on the basis of sequence comparison in core and NS-5 (n = 9) or provisionally using core alone (n = 8). The remaining sequences from Thailand made up two groups showing no close similarity to any of the six major genotypes classified to date, although one corresponded to an as yet unclassified variant of HCV also found in Thailand. However, phylogenetic analysis of the core and NS-5 regions indicated a distant relationship between these sequences with variants found in Vietnam and with type 6a, and collectively they formed a diverse single phylogenetic group. The existence of great diversity within a single genotype was also found amongst type 3 sequences in the Indian subcontinent, amongst type 4 variants in Central Africa and the Middle East, and amongst type variants in Nigeria. These findings may provide clues for understanding the origins and mechanisms of transmission of HCV.


Design of a controlled-release ergometer for the measurement of musculotendinous stiffness of the knee flexors.
The stiffness of muscle-tendon units (MTUs) influences many aspects of human movement from athletic performance to injury risk. Presently the controlled-release technique of measuring MTU stiffness has been applied almost exclusively to the distal joints of the body, i.e., the ankle. This is primarily because of the mechanical limitations of implementing this technique. However, in order to better understand how the elastic properties of the MTU affect both performance and injury potential, measurements of MTU stiffness of the more proximal joints must be made. The knee flexors are a logical choice because of the integral role of MTU stiffness of this muscle group in both hamstring strains and knee injury. The purpose of this study was to modify a commercial ergometer so that it could be used to measure the musculotendinous stiffness of the knee flexors. Data are presented for a representative participant to illustrate the feasibility and capability of this ergometer, and the measured MTU stiffness was 519 N.m.rad(-1) at a knee flexion moment of 100 N.m. Our results indicate that it is indeed possible to modify a commercial ergometer and measure musculotendinous stiffness of large muscle groups crossing proximal joints.


Brain metastases in patients with non-small cell lung cancer: the role of mutated-EGFRs with an exon 19 deletion or L858R point mutation in cancer cell dissemination.
Non-small cell lung cancer (NSCLC) patients tend to develop brain metastases (BM), but the link between BM occurrence and driver mutations in NSCLC is not very clear. We explored whether activating mutations of epidermal growth factor receptors (EGFRs) in exon 19 deletion or L858R predict BM in NSCLC. A retrospective multivariable logistic regression analysis of 384 patients demonstrated that the presence of mutated-EGFRs was associated with overall BM (OR=2.24, P=0.001) compared to that of wild-type EGFR (WT-EGFR). Moreover, the time-to-event analysis model considering death as a competing risk revealed that, irrespective of survival, mutated-EGFRs predicted subsequent BM (SBM) in stage IIIB-IV patients (37.1% vs. 10.6%, HR=2.98, P=0.002) after adjusting for age (HR=2.00, P=0.012), gender, histological subtype, and smoking history. Notably, the younger mutated-EGFR subgroup was at a higher risk for SBM compared to the older WT-EGFR one (58.1% vs.10.9%, HR=6.57, P<0.001). Additionally, EGFR exon 19 deletion, despite having a slightly longer overall survival (20.6 vs. 14.2 months, P=0.368), was comparable to L858R mutation in predicting SBM (39.5% vs. 34.5%, HR=0.91, P=0.770). In vitro, the overexpression of mutated-EGFRs induced morphological changes towards a mesenchymal-like phenotype and promoted mobility in lung cancer cells. Clinically, mutated-EGFR NSCLC displayed a higher proportion of vimentin-positive expression (75.3% vs. 51.2%; P=0.007) and a shorter median time to SBM (23.5 months vs. not reached, P=0.017) than WT-EGFR NSCLC. These results suggest that NSCLC patients carrying mutated-EGFRs may require a higher frequency of brain imaging assessments than those with WT-EGFR to facilitate earlier SBM detection during follow-up.


