Alprazolam for depression.
The 'off-label' effect of alprazolam on depression has not been systematically evaluated. To determine the antidepressant effect, including tolerability and acceptability, of alprazolam as monotherapy for major depression, when compared to placebo and conventional antidepressants in outpatients and patients in primary care. We searched the Cochrane Central Register of Controlled Trials and the Cochrane Depression, Anxiety and Neurosis Group Register, which includes relevant randomised controlled trials from the following bibliographic databases: The Cochrane Library (all years to February 2012); EMBASE (1970 to February 2012); MEDLINE (1950 to February 2012) and PsycINFO (1960 to February 2012). Two review authors identified relevant trials by assessing the abstracts of all possible studies. We applied no language restrictions. We selected randomised controlled trials (RCTs) of alprazolam versus placebo or conventional antidepressants for depression in adults, excluding studies with inpatients only. Two review authors performed the data extraction and 'Risk of bias' assessment independently with disagreements resolved through discussion with a third review author. Primary outcomes included the mean difference (MD) in reduction of depression on a continuous measure of depression symptoms, and the risk ratio (RR) of the clinical response based on a dichotomous measure, with 95% confidence intervals (CI). We identified 21 alprazolam studies (22 reports) with a total of 2693 participants. Seven studies used a placebo (n = 771) and 20 used cyclic antidepressants (n = 1765). The typical duration of the studies was four to six weeks. We considered six studies to have a high risk of bias.When alprazolam was compared with placebo for reduction in symptoms all estimates indicated a positive effect for alprazolam. Pooled estimates of efficacy data showed a moderately large continuous mean difference (MD) at the end of trial (-5.34, 95% CI -7.48 to -3.20; I(2) = 68%). The risk difference (RD) for the dichotomous measure of clinical response (50% improvement) was 0.32 in favour of alprazolam (95% CI 0.22 to 0.42; I(2) = 0%), with a number needed to treat to benefit (NNTB) of 3 (95% CI 2 to 5). The RD of all-cause withdrawals did not differ between alprazolam and placebo.When depression severity was measured as a continuum the effect of alprazolam did not differ statistically or clinically from the effects of any of the conventional antidepressants combined (MD 0.25, 95% CI -0.93 to 1.43; I(2) = 55%). However, for dichotomised depression severity, alprazolam had less effect than antidepressants (RR 0.86, 95% CI 0.75 to 0.99; I(2) = 37%; RD -0.11, 95% CI -0.24 to 0.01; I(2) = 58%; NNTB 9, 95% CI 4 to 100). The RD of all-cause withdrawals was -0.04 (95% CI -0.07 to 0.00; I(2) = 35%), in favour of alprazolam. Alprazolam appears to reduce depressive symptoms more effectively than placebo and as effectively as tricyclic antidepressants. However, the studies included in the review were heterogeneous, of poor quality and only addressed short-term effects, thus limiting our confidence in the findings. Whilst the rate of all-cause withdrawals did not appear to differ between alprazolam and placebo, and withdrawals were less frequent in the alprazolam group than in any of the conventional antidepressants combined group, these findings should be interpreted with caution, given the dependency properties of benzodiazepines.


A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor.
The main protease, Mpro, is critical for SARS-CoV-2 replication and an appealing target for designing anti-SARS-CoV-2 agents. Therefore, there is a demand for the development of improved sensors to monitor its activity. Here, we report a pair of genetically encoded, bioluminescence resonance energy transfer (BRET)-based sensors for detecting Mpro proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with Mpro in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in Mpro completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensors developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.


A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids.
In planta, H2O2 is produced as a by-product of enzymatic reactions and during defense responses. Ascorbate peroxidase (APX) is a key enzyme involved in scavenging cytotoxic H2O2. Here, we report the crystal structure of cytosolic APX from sorghum (Sorghum bicolor) (Sobic.001G410200). While the overall structure of SbAPX was similar to that of other APXs, SbAPX uniquely displayed four bound ascorbates rather than one. In addition to the ɣ-heme pocket identified in other APXs, ascorbates were bound at the δ-meso and two solvent-exposed pockets. Consistent with the presence of multiple binding sites, our results indicated that the H2O2-dependent oxidation of ascorbate displayed positive cooperativity. Bound ascorbate at two surface sites established an intricate proton network with ascorbate at the ɣ-heme edge and δ-meso sites. Based on crystal structures, steady-state kinetics, and site-directed mutagenesis results, both ascorbate molecules at the ɣ-heme edge and the one at the surface are expected to participate in the oxidation reaction. We provide evidence that the H2O2-dependent oxidation of ascorbate by APX produces a C2-hydrated bicyclic hemiketal form of dehydroascorbic acid at the ɣ-heme edge, indicating two successive electron transfers from a single-bound ascorbate. In addition, the δ-meso site was shared with several organic compounds, including p-coumaric acid and other phenylpropanoids, for the potential radicalization reaction. Site-directed mutagenesis of the critical residue at the ɣ-heme edge (R172A) only partially reduced polymerization activity. Thus, APX removes stress-generated H2O2 with ascorbates, and also uses this same H2O2 to potentially fortify cell walls via oxidative polymerization of phenylpropanoids in response to stress.


Fruit, vegetable, and antioxidant intake and all-cause, cancer, and cardiovascular disease mortality in a community-dwelling population in Washington County, Maryland.
Higher intake of fruits, vegetables, and antioxidants may help protect against oxidative damage, thus lowering cancer and cardiovascular disease risk. This Washington County, Maryland, prospective study examined the association of fruit, vegetable, and antioxidant intake with all-cause, cancer, and cardiovascular disease death. CLUE participants who donated a blood sample in 1974 and 1989 and completed a food frequency questionnaire in 1989 (N = 6,151) were included in the analysis. Participants were followed to date of death or January 1, 2002. Compared with those in the bottom fifth, participants in the highest fifth of fruit and vegetable intake had a lower risk of all-cause (cases = 910; hazard ratio (HR) = 0.63, 95% confidence interval (CI): 0.51, 0.78; p-trend = 0.0004), cancer (cases = 307; HR = 0.65, 95% CI: 0.45, 0.93; p-trend = 0.08), and cardiovascular disease (cases = 225; HR = 0.76, 95% CI: 0.54, 1.06; p-trend = 0.15) mortality. Higher intake of cruciferous vegetables was associated with lower risk of all-cause mortality (HR = 0.74, 95% CI: 0.60, 0.91; p-trend = 0.04). No statistically significant associations were observed between dietary vitamin C, vitamin E, and beta-carotene intake and mortality. Overall, greater intake of fruits and vegetables was associated with lower risk of all-cause, cancer, and cardiovascular disease death. These findings support the general health recommendation to consume multiple servings of fruits and vegetables (5-9/day).