Spatio-temporal analyses of Symbiodinium physiology of the coral Pocillopora verrucosa along large-scale nutrient and temperature gradients in the Red Sea.
Algal symbionts (zooxanthellae, genus Symbiodinium) of scleractinian corals respond strongly to temperature, nutrient and light changes. These factors vary greatly along the north-south gradient in the Red Sea and include conditions, which are outside of those typically considered optimal for coral growth. Nevertheless, coral communities thrive throughout the Red Sea, suggesting that zooxanthellae have successfully acclimatized or adapted to the harsh conditions they experience particularly in the south (high temperatures and high nutrient supply). As such, the Red Sea is a region, which may help to better understand how zooxanthellae and their coral hosts successfully acclimatize or adapt to environmental change (e.g. increased temperatures and localized eutrophication). To gain further insight into the physiology of coral symbionts in the Red Sea, we examined the abundance of dominant Symbiodinium types associated with the coral Pocillopora verrucosa, and measured Symbiodinium physiological characteristics (i.e. photosynthetic processes, cell density, pigmentation, and protein composition) along the latitudinal gradient of the Red Sea in summer and winter. Despite the strong environmental gradients from north to south, our results demonstrate that Symbiodinium microadriaticum (type A1) was the predominant species in P. verrucosa along the latitudinal gradient. Furthermore, measured physiological characteristics were found to vary more with prevailing seasonal environmental conditions than with region-specific differences, although the measured environmental parameters displayed much higher spatial than temporal variability. We conclude that our findings might present the result of long-term acclimatization or adaptation of S. microadriaticum to regionally specific conditions within the Red Sea. Of additional note, high nutrients in the South correlated with high zooxanthellae density indicating a compensation for a temperature-driven loss of photosynthetic performance, which may prove promising for the resilience of these corals under increase of temperature increase and eutrophication.


Comparison of flat-panel digital to conventional film-screen radiography in detection of experimentally created lesions of the equine third metacarpal bone.
Radiographic diagnosis of equine bone disease using digital radiography is prevalent in veterinary practice. However, the diagnostic quality of digital vs. conventional radiography has not been compared systematically. We hypothesized that digital radiography would be superior to film-screen radiography for detection of subtle lesions of the equine third metacarpal bone. Twenty-four third metacarpal bones were collected from horses euthanized for reasons other than orthopedic disease. Bones were dissected free of soft tissue and computed tomography was performed to ensure that no osseous abnormalities were present. Subtle osseous lesions were produced in the dorsal cortex of the third metacarpal bones, and the bones were radiographed in a soft tissue phantom using indirect digital and conventional radiography at standard exposures. Digital radiographs were printed onto film. Three Diplomates of the American College of Veterinary Radiology evaluated the radiographs for the presence or absence of a lesion. Receiver operator characteristic curves were constructed, and the area under these curves were compared to assess the ability of the digital and film-screen radiographic systems to detect lesions. The area under the ROC curves for film-screen and digital radiography were 0.87 and 0.90, respectively (P = 0.59). We concluded that the digital radiographic system was comparable to the film-screen system for detection of subtle lesions of the equine third metacarpal bone.


Acetal-linked branched poly(dimethyl-aminoethyl methacrylate) as an acid cleavable gene vector with reduced cytotoxicity.
An acid labile branched PDMAEMA/acetal copolymer with amino group was synthesized by the DE-ATRP and followed by Michael addition. The degradation of the polymer was strongly pH-dependent. High nucleic acid transfection efficiency with low cytotoxicity was observed compared to its non-degradable copolymer counterpart.


What drives 'first-time testers' to test for HIV at community-based HIV testing services?
Drivers of and barriers to testing are not well understood for those who have never been tested previously and now self-initiate at a community-based human immuno-deficiency virus (HIV) testing service (CB-HTS). This descriptive study enrolled 229 first-time testers. Participants completed an electronic questionnaire. The majority reported fear and (non) accessibility of HTS as barriers to testing (40% and 24%, respectively). Wanting 'to know my status' and the immediate opportunity to test were reported as drivers of testing (41% and 35%, respectively). Addressing fear of testing and providing an easily accessible opportunity to test may go some way to encouraging those previously untested individuals to test.