A Comparison of Three Different Positioning Techniques on Surgical Corrections and Postoperative Alignment in Cervical Spinal Deformity (CD) Surgery.
Retrospective review of a prospective multicenter cervical deformity database. To examine the differences in sagittal alignment correction between three positioning methods in cervical spinal deformity surgery (CD). Surgical correction for CD is technically demanding and various techniques are utilized to achieve sagittal alignment objectives. The effect of different patient positioning techniques on sagittal alignment correction following CD remains unknown. Patients with sagittal deformity who underwent a posterior approach (with and without anterior approach) with an upper instrumented vertebra of C6 or above. Patients with Grade 5, 6, or 7 osteotomies were excluded. Positioning groups were Mayfield skull clamp, bivector traction, and halo ring. Preoperative lower surgical sagittal curve (C2-C7), C2-C7 sagittal vertical axis (cSVA), cervical scoliosis, T1 slope minus cervical lordosis (TS-CL), T1 slope (T1S), chin-brow vertebral angle (CBVA), C2-T3 curve, and C2-T3 SVA was assessed and compared with postoperative radiographs. Segmental changes were analyzed using the Fergusson method. Eighty patients (58% female) with a mean age of 60.6 ± 10.5 years (range, 31-83) were included. The mean postoperative C2-C7 lordosis was 7.8° ± 14 and C2-C7 SVA was 34.1 mm ± 15. There were overall significant changes in cervical alignment across the entire cohort, with improvements in T1 slope (P < 0.001), C2-C7 (P < 0.001), TS-CL (P < 0.001), and cSVA (P = 0.006). There were no differences postoperatively of any radiographic parameter between positioning groups (P > 0.05). The majority of segmental lordotic correction was achieved at C4-5-6 (mean 6.9° ± 11). Additionally, patients who had bivector traction applied had had significantly more segmental correction at C7-T1-T2 compared with Mayfield and halo traction (4.2° vs. 0.3° vs. -1.7° respectively, P < 0.027). Postoperative cervical sagittal correction or alignment was not affected by patient position. The majority of segmental correction occurred at C4-5-6 across all positioning methods, while bivector traction had the largest corrective ability at the cervicothoracic junction.Level of Evidence: 4.


Candidate variants at 6p21.33 and 6p22.1 and risk of non-small cell lung cancer in a Chinese population.
Chromosome 6p21.33, containing BAT3 and MSH5 genes, together with chromosome 6p22.1 were recently identified as susceptible regions for lung cancer in Caucasian populations. These findings interest us in assessing whether genetic variants in these regions also contribute to lung cancer risk in Chinese populations. We genotyped the most significant single nucleotide polymorphism (SNP) (rs9295740) reported in Caucasian populations at Chromosome 6p22.1 and one common potentially functional variant (rs2075789) located at exon 2 of MSH5 in a case-control study including 1009 histologically confirmed non-small cell lung cancer (NSCLC) cases and 1127 cancer-free controls in a Chinese population. We found that the distributions of genotypes of both SNPs between cases and controls were not significantly different (P = 0.624 for rs9295740 and P = 0.937 for rs2075789). Logistic regression analyses revealed neither of the two SNPs was significantly associated with altered risk of NSCLC in dominant or recessive genetic models. When we compared the combined variant genotypes (GA+AA) with the common homozygote GG, assuming a dominant genetic model, the adjusted ORs were 1.03 (95% CI = 0.86-1.25) for rs9295740 and 1.03 (95% CI = 0.85-1.25) for rs2075789. In addition, no significant associations were observed in subgroups stratified by age, gender, smoking status or histologic types. Our results indicate that the most significant SNP rs9295740 identified in Caucasians in 6p22.1 and the potentially functional SNP rs2075789 in 6p21.33, seem not applicable to Chinese populations as susceptible markers for lung cancer. Re-sequencing and fine-mapping this region, along with extensive functional evaluations, is required.


Dolomitization: Observations on the Island of Bonaire, Netherlands Antittes.
Evaporation of sea water and gypsum precipitation on south Bonaire produce dense brines having a high ratio of magnesium to calcium. Recent dolomite is a common replacement product in the associated carbonate sediments. A mass balance indicates that brine is being refluxed downward today, and the Pleistocene dolomite on north Bonaire could have been produced by similar reflux of brine.


A qualitative analysis of gestational surrogates' healthcare experiences during the COVID-19 pandemic.
No empirical data are available on the healthcare experiences of surrogates during the COVID-19 pandemic. This study aimed to examine the impact of pandemic-control measures on surrogates' fertility, pregnancy and birthing experiences. Sampling frame included eligible surrogates who were actively involved in a surrogacy process at an academic IVF centre during the pandemic (03/2020 to 02/2022). Data were collected between 29/04/2022 and 31/07/2022 using an anonymous 85-item online survey that included twelve open-ended questions. Free-text comments were analysed by thematic analysis. The response rate was 50.7% (338/667). Of the 320 completed surveys used for analysis, 609 comments were collected from 206 respondents. Twelve main themes and thirty-six sub-themes grouped under 'vaccination', 'fertility treatment', 'pregnancy care', and 'surrogacy birth' were identified. Three in five surrogates found the control measures highly or moderately affected their surrogacy experiences. Themes involving loneliness and isolation frequently emerged when essential surrogacy support was restricted by the visitor protocols implemented at healthcare facilities. Our findings show that restricting or limiting intended parents' in-person involvement increased surrogates' feelings of isolation and made the overall surrogacy experience less rewarding and fulfilling. Furthermore, the childbirth experiences of surrogates were mostly negative, suggesting that hospitals were ill-equipped to manage all births, including surrogacy births, during the pandemic. Our findings highlight the needs to rethink how surrogacy care and maternity services could be strengthened to better serve the needs of surrogates during times of public health crises, such as COVID-19, while still allowing for risk mitigation and maximising patient safety.


Age-related differences in functional network segregation in the context of sex and reproductive stage.
Age is accompanied by differences in the organization of functional brain networks, which impact behavior in adulthood. Functional networks become less segregated and more integrated with age. However, sex differences in network segregation declines with age are not well-understood. Further, network segregation in the context of female reproductive stage is relatively understudied, though unmasking such relationships would be informative for elucidating biological mechanisms that contribute to sex-specific differences in aging. In the current work, we used data from the Cambridge Centre for Ageing and Neuroscience (Cam-CAN) repository to evaluate differences in resting-state network segregation as a product of sex and reproductive stage. Reproductive stage was categorized using the Stages of Reproductive Aging Workshop (STRAW+10) criteria. Replicating prior work, we investigated the following functional networks: auditory, cerebellar-basal ganglia, cingulo-opercular task control, default mode, dorsal attention, fronto-parietal task control, salience, sensory somatomotor mouth, sensory somatomotor hand, ventral attention, and visual. First, our results mirror findings from previous work indicating that network segregation is lower with increasing age. Second, when analyzing associations between network segregation and age within each sex separately, we find qualitative differences between females and males. Finally, we report significant effects of reproductive stage on network segregation, though these findings are likely driven by age. Broadly, our results suggest that impacts of sex may be important to evaluate when investigating network segregation differences across adulthood, though further work is needed to determine the unique role of menopause and sex hormones on the organization of functional brain networks within aging females.