Promoting universal financial protection: how the Thai universal coverage scheme was designed to ensure equity.
Empirical evidence demonstrates that the Thai Universal Coverage Scheme (UCS) has improved equity of health financing and provided a relatively high level of financial risk protection. Several UCS design features contribute to these outcomes: a tax-financed scheme, a comprehensive benefit package and gradual extension of coverage to illnesses that can lead to catastrophic household costs, and capacity of the National Health Security Office (NHSO) to mobilise adequate resources. This study assesses the policy processes related to making decisions on these features. The study employs qualitative methods including reviews of relevant documents, in-depth interviews of 25 key informants, and triangulation amongst information sources. Continued political and financial commitments to the UCS, despite political rivalry, played a key role. The Thai Rak Thai (TRT)-led coalition government introduced UCS; staying in power 8 of the 11 years between 2001 and 2011 was long enough to nurture and strengthen the UCS and overcome resistance from various opponents. Prime Minister Surayud's government, replacing the ousted TRT government, introduced universal renal replacement therapy, which deepened financial risk protection.Commitment to their manifesto and fiscal capacity pushed the TRT to adopt a general tax-financed universal scheme; collecting premiums from people engaged in the informal sector was neither politically palatable nor technically feasible. The relatively stable tenure of NHSO Secretary Generals and the chairs of the Financing and the Benefit Package subcommittees provided a platform for continued deepening of financial risk protection. NHSO exerted monopsonistic purchasing power to control prices, resulting in greater patient access and better systems efficiency than might have been the case with a different design.The approach of proposing an annual per capita budget changed the conventional line-item programme budgeting system by basing negotiations between the Bureau of Budget, the NHSO and other stakeholders on evidence of service utilization and unit costs. Future success of Thai UCS requires coverage of effective interventions that address primary and secondary prevention of non-communicable diseases and long-term care policies in view of epidemiologic and demographic transitions. Lessons for other countries include the importance of continued political support, evidence informed decisions, and a capable purchaser organization.


Endothelium-derived hyperpolarizing factor determines resting and stimulated forearm vasodilator tone in health and in disease.
We assessed the contribution of endothelium-derived hyperpolarizing factors to resting and agonist-stimulated vasodilator tone in health and disease. Tetraethylammonium chloride (TEA) was used to inhibit K(+)(Ca) channel activation and fluconazole was used to inhibit cytochrome P450 2C9-mediated epoxyeicosatrienoic acid synthesis. We hypothesized that endothelium-derived hyperpolarizing factors contribute to resting vascular tone by K(+)(Ca) channel activation and epoxyeicosatrienoic acid release and that endothelium-derived hyperpolarizing factors compensate for reduced nitric oxide bioavailability at rest and with endothelium-dependent vasodilators. In 103 healthy subjects and 71 nonhypertensive subjects with multiple risk factors, we measured resting forearm blood flow (FBF) using venous occlusion plethysmography before and after intra-arterial infusions of N(G)-monomethyl-l-arginine (L-NMMA), TEA, fluconazole, and their combination. The effects of these antagonists on resting FBF and on bradykinin- and acetylcholine-mediated vasodilation were studied. Resting FBF decreased with TEA and L-NMMA in all subjects (P<0.001); however, the vasoconstrictor response to L-NMMA was greater (P=0.04) and to TEA was lower (P=0.04) in healthy subjects compared with those with risk factors. Fluconazole decreased resting FBF in all subjects, and the addition of TEA further reduced FBF after fluconazole, suggesting that cytochrome P450 metabolites and other hyperpolarizing factor(s) activate K(+)(Ca) channels. Both L-NMMA and TEA attenuated bradykinin-mediated vasodilation in healthy and hypercholesterolemic subjects (P<0.001). In contrast, acetylcholine-mediated vasodilation remained unchanged with TEA in healthy subjects but was significantly attenuated in hypercholesterolemia (P<0.04). First, by activating TEA-inhibitable K(+)(Ca) channels, endothelium-derived hyperpolarizing factors, together with nitric oxide, contribute to resting microvascular dilator tone. The contribution of K(+)(Ca) channel activation compared with nitric oxide is greater in those with multiple risk factors compared with healthy subjects. Second, activation of K(+)(Ca) channels is only partly through epoxyeicosatrienoic acid release, indicating the presence of other hyperpolarizing mechanisms. Third, bradykinin, but not acetylcholine, stimulates K(+)(Ca) channel-mediated vasodilation in healthy subjects, whereas in hypercholesterolemia, K(+)(Ca) channel-mediated vasodilation compensates for the reduced nitric oxide activity. Thus, enhanced endothelium-derived hyperpolarizing factor activity in conditions of nitric oxide deficiency contributes to maintenance of resting and agonist-stimulated vasodilation. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT00166166.


Potassium bromate as positive assay control for the Fpg-modified comet assay.
The comet assay is a popular assay in biomonitoring studies. DNA strand breaks (or unspecific DNA lesions) are measured using the standard comet assay. Oxidative stress-generated DNA lesions can be measured by employing DNA repair enzymes to recognise oxidatively damaged DNA. Unfortunately, there has been a tendency to fail to report results from assay controls (or maybe even not to employ assay controls). We believe this might have been due to uncertainty as to what really constitutes a positive control. It should go without saying that a biomonitoring study cannot have a positive control group as it is unethical to expose healthy humans to DNA damaging (and thus potentially carcinogenic) agents. However, it is possible to include assay controls in the analysis (here meant as a cryopreserved sample of cells i.e. included in each experiment as a reference sample). In the present report we tested potassium bromate (KBrO3) as a positive comet assay control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. Ten laboratories used the same procedure for treatment of monocytic THP-1 cells with KBrO3 (0.5, 1.5 and 4.5 mM for 1 h at 37°C) and subsequent cryopreservation. Results from one laboratory were excluded in the statistical analysis because of technical issues in the Fpg-modified comet assay. All other laboratories found a concentration-response relationship in cryopreserved samples (regression coefficients from 0.80 to 0.98), although with different slopes ranging from 1.25 to 11.9 Fpg-sensitive sites (%DNA in tail) per 1 mM KBrO3. Our results demonstrate that KBrO3 is a suitable positive comet assay control.


Dose of early intervention treatment during children's first 36 months of life is associated with developmental outcomes: an observational cohort study in three low/low-middle income countries.
The positive effects of early developmental intervention (EDI) on early child development have been reported in numerous controlled trials in a variety of countries. An important aspect to determining the efficacy of EDI is the degree to which dosage is linked to outcomes. However, few studies of EDI have conducted such analyses. This observational cohort study examined the association between treatment dose and children's development when EDI was implemented in three low and low-middle income countries as well as demographic and child health factors associated with treatment dose. Infants (78 males, 67 females) born in rural communities in India, Pakistan, and Zambia received a parent-implemented EDI delivered through biweekly home visits by trainers during the first 36 months of life. Outcome was measured at age 36 months with the Mental (MDI) and Psychomotor (PDI) Development Indices of the Bayley Scales of Infant Development-II. Treatment dose was measured by number of home visits completed and parent-reported implementation of assigned developmental stimulation activities between visits. Sociodemographic, prenatal, perinatal, and child health variables were measures as correlates. Average home visits dose exceeded 91% and mothers engaged the children in activities on average 62.5% of days. Higher home visits dose was significantly associated with higher MDI (mean for dose quintiles 1-2 combined = 97.8, quintiles 3-5 combined = 103.4, p = 0.0017). Higher treatment dose was also generally associated with greater mean PDI, but the relationships were non-linear. Location, sociodemographic, and child health variables were associated with treatment dose. Receiving a higher dose of EDI during the first 36 months of life is generally associated with better developmental outcomes. The higher benefit appears when receiving ≥91% of biweekly home visits and program activities on ≥67% of days over 3 years. It is important to ensure that EDI is implemented with a sufficiently high dose to achieve desired effect. To this end groups at risk for receiving lower dose can be identified and may require special attention to ensure adequate effect.


TRPV1 receptors mediate particulate matter-induced apoptosis.
Exposure to airborne particulate matter (PM) is a world-wide health problem mainly because it produces adverse cardiovascular and respiratory effects that frequently result in morbidity. Despite many years of epidemiological and basic research, the mechanisms underlying PM toxicity remain largely unknown. To understand some of these mechanisms, we measured PM-induced apoptosis and necrosis in normal human airway epithelial cells and sensory neurons from both wild-type mice and mice lacking TRPV1 receptors using Alexa Fluor 488-conjugated annexin V and propidium iodide labeling, respectively. Exposure of environmental PMs containing residual oil fly ash and ash from Mount St. Helens was found to induce apoptosis, but not necrosis, as a consequence of sustained calcium influx through TRPV1 receptors. Apoptosis was completely prevented by inhibiting TRPV1 receptors with capsazepine or by removing extracellular calcium or in sensory neurons from TRPV1(-/-) mice. Binding of either one of the PMs to the cell membrane induced a capsazepine-sensitive increase in cAMP. PM-induced apoptosis was augmented upon the inhibition of PKA. PKA inhibition on its own also induced apoptosis, thereby suggesting that this pathway may be endogenously protective against apoptosis. In summary, it was found that inhibiting TRPV1 receptors prevents PM-induced apoptosis, thereby providing a potential mechanism to reduce their toxicity.