Improving satellite-driven PM2.5 models with Moderate Resolution Imaging Spectroradiometer fire counts in the southeastern U.S.
Multiple studies have developed surface PM2.5 (particle size less than 2.5 µm in aerodynamic diameter) prediction models using satellite-derived aerosol optical depth as the primary predictor and meteorological and land use variables as secondary variables. To our knowledge, satellite-retrieved fire information has not been used for PM2.5 concentration prediction in statistical models. Fire data could be a useful predictor since fires are significant contributors of PM2.5. In this paper, we examined whether remotely sensed fire count data could improve PM2.5 prediction accuracy in the southeastern U.S. in a spatial statistical model setting. A sensitivity analysis showed that when the radius of the buffer zone centered at each PM2.5 monitoring site reached 75 km, fire count data generally have the greatest predictive power of PM2.5 across the models considered. Cross validation (CV) generated an R2 of 0.69, a mean prediction error of 2.75 µg/m3, and root-mean-square prediction errors (RMSPEs) of 4.29 µg/m3, indicating a good fit between the dependent and predictor variables. A comparison showed that the prediction accuracy was improved more substantially from the nonfire model to the fire model at sites with higher fire counts. With increasing fire counts, CV RMSPE decreased by values up to 1.5 µg/m3, exhibiting a maximum improvement of 13.4% in prediction accuracy. Fire count data were shown to have better performance in southern Georgia and in the spring season due to higher fire occurrence. Our findings indicate that fire count data provide a measurable improvement in PM2.5 concentration estimation, especially in areas and seasons prone to fire events.


Cross talk between the response regulators PhoB and TctD allows for the integration of diverse environmental signals in Pseudomonas aeruginosa.
Two-component systems (TCS) serve as stimulus-response coupling mechanisms to allow organisms to adapt to a variety of environmental conditions. The opportunistic pathogen Pseudomonas aeruginosa encodes for more than 100 TCS components. To avoid unwanted cross-talk, signaling cascades are very specific, with one sensor talking to its cognate response regulator (RR). However, cross-regulation may provide means to integrate different environmental stimuli into a harmonized output response. By applying a split luciferase complementation assay, we identified a functional interaction of two RRs of the OmpR/PhoB subfamily, namely PhoB and TctD in P. aeruginosa. Transcriptional profiling, ChIP-seq analysis and a global motif scan uncovered the regulons of the two RRs as well as a quadripartite binding motif in six promoter regions. Phosphate limitation resulted in PhoB-dependent expression of the downstream genes, whereas the presence of TctD counteracted this activation. Thus, the integration of two important environmental signals e.g. phosphate availability and the carbon source are achieved by a titration of the relative amounts of two phosphorylated RRs that inversely regulate a common subset of genes. In conclusion, our results on the PhoB and TctD mediated two-component signal transduction pathways exemplify how P. aeruginosa may exploit cross-regulation to adapt bacterial behavior to complex environments.


Targeting global conservation funding to limit immediate biodiversity declines.
Inadequate funding levels are a major impediment to effective global biodiversity conservation and are likely associated with recent failures to meet United Nations biodiversity targets. Some countries are more severely underfunded than others and therefore represent urgent financial priorities. However, attempts to identify these highly underfunded countries have been hampered for decades by poor and incomplete data on actual spending, coupled with uncertainty and lack of consensus over the relative size of spending gaps. Here, we assemble a global database of annual conservation spending. We then develop a statistical model that explains 86% of variation in conservation expenditures, and use this to identify countries where funding is robustly below expected levels. The 40 most severely underfunded countries contain 32% of all threatened mammalian diversity and include neighbors in some of the world's most biodiversity-rich areas (Sundaland, Wallacea, and Near Oceania). However, very modest increases in international assistance would achieve a large improvement in the relative adequacy of global conservation finance. Our results could therefore be quickly applied to limit immediate biodiversity losses at relatively little cost.


Once weekly treatment with epoetin-beta.
Studies have shown that both intravenous (i.v.) and subcutaneous (s.c.) administration of epoetin-beta therapy are effective and well tolerated in the treatment of renal anaemia; however, the s.c. route provides enhanced efficacy with a lower dose compared with the i.v. route and it is more cost-effective. Epoetin dosing frequency is an important issue for health care professionals and patients. Recent studies have shown that epoetin-beta administered once weekly and once every 2 weeks can maintain stable target haemoglobin and haematocrit levels in dialysis patients. Such reduced dosing frequencies may improve patient satisfaction and compliance with treatment, and encourage patients to self-administer. Furthermore, less frequent dosing administration would be associated with economic benefits in terms of reduced nursing time in the clinic or out-patient setting. Where this is clinical practice, fewer injections and visits to the clinic should also improve patients' quality of life. A range of effective dosing regimens with epoetin-beta administered via either pre-filled syringes, multidose vials or injector pens allows physicians to tailor treatment to an individual patient's preference.


Barriers to Disease Monitoring and Liver Cancer Surveillance Among Patients with Chronic Hepatitis B in the United States.
Chronic hepatitis B infection (CHB) is a condition that needs ongoing care such as monitoring for liver enzymes (ALT) and HBV DNA tests in treated and untreated patients, and annual imaging evaluation for liver cancer. Although follow-up care and treatment might seem straight forward, an estimated two-thirds of those who are aware of their infection are not seeing a health care provider, and more than half of those who are eligible for treatment do not receive it. This study aimed to compile and examine studies related to the barriers of disease monitoring, treatment, and liver cancer surveillance for CHB patients in the United States (US). A total of 4439 studies on monitoring and surveillance of CHB published between 2007 and 2018 were identified through a search of electronic databases. After critical assessment, the authors included 42 studies, divided into categories: 'patient-related barriers'; 'provider-related barriers'; and 'system-related barriers'. Among the patient-related barriers, one of the most frequent factors invoked in failing to have adequate surveillance was lack of patient's knowledge. In the provider-related barrier category, a lack of disease knowledge and adherence to guidelines was frequently reported. For the system-related barrier category, the only recurrent mention was a lack of clarity in guidelines or lack of guidelines from certain national institutions. This review summarizes and highlights the need for long-term disease management improvement of chronic hepatitis B infection for patients and healthcare providers that care for them.


Growth retardation in preterm infants.
This paper combines earlier results on the relation between birthweight and gestational age, and the relation between fetal weight and ultrasound measurements of the fetal biparietal diameter (BPD) and mean abdominal diameter (AD) to investigate whether preterm infants (viewed as a group) are smaller than unborn fetuses of the same gestational age. The birthweight distribution for each sex at 223 and 258 days gestation was derived from the birthweight-for-gestational age charts based on 3888 newborn infants. The sex-specific intrauterine weight distribution was estimated from ultrasound measurement of the fetal BPD and AD performed on randomly selected fetuses of gestational age 223 and 258 days. The birthweights were lower than the intrauterine weights, especially early in pregnancy and for female infants. Thus, the 10th birthweight centile for girls at day 223 corresponds to the 4th centile of the 'true' intrauterine weight, and the 'true' intrauterine 10th centile corresponds to the 25th centile birthweight at day 223.


Sealer penetration and adaptation in root canals with the butterfly effect.
The butterfly effect is a phenomenon seen in some roots and is related to density of dentinal tubules. The aim was to investigate penetration depth and adaptation quality of root canal sealers and ProRoot MTA into bucco-lingual and mesio-distal aspects of roots with and without the effect. One hundred and twenty teeth were decoronated at the cemento-enamel junction. Canals were prepared and assigned to obturation groups: gutta-percha with a sealer (AH Plus, EndoREZ, Kerr Pulp Canal Sealer, MTA Fillapex) or ProRoot MTA alone (each containing 10 butterfly and 10 non-butterfly roots). Root sectioning yielded coronal and middle samples. Confocal laser scanning and scanning electron microscopy were used to assess penetration and adaptation. Teeth with the effect had greater mean penetration bucco-lingually (766 μm) than mesio-distally (184 μm, P = 0.003). Coronal sections had greater penetration (430 μm) compared with middle (247 μm, P = 0.006). In conclusion, greater penetration in roots with the effect may improve treatment outcomes.


Clostridium difficile: clinical disease and diagnosis.
Clostridium difficile is an opportunistic pathogen that causes a spectrum of disease ranging from antibiotic-associated diarrhea to pseudomembranous colitis. Although the disease was first described in 1893, the etiologic agent was not isolated and identified until 1978. Since clinical and pathological features of C. difficile-associated disease are not easily distinguished from those of other gastrointestinal diseases, including ulcerative colitis, chronic inflammatory bowel disease, and Crohn's disease, diagnostic methods have relied on either isolation and identification of the microorganism or direct detection of bacterial antigens or toxins in stool specimens. The current review focuses on the sensitivity, specificity, and practical use of several diagnostic tests, including methods for culture of the etiologic agent, cellular cytotoxicity assays, latex agglutination tests, enzyme immunoassay systems, counterimmunoelectrophoresis, fluorescent-antibody assays, and polymerase chain reactions.


Standing lateral thoracotomy in horses: indications, complications, and outcomes.
To describe the indications for, complications arising from, and outcome of horses that had standing lateral thoracotomy for pleural or pericardial disease. Case series. Horses (n=16). Medical records (January 1990-December 2008) of sedated standing horses that had lateral thoracotomy were reviewed. Clinical and surgical findings, perioperative and short-term complications were recorded. Long-term (>6 months) outcome was determined through telephone conversations with owners and veterinarians. Mean (±SD) horse age was 6.6±5.3 years (range, 1-15 years). Thoracotomy was most commonly for treatment of recurrent or chronic pleural infection (94%). Anaerobic bacteria were frequently isolated from pleural effusion associated with pleuropneumonia (63%). Right lateral intercostal thoracotomy was performed in 13 horses (82%); intercostal muscle myectomy in 5 horses; and rib resection in 1. Perioperative complications were hemipneumothorax (2 horses) and short-term complications included cellulitis and abscessation of the thoracotomy site (6 horses). Fourteen (88%) horses survived to discharge and 46% of horses that survived returned to their previous level of athletic activity. Lateral thoracotomy is well tolerated by standing sedated horses with minimal perioperative and short-term complications. Standing lateral thoracotomy should be considered for the treatment of complicated pleuropneumonia in horses.


Intramedullary metastasis.
Three cases of intramedullary metastases and one of a metastasis into the medulla oblongata are described. In two cases the primary tumour was a bronchial carcinoma and in one case a carcinoma of the breast. In one patient a primary tumour could not be found. The literature on this condition is reviewed and the difficulties of clinical diagnosis are discussed. The question remains unanswered as to the mechanism by which these tumour-cells reach the spinal cord and there is, as yet, no satisfactory explanation for the relative rare occurrence of these metastases.


Functional biodiversity loss along natural CO2 gradients.
The effects of environmental change on biodiversity are still poorly understood. In particular, the consequences of shifts in species composition for marine ecosystem function are largely unknown. Here we assess the loss of functional diversity, i.e. the range of species biological traits, in benthic marine communities exposed to ocean acidification (OA) by using natural CO2 vent systems. We found that functional richness is greatly reduced with acidification, and that functional loss is more pronounced than the corresponding decrease in taxonomic diversity. In acidified conditions, most organisms accounted for a few functional entities (i.e. unique combination of functional traits), resulting in low functional redundancy. These results suggest that functional richness is not buffered by functional redundancy under OA, even in highly diverse assemblages, such as rocky benthic communities.


Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus.
The template for synthesis of hepadnaviral RNAs is a covalently closed circular (ccc) DNA located in the nucleus of the infected hepatocyte. Hepatocytes are normally long-lived and nondividing, and antiviral therapies in chronically infected individuals face the problem of eliminating not only the replicative forms of viral DNA found in the cytoplasm but also the cccDNA from the nucleus. Because cccDNA does not replicate semiconservatively, it is not an obvious target for antiviral therapy. However, elimination of cccDNA might be facilitated if its half-life were short in comparison to the generation time of hepatocytes and if new cccDNA formation were effectively blocked. We have therefore measured cccDNA levels in woodchuck hepatocyte cultures following in vitro infection with woodchuck hepatitis virus and treatment with inhibitors of viral DNA synthesis. The viral reverse transcriptase inhibitors lamivudine (3TC) [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine), FTC (5-fluoro-2',3'-dideoxy-3'-thiacytidine) and ddC (2',3'-dideoxycytidine) were added to the cultures beginning at 4 days postinfection. Treatment for up to 36 days with 3TC reduced the amount of cccDNA in the cultures not more than twofold compared to that of an untreated control. Treatment with ddC for 36 days and with FTC for 12 days resulted in effects similar to that of treatment with 3TC. Moreover, the declines in cccDNA appeared to reflect the loss of hepatocytes from the cultures rather than of cccDNA from hepatocytes. These results emphasize the important role of the longevity of the infected hepatocytes in the persistence of an infection.


Silicon-Based Biomaterials Modulate the Adaptive Immune Response of T Lymphocytes to Promote Osteogenesis/Angiogenesis via Epigenetic Regulation.
Silicon (Si)-based biomaterials are widely applied for bone regeneration. However, the underlying mechanisms of the materials function remain largely unknown. T lymphocyte-mediated adaptive immune response plays a vital role in the process of bone regeneration. In the current study, mesoporous silica (MS) is used as a model material of Si-based biomaterials. It shows that the supernatant of CD4+ T lymphocytes pretreated with MS extract significantly promotes the vascularized bone regeneration. The potential mechanism is closely related to the fact that MS extract can reduce the expression of regulatory factor X-1 (RFX-1) in CD4+ T lymphocytes. This may result in the overexpression of interleukin-17A (IL-17A) by boosting histone H3 acetylation and lowering DNA methylation and H3K9 trimethylation. Importantly, the in vivo experiments further reveal that MS particles significantly enhance bone regeneration with improved angiogenesis in the critical-sized calvarial defect mouse model accompanied by upregulation of IL-17A in peripheral blood and the proportion of Th17 cells. This study suggests that modulation of the adaptive immune response of T lymphocytes by silicate-based biomaterials plays an important role for bone regeneration.


Forced Intercalation (FIT)-Aptamers.
Aptamers are oligonucleotide sequences that can be evolved to bind to various analytes of interest. Here, we present a general design strategy that transduces an aptamer-target binding event into a fluorescence readout via the use of a viscosity-sensitive dye. Target binding to the aptamer leads to forced intercalation (FIT) of the dye between oligonucleotide base pairs, increasing its fluorescence by up to 20-fold. Specifically, we demonstrate that FIT-aptamers can report target presence through intramolecular conformational changes, sandwich assays, and target-templated reassociation of split-aptamers, showing that the most common aptamer-target binding modes can be coupled to a FIT-based readout. This strategy also can be used to detect the formation of a metallo-base pair within a duplexed strand and is therefore attractive for screening for metal-mediated base pairing events. Importantly, FIT-aptamers reduce false-positive signals typically associated with fluorophore-quencher based systems, quantitatively outperform FRET-based probes by providing up to 15-fold higher signal to background ratios, and allow rapid and highly sensitive target detection (nanomolar range) in complex media such as human serum. Taken together, FIT-aptamers are a new class of signaling aptamers which contain a single modification, yet can be used to detect a broad range of targets.


Modulation of cellular and humoral immune responses during exposure of healthy individuals to an aqueous mistletoe extract.
Mistletoe extracts have been shown to exert immunomodulatory properties. However, little is known about changes in different immunological parameters during therapy. Aim of our study was, therefore, to investigate the influence of subcutaneous treatment in 8 volunteers who were exposed to an aqueous mistletoe extract (Viscum album Mali; VM) for 8 weeks. During in vivo exposure, VM induced the production of anti-ML-1 antibodies of the IgG-type. A pronounced proliferation of peripheral blood mononuclear cells (PBMC) could be observed in 6 volunteers. However, the activity and time of maximal response varied from one individual to the other. There was no correlation between the cellular and the humoral reactivity and different antigens present in this mistletoe extract may be operative at different levels of the immune system. In order to define more precisely the type of immune response, cytokines were measured in the supernatants of VM-stimulated cell cultures. VM induced the secretion of Th1- (IFN-gamma) or Th2- (IL-4) related cytokines and also the release of TNF-alpha and IL-6. All these different immunological phenomena can be best explained by presentation of mistletoe-related antigens to T- and B-cells via antigen presenting cells in the draining lymph nodes.


Genome-wide analyses of carboxyl-terminal sequences.
Sequence motifs at the protein carboxyl termini in linear polypeptides are uniquely positioned and functionally capable of serving as recognition signatures for a variety of cellular and biochemical processes. At the proteome level, it is unknown whether and what carboxyl-terminal sequences might be particularly conserved, which may be directly related to specific biological functions shared among certain groups of proteins. To investigate this question, we analyzed the terminal sequences of reported yeast open reading frames, which presumably constitute the predicted, entire proteome of Saccharomyces cerevisiae. The results show that there are both known and novel terminal sequences. They are conserved at a frequency similar to that of functionally important, experimentally confirmed signals such as the HDEL sequence that mediates the endoplasmic reticulum retention and/or retrieval. The findings support the notion that there may be additional carboxyl-terminal signals, and the conserved motifs could be experimentally tested for currently unknown biological functions. Similar analyses were also applied to the limited proteome databases of other organisms with overall consistent findings. Therefore, indexing a proteome according to its carboxyl-terminal sequences may provide a means for functional classification and determination of proteins.


Potential mechanisms and environmental controls of TiO2 nanoparticle effects on soil bacterial communities.
It has been reported that engineered nanoparticles (ENPs) alter soil bacterial communities, but the underlying mechanisms and environmental controls of such effects remain unknown. Besides direct toxicity, ENPs may indirectly affect soil bacteria by changing soil water availability or other properties. Alternatively, soil water or other environmental factors may mediate ENP effects on soil bacterial communities. To test, we incubated nano-TiO2-amended soils across a range of water potentials for 288 days. Following incubation, the soil water characteristics, organic matter, total carbon, total nitrogen, and respiration upon rewetting (an indicator of bioavailable organic carbon) were measured. Bacterial community shifts were characterized by terminal restriction fragment length polymorphism (T-RFLP). The endpoint soil water holding had been reported previously as not changing with this nano-TiO2 amendment; herein, we also found that some selected soil properties were unaffected by the treatments. However, we found that nano-TiO2 altered the bacterial community composition and reduced diversity. Nano-TiO2-induced community dissimilarities increased but tended to approach a plateau when soils became drier. Taken together, nano-TiO2 effects on soil bacteria appear to be a result of direct toxicity rather than indirectly through nano-TiO2 affecting soil water and organic matter pools. However, such directs effects of nano-TiO2 on soil bacterial communities are mediated by soil water.


Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway.
The ubiquitin proteasome system (UPS) comprises hundreds of different conjugation/deconjugation enzymes and multiple receptors that recognize ubiquitylated proteins. A formidable challenge to deciphering the biology of ubiquitin is to map the networks of substrates and ligands for components of the UPS. Several different receptors guide ubiquitylated substrates to the proteasome, and neither the basis for specificity nor the relative contribution of each pathway is known. To address how broad of a role the ubiquitin receptor Rpn10 (S5a) plays in turnover of proteasome substrates, we implemented a method to perform quantitative analysis of ubiquitin conjugates affinity-purified from experimentally perturbed and reference cultures of Saccharomyces cerevisiae that were differentially labeled with 14N and 15N isotopes. Shotgun mass spectrometry coupled with relative quantification using metabolic labeling and statistical analysis based on q values revealed ubiquitylated proteins that increased or decreased in level in response to a particular treatment. We first identified over 225 candidate UPS substrates that accumulated as ubiquitin conjugates upon proteasome inhibition. To determine which of these proteins were influenced by Rpn10, we evaluated the ubiquitin conjugate proteomes in cells lacking either the entire Rpn10 (rpn10delta) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uimdelta)). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10delta cells, whereas only one-fifth as many accumulated in uimdelta cells. These findings underscore a broad role for Rpn10 in turnover of ubiquitylated substrates but a relatively modest role for its ubiquitin-binding UIM domain. This approach illustrates the feasibility of systems-level quantitative analysis to map enzyme-substrate networks in the UPS.


Effects of a multicomponent behavioral intervention on impulsivity and cognitive deficits in adolescents with excess weight.
The aim of this study was to explore the effects of a multidisciplinary behavioral intervention including cognitive behavioral therapy, structured physical activity, and dietary counseling on impulsive personality and cognitive skills and subsequent BMI loss in excess weight adolescents. Forty-two adolescents with excess weight (14 males and 28 females, range 12-17 years), as defined by the International Obesity Task Force Criteria, participated in our study. We used a longitudinal observational design with two assessments: before and after treatment. We collected baseline measures of impulsive personality (UPPS-P scale), cognitive performance (letter number sequencing, Stroop and Iowa gambling task), and biometric parameters. After 12 weeks of intervention, parallel measures were used to determine whether treatment-induced changes in impulsivity and cognition predicted changes in BMI. BMI showed a statistically significant reduction after treatment [from mean (SD) 29.36 (4.51) to 27.31 (4.41), Cohen's d=0.5]. Greater reductions in negative urgency (negative-emotion-driven impulsivity) and greater improvement in cognitive inhibitory control skills were associated with greater reductions in BMI. Because the design was correlational and lacked a control group, future studies should clarify whether these associations reflect a causal effect or just overlapping improvements associated with a third variable (e.g. increases in attention procurement or motivation).


Dose-dependent stimulation of renal prostaglandin synthesis by deamino-8-D-arginine vasopressin in rats with hereditary diabetes insipidus.
It has been postulated that renal prostaglandins (PGs) function as negative feedback inhibitors of the action of antidiuretic hormone (ADH), implying a correlation between levels of ADH and the rate of renal PG synthesis. These studies have evaluated the relationship between renal PG synthesis and hormone levels in rats with hereditary diabetes insipidus, a species devoid of circulating ADH. Since vasoconstrictor agents can stimulate renal PG synthesis by mechanisms unrelated to antidiuretic activity, deamino-8-D-arginine vasopressin (dDAVP) was utilized for replacement therapy instead of arginine vasopressin, which has considerable pressor activity. dDAVP was administered by S.C. implanted osmotic minipumps to obtain steady states of dDAVP at different dose levels. As indices of renal PG synthesis, urinary excretion of PGE2 and PGF2 alpha were measured by gas chromatography-mass spectrometry. PGE2 excretion, although increased by dDAVP treatment, was not correlated with dose of dDAVP. However, PGF2 alpha excretion was highly correlated with dose of dDAVP (r = 0.97, P less than .01). The sum (PGE2 + PGF2 alpha), which may more accurately reflect total medullary PG synthesis, was also significantly correlated with dose of dDAVP (r = 0.98, P less than .001). It is concluded that dDAVP stimulates renal PG synthesis in a dose-related fashion. This occurs at doses which bring urine osmolality into the normal physiological range. Furthermore, it is shown that stimulation of renal PG synthesis by arginine vasopressin is not due primarily to its pressor action. These experiments also provide evidence that urinary PGE2 and PGF2 alpha excretion can vary independently.


DNA-dependent protein kinase is a molecular target for the development of noncytotoxic radiation-sensitizing drugs.
DNA-dependent protein kinase (DNA-PK)-defective severe combined immunodeficient (SCID) mice have a greater sensitivity to ionizing radiation compared with wild-type mice due to deficient repair of DNA double-strand break. SCID cells were therefore studied to determine whether radiosensitization by the specific inhibitor of DNA-PK, IC87361, is eliminated in the absence of functional DNA-PK. IC87361 enhanced radiation sensitivity in wild-type C57BL6 endothelial cells but not in SCID cells. The tumor vascular window model was used to assess IC87361-induced radiosensitization of SCID and wild-type tumor microvasculature. Vascular density was 5% in irradiated SCID host compared with 50% in C57BL6 mice (P < 0.05). IC87361 induced radiosensitization of tumor microvasculature in wild-type mice that resembled the radiosensitive phenotype of tumor vessels in SCID mice. Radiosensitization by IC87361 was eliminated in SCID tumor vasculature, which lack functional DNA-PK. Irradiated LLC and B16F0 tumors implanted into SCID mice showed greater tumor growth delay compared with tumors implanted into either wild-type C57BL6 or nude mice. Furthermore, LLC tumors treated with radiation and IC87361 showed tumor growth delay that was significantly greater than tumors treated with radiation alone (P < 0.01 for 3 Gy alone versus 3 Gy + IC87361). DNA-PK inhibitors induced no cytotoxicity and no toxicity in mouse normal tissues. Mouse models deficient in enzyme activity are useful to assess the specificity of novel kinase inhibitors. DNA-PK is an important target for the development of novel radiation-sensitizing drugs that have little intrinsic cytotoxicity.


A neurotrophic model of the development of the retinogeniculocortical pathway induced by spontaneous retinal waves.
The development of the retinogeniculate pathway or the geniculocortical pathway, or both, occurs either before birth or before eye opening in many species. It is widely believed that spontaneous retinal activity could drive the segregation of afferents into eye-specific laminae or columns and the refinement of initially diffuse receptive fields and the emergence of orderly, retinotopic organization. We show that a recent computational model that generates a phenomenologically accurate representation of spontaneous retinal activity can indeed drive afferent segregation and, more particularly, topographic and receptive field refinement in the retinogeniculocortical system. We use a model of anatomical synaptic plasticity based on recent data suggesting that afferents might compete for limited amounts of retrograde neurotrophic factors (NTFs). We find that afferent segregation and receptive field formation are disrupted in the presence of exogenous NTFs. We thus predict that infusion of NTFs into the lateral geniculate nucleus should disrupt normal development and that the infusion of such factors into the striate cortex should disrupt receptive field refinement in addition to the well known disruption of ocular dominance column (ODC) formation. To demonstrate that the capacity of our model of plasticity to drive normal development is not restricted just to spontaneous retinal activity, we also use a coarse representation of visually evoked activity in some simulations. We find that such simulations can exhibit the formation of ODCs followed by their disappearance, reminiscent of the New World marmoset. A decrease in interocular correlations stabilizes these ODCs. Thus we predict that divergent strabismus should render marmoset ODCs stable into adulthood.


Regulation of Posttranslational Modifications of HMGB1 During Immune Responses.
High-mobility group protein 1 (HMGB1) is an evolutionarily conserved and multifunctional protein. The biological function of HMGB1 depends on its cellular locations, binding partners, and redox states. Extracellular HMGB1 is a mediator of inflammation during infection or tissue injury. Immune cells actively release HMGB1 in response to infection, which in turn orchestrates both innate and adaptive immune responses. Hyperacetylation of HMGB1 within its nuclear localization sequences mobilizes HMGB1 from the nucleus to the cytoplasm and subsequently promotes HMGB1 release. The redox states of the cysteines in positions 23, 45, and 106 determine the biological activity of the extracellular HMGB1. The full picture and the detailed molecular mechanisms of how cells regulate the posttranslational modifications and the redox status of HMGB1 during immune responses or under stress not only unravel the molecular mechanisms by which cells regulate the release and the biological function of HMGB1 but may also provide novel therapeutic targets to treat inflammatory diseases. It is important to identify the signaling pathways that regulate the posttranslational modifications and the redox status of HMGB1 and find their roles in host immune responses and pathogenesis of diseases.


Verification of chicken Nanog as an epiblast marker and identification of chicken PouV as Pou5f3 by newly raised antibodies.
Pluripotency is an important feature of early embryonic cells of multicellular organisms. Recent advances in stem cell research have shown that Nanog and Pou5f1 (Oct3/4) play important roles in mammalian pluripotency. However, whether these molecules exert conserved functions in other species remains unknown. Although the epiblast of the early chicken embryo would provide a useful experimental model, a lack of antibodies against chicken Nanog (cNanog) and chicken PouV/Pou5f3 (cPouV) proteins has hampered intensive investigation. Here we report newly raised polyclonal antibodies that specifically recognize cNanog and cPouV proteins. The specificity and sensitivity of the antibodies were validated by both western blotting and immunostaining with transfected 293T cells and chicken embryonic tissues. Immunohistochemistry using these antibodies revealed that cNanog protein was specifically localized in epiblastic cells and germ cells. In contrast, cPouV expression was seen almost ubiquitously. We also found that chicken epiblast-derived colony-forming cells that morphologically resemble mouse embryonic stem cells were cNanog-positive, implying that these colony-forming cells possess pluripotency. The anti-cPouV antibody further enabled us to identify a previously unknown region at the N-terminus of the cPouV protein containing a characteristic motif that is absent in mammalian Pou5f1. Thus, the antibodies raised in this study are useful tools for studying the functions of cNanog and cPouV at the protein level and the molecular mechanisms of chicken pluripotency.


Weighted Graph Regularized Sparse Brain Network Construction for MCI Identification.
Brain functional networks (BFNs) constructed from resting-state functional magnetic resonance imaging (rs-fMRI) have been widely applied to the analysis and diagnosis of brain diseases, such as Alzheimer's disease and its prodrome, namely mild cognitive impairment (MCI). Constructing a meaningful brain network based on, for example, sparse representation (SR) is the most essential step prior to the subsequent analysis or disease identification. However, the independent coding process of SR fails to capture the intrinsic locality and similarity characteristics in the data. To address this problem, we propose a novel weighted graph (Laplacian) regularized SR framework, based on which BFN can be optimized by considering both intrinsic correlation similarity and local manifold structure in the data, as well as sparsity prior of the brain connectivity. Additionally, the non-convergence of the graph Laplacian in the self-representation model has been solved properly. Combined with a pipeline of sparse feature selection and classification, the effectiveness of our proposed method is demonstrated by identifying MCI based on the constructed BFNs.


Filamentous hemagglutinin has a major role in mediating adherence of Bordetella pertussis to human WiDr cells.
[35S]methionine-labeled Bordetella pertussis adhered to monolayers of WiDr cells, an epitheliumlike cell line from a human intestinal carcinoma. Adherence was proportional to the density of the WiDr cells and to the concentration of B. pertussis in the assay. Adherence of virulent phase I strains Tohama phase I, 114, and BP338 was much greater than adherence of avirulent strains Tohama phase III and 423 phase IV. Mutants deficient in the production of the filamentous hemagglutinin (FHA) were hemagglutination negative and adhered to WiDr cells much less efficiently than the parent strains. Preincubation of B. pertussis cells with FHA increased their hemagglutination activity and adherence to WiDr cells. Goat antibody to FHA inhibited, in a dose-dependent manner, the adherence of strain Tohama I but not the adherence of FHA-deficient mutant Tohama 325. At similar protein concentrations, normal goat antibody, goat antibody to pertussis toxin, or the Fab fragments of goat antibody to serotype 2 fimbriae had no effect on adherence. Also, an FHA-positive strain without fimbriae showed high adherence, while a fimbriated FHA-deficient mutant adhered poorly. Our data indicate that FHA plays a major role in adherence of B. pertussis to human WiDr cells. Fimbriae do not appear to mediate attachment of B. pertussis to WiDr cells.


A Governance Framework to Integrate Longitudinal Clinical and Community Data in a Distributed Data Network: The Childhood Obesity Data Initiative.
Integrating longitudinal data from community-based organizations (eg, physical activity programs) with electronic health record information can improve capacity for childhood obesity research. A governance framework that protects individual privacy, accommodates organizational data stewardship requirements, and complies with laws and regulations was developed and implemented to support the harmonization of data from disparate clinical and community information systems. Through the Childhood Obesity Data Initiative (CODI), 5 Colorado-based organizations collaborated to expand an existing distributed health data network (DHDN) to include community-generated data and assemble longitudinal patient records for research. A governance work group expanded an existing DHDN governance infrastructure with CODI-specific data use and exchange policies and procedures that were codified in a governance plan and a delegated-authority, multiparty, reciprocal agreement. A CODI governance work group met from January 2019 to March 2020 to conceive an approach, develop documentation, and coordinate activities. Governance requirements were synthesized from the CODI use case, and a customized governance approach was constructed to address governance gaps in record linkage, a procedure to request data, and harmonizing community and clinical data. A Master Sharing and Use Agreement (MSUA) and Memorandum of Understanding were drafted and executed to support creation of linked longitudinal records of clinical- and community-derived childhood obesity data. Furthermore, a multiparty infrastructure protocol was approved by the local institutional review board (IRB) to expedite future CODI research by simplifying IRB research applications. CODI implemented a clinical-community governance strategy that built trust between organizations and allowed efficient data exchange within a DHDN. A thorough discovery process allowed CODI stakeholders to assess governance capacity and reveal regulatory and organizational obstacles so that the governance infrastructure could effectively leverage existing knowledge and address challenges. The MSUA and complementary governance documents can inform similar efforts.


Soil chemistry dynamics of Sus scrofa carcasses with and without delayed Diptera colonization.
Under normal circumstances, insects such as blow flies will oviposit and larvae will colonize a carcass as soon as possible. However, insect colonization on a carcass may be delayed due to the effects of wrapping, shallow burial, addition of lime derivatives to mitigate scavenging and odor, or extreme weather. The impacts of delayed insect colonization on carcass decomposition and its subsequent effect on soil chemistry profiles have not been examined to date. The objectives of this study were to determine soil chemistry dynamics associated with porcine carcasses experiencing delayed insect colonization for 7-day or 14-day. Soil chemistry profiles such as ammonium-N (NH4 -N), orthophosphate-P (PO4 -P), and dissolved organic carbon (DOC) were significantly different among treatments: insect inclusion (immediate access of blow fly colonization on porcine carcasses), 7-day insect exclusion and 14-day insect exclusion (blow fly access was delayed up to 7-day and 14-day). Furthermore, significant differences of soil chemical profiles were detected between days of decomposition and soil regions. Soil moisture, NH4 -N, PO4 -P, and DOC were significantly higher when insects were excluded from the porcine carcass suggesting loss of tissue from larval feeding reduced the mass of nutrients entering the soil. This study provides useful information for forensic science in cases where insect colonization is delayed for a period of time postmortem and soil chemistry in the cadaver decomposition island is considered for estimating postmortem interval.


Differential Assimilation of Inorganic Carbon and Leucine by Prochlorococcus in the Oligotrophic North Pacific Subtropical Gyre.
The light effect on photoheterotrophic processes in Prochlorococcus, and primary and bacterial productivity in the oligotrophic North Pacific Subtropical Gyre was investigated using (14)C-bicarbonate and (3)H-leucine. Light and dark incubation experiments were conducted in situ throughout the euphotic zone (0-175 m) on nine expeditions to Station ALOHA over a 3-year period. Photosynthetrons were also used to elucidate rate responses in leucine and inorganic carbon assimilation as a function of light intensity. Taxonomic group and cell-specific rates were assessed using flow cytometric sorting. The light:dark assimilation rate ratios of leucine in the top 150 m were ∼7:1 for Prochlorococcus, whereas the light:dark ratios for the non-pigmented bacteria (NPB) were not significant different from 1:1. Prochlorococcus assimilated leucine in the dark at per cell rates similar to the NPB, with a contribution to the total community bacterial production, integrated over the euphotic zone, of approximately 20% in the dark and 60% in the light. Depth-resolved primary productivity and leucine incorporation showed that the ratio of Prochlorococcus leucine:primary production peaked at 100 m then declined steeply below the deep chlorophyll maximum (DCM). The photosynthetron experiments revealed that, for Prochlorococcus at the DCM, the saturating irradiance (E k) for leucine incorporation was reached at approximately half the light intensity required for light saturation of (14)C-bicarbonate assimilation. Additionally, high and low red fluorescing Prochlorococcus populations (HRF and LRF), co-occurring at the DCM, had similar E k values for their respective substrates, however, maximum assimilation rates, for both leucine and inorganic carbon, were two times greater for HRF cells. Our results show that Prochlorococcus contributes significantly to bacterial production estimates using (3)H-leucine, whether or not the incubations are conducted in the dark or light, and this should be considered when making assessments of bacterial production in marine environments where Prochlorococcus is present. Furthermore, Prochlorococcus primary productivity showed rate to light-flux patterns that were different from its light enhanced leucine incorporation. This decoupling from autotrophic growth may indicate a separate light stimulated mechanism for leucine acquisition.


Lysosomal Acid Lipase Deficiency: Genetics, Screening, and Preclinical Study.
Lysosomal acid lipase (LAL) is a lysosomal enzyme essential for the degradation of cholesteryl esters through the endocytic pathway. Deficiency of the LAL enzyme encoded by the LIPA gene leads to LAL deficiency (LAL-D) (OMIM 278000), one of the lysosomal storage disorders involving 50-60 genes. Among the two disease subtypes, the severe disease subtype of LAL-D is known as Wolman disease, with typical manifestations involving hepatomegaly, splenomegaly, vomiting, diarrhea, and hematopoietic abnormalities, such as anemia. In contrast, the mild disease subtype of this disorder is known as cholesteryl ester storage disease, with hypercholesterolemia, hypertriglyceridemia, and high-density lipoprotein disappearance. The prevalence of LAL-D is rare, but several treatment options, including enzyme replacement therapy, are available. Accordingly, a number of screening methodologies have been developed for this disorder. This review summarizes the current discussion on LAL-D, covering genetics, screening, and the tertiary structure of human LAL enzyme and preclinical study for the future development of a novel therapy.


Nasal surgery for contact point headaches.
Headache due to the pressure on nasal mucosa of anatomical variations, nasal polyps, or mucosal swelling in the absence of inflammation of the paranasal sinuses is a clinical entity that has gained wide acceptance. In this paper, the outcomes of surgical treatment in 30 patients whose headaches were believed to be the result of intranasal contact points are presented. Total relief of the headache and significant improvement were achieved in 43% and 47% of the patients, respectively, after endoscopic endonasal surgery. The intensity of the headache was the same as preoperatively in 10% of the patients. In the absence of any other identifiable etiological factors, intranasal mucosal contacts must be kept in mind as a cause of the headache.


Rapid plasma membrane isolation via intracellular polymerization-mediated biomolecular confinement.
Plasma membrane isolation is a foundational process in membrane proteomic research, cellular vesicle studies, and biomimetic nanocarrier development, yet separation processes for this outermost layer are cumbersome and susceptible to impurities and low yield. Herein, we demonstrate that cellular cytosol can be chemically polymerized for decoupling and isolation of plasma membrane within minutes. A rapid, non-disruptive in situ polymerization technique is developed with cell membrane-permeable polyethyleneglycol-diacrylate (PEG-DA) and a blue-light-sensitive photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The photopolymerization chemistry allows for precise control of intracellular polymerization and tunable confinement of cytosolic molecules. Upon cytosol solidification, plasma membrane proteins and vesicles are rapidly derived and purified as nucleic acids and intracellular proteins as small as 15 kDa are stably entrapped for removal. The polymerization chemistry and membrane derivation technique are broadly applicable to primary and fragile cell types, enabling facile membrane vesicle extraction from shorted-lived neutrophils and human primary CD8 T cells. The study demonstrates tunable intracellular polymerization via optimized live cell chemistry, offers a robust membrane isolation methodology with broad biomedical utility, and reveals insights on molecular crowding and confinement in polymerized cells. STATEMENT OF SIGNIFICANCE: Isolating the minute fraction of plasma membrane proteins and vesicles requires extended density gradient ultracentrifugation processes, which are susceptible to low yield and impurities. The present work demonstrates that the membrane isolation process can be vastly accelerated via a rapid, non-disruptive intracellular polymerization approach that decouples cellular cytosols from the plasma membrane. Following intracellular polymerization, high-yield plasma membrane proteins and vesicles can be derived from lysis buffer and sonication treatment, respectively. And the intracellular content entrapped within the polymerized hydrogel is readily removed within minutes. The technique has broad utility in membrane proteomic research, cellular vesicle studies, and biomimetic materials development, and the work offers insights on intracellular hydrogel-mediated molecular confinement.


Identification of a novel mammalian post-translational modification, phosphocholine, on placental secretory polypeptides.
Placental neurokinin B appears to be post-translationally modified by phosphocholine (PC) attached to the aspartyl side chain at residue 4 of the mature peptide. Corticotrophin releasing factor (CRF) was found to be expressed by the rat placenta with the main secreted forms being phosphocholinated proCRF+/- one or two polysaccharide moieties. A combination of high-pressure liquid chromatography (HPLC) and two-site immunometric analysis suggested that PC was also attached to the placental precursors of adrenocorticotrophin, hemokinin, activin and follistatin. However, the fully processed forms of rat placental activin and CRF were free of PC. Formerly, the parasitic filarial nematodes have used PC as a post-translational modification, attached via the polysaccharide moiety of certain secretory glycoproteins to attenuate the host immune system allowing parasite survival, but it is the PC group itself which endows the carrier with the biological activity. The fact that treatment of proCRF peptides with phospholipase C but not endoglycosidase destroyed PC immunoreactivity suggested a simpler mode of attachment of PC to placental peptides than that used by nematodes. Thus, it is possible that by analogy the placenta uses its secreted phosphocholinated hormones to modulate the mother's immune system and help protect the placenta from rejection.


Characterization of a PLDζ2 Homology Gene from Developing Castor Bean Endosperm.
Castor oil contains approximately 90% ricinoleic acid (RA) which is stored mainly in the form of tri-ricinoleic acid containing triacylglycerols