Outcomes of early language delay: II. Etiology of transient and persistent language difficulties.
Genes are known to play an important role in causing specific language impairment, but it is unclear how far a similar etiology is implicated in transient language delay in early childhood. Two-year-old children with vocabulary scores below the 10th centile were selected from a cohort of over 2,800 same-sex twin pairs whose language was assessed by parental report at 2, 3, and 4 years of age. These children with early language delay (ELD) were divided into cases of transient and persistent language difficulties on the basis of outcome at 3 and 4 years. A DeFries-Fulker analysis (J. C. DeFries & D. W. Fulker, 1985) was used to compute group heritability (h2g) of 2-year vocabulary delay separately for those with transient and persistent difficulties. When 3-year and 4-year language attainments were used to categorize outcomes, h2g was similar and modest (.25 or less) for both transient and persistent difficulties. However, when persistent difficulties were defined according to whether parents expressed concern about language at 3 years or according to whether a professional had been consulted about language difficulties at 4 years, heritability was significantly higher. For 289 children with no professional involvement at 4 years, heritability of 2-year vocabulary delay was close to zero, whereas for 134 children with professional involvement, a significant h2g of .41 (SE = .127) was found. Early language delay appears largely environmental in origin for 2-year-olds whose parents do not go on to seek professional help.


Atypical Femoral Fractures: A Teaching Perspective.
This article provides an overview of atypical femoral fractures with a highlight on their radiographic findings. Potent antiresorptive agents such as bisphosphonates or denosumab have been associated with the development of such fractures. However, at this time, a causal association has not been conclusively established. Atypical femoral fractures are insufficiency fractures, which frequently present with bone pain. Early identification of characteristic radiographic features and withdrawal of antiresorptive therapy may prevent the development of completed atypical femoral fractures.


Ticagrelor compared with clopidogrel by geographic region in the Platelet Inhibition and Patient Outcomes (PLATO) trial.
In the Platelet Inhibition and Patient Outcomes (PLATO) trial, a prespecified subgroup analysis showed a significant interaction between treatment and region (P=0.045), with less effect of ticagrelor in North America than in the rest of the world. Reasons for the interaction were explored independently by 2 statistical groups. Systematic errors in trial conduct were investigated. Statistical approaches evaluated the likelihood of play of chance. Cox regression analyses were performed to quantify how much of the regional interaction could be explained by patient characteristics and concomitant treatments, including aspirin maintenance therapy. Landmark Cox regressions at 8 time points evaluated the association of selected factors, including aspirin dose, with outcomes by treatment. Systematic errors in trial conduct were ruled out. Given the large number of subgroup analyses performed and that a result numerically favoring clopidogrel in at least 1 of the 4 prespecified regions could occur with 32% probability, chance alone cannot be ruled out. More patients in the United States (53.6%) than in the rest of the world (1.7%) took a median aspirin dose ≥300 mg/d. Of 37 baseline and postrandomization factors explored, only aspirin dose explained a substantial fraction of the regional interaction. In adjusted analyses, both Cox regression with median maintenance dose and landmark techniques showed that, in patients taking low-dose maintenance aspirin, ticagrelor was associated with better outcomes compared with clopidogrel, with statistical superiority in the rest of the world and similar outcomes in the US cohort. The regional interaction could arise from chance alone. Results of 2 independently performed analyses identified an underlying statistical interaction with aspirin maintenance dose as a possible explanation for the regional difference. The lowest risk of cardiovascular death, myocardial infarction, or stroke with ticagrelor compared with clopidogrel is associated with a low maintenance dose of concomitant aspirin. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00391872.


Impact of different partitioned solvents on chemical composition and bioavailability of Sasa quelpaertensis Nakai leaf extract.
The leaves of Sasa quelpaertensis Nakai were extracted with 80% ethanol and further partitioned with n-hexane, chloroform, ethyl acetate, n-butanol, and aqueous fractions to evaluate the biological activity through assessment via various in vitro assays, including total phenol content; 1,1-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazothiazoline-6-sulfornic acid (ABTS) radical scavenging; reducing power; α-glucosidase and tyrosinase inhibitory; and alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity assays. The highest activity was found in the ethyl acetate fraction for all assays and showed stronger DPPH radical scavenging, reducing power, and tyrosinase inhibitory activity than the positive controls (butylated hydroxytoluene, α-tocopherol, and arbutin, respectively). When compared to the ethyl acetate fraction, the n-butanol fraction had lower rates, but it still demonstrated relatively high activity. The activity of the n-hexane fraction was high for DPPH and ABTS radical scavenging activity and contained significant amounts of phenol content, whereas the chloroform fraction possessed the highest reducing power, tyrosinase inhibitory, and ADH and ALDH activity, despite having the lowest phenol content when compared to the other fractions. These findings clearly indicate that S. quelpaertensis Nakai leaves can be a good natural source of antioxidants and tyrosinase inhibitors, as well as ADH and ALDH activity inducers, suggesting that may have potential for treating various diseases and improving human health.


Diversity of nitrite reductase genes in "Candidatus Accumulibacter phosphatis"-dominated cultures enriched by flow-cytometric sorting.
"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.


Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes.
The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby plants. Microbes in the rhizosphere can provide nutrients, secrete signaling compounds, and inhibit pathogens. These processes could be manipulated with synthetic biology to enhance the agricultural performance of crops grown for food, energy, or environmental remediation, if methods can be implemented in these nonmodel microbes. A common first step for domesticating nonmodel organisms is the development of a set of genetic engineering tools, termed a synthetic biology toolbox. A toolbox comprises transformation protocols, replicating vectors, genome engineering (e.g., CRISPR/Cas9), constitutive and inducible promoter systems, and other gene expression control elements. This work validated synthetic biology toolboxes in three nitrogen-fixing soil bacteria: Azotobacter vinelandii, Stutzerimonas stutzeri (Pseudomonas stutzeri), and a new isolate of Klebsiella variicola. All three organisms were amenable to transformation and reporter protein expression, with several functional inducible systems available for each organism. S. stutzeri and K. variicola showed more reliable plasmid-based expression, resulting in successful Cas9 recombineering to create scarless deletions and insertions. Using these tools, we generated mutants with inducible nitrogenase activity and introduced heterologous genes to produce resorcinol products with relevant biological activity in the rhizosphere.


Autoimmune disease and HTLV-1 infection.
We describe two patients who presented with mixed connective tissue disease. Both had antinuclear antibodies and antibodies to the extractable nuclear antigen U1RNP, and both were found to be seropositive for HTLV-1 infection. We found no evidence of HTLV-1 infection in 20 other patients known to have antibodies to U1RNP or in 36 British and West German patients with idiopathic adult polymyositis. In addition the serum from 20 anti-HTLV-1 positive patients did not contain antinuclear antibodies or antibodies to U1RNP. We conclude that rheumatological disease is associated with HTLV-1 through geographical rather than aetiological means.


Three-month stability of the CogState brief battery in healthy older adults, mild cognitive impairment, and Alzheimer's disease: results from the Australian Imaging, Biomarkers, and Lifestyle-rate of change substudy (AIBL-ROCS).
Large prospective studies of Alzheimer's disease (AD) have sought to understand the pathological evolution of AD and factors that may influence the rate of disease progression. Estimates of rates of cognitive change are available for 12 or 24 months, but not for shorter time frames (e.g., 3 or 6 months). Most clinical drug trials seeking to reduce or modify AD symptoms have been conducted over 12- or 24-week periods. As such, we aimed to characterize the performance of a group of healthy older adults, adults with amnestic mild cognitive impairment (aMCI), and adults with AD on the CogState battery of tests over short test-retest intervals. This study recruited 105 healthy older adults, 48 adults with aMCI, and 42 adults with AD from the Australian Imaging, Biomarkers, and Lifestyle study and administered the CogState battery monthly over 3 months. The CogState battery of tests showed high test-retest reliability and stability in all clinical groups when participants were assessed over 3 months. When considered at baseline, the CogState battery of tests was able to detect AD-related cognitive impairment. The data provide important estimates of the reliability, stability, and variability of each cognitive test in healthy older adults, adults with aMCI, and adults with AD. This may potentially be used to inform future estimates of cognitive change in clinical trials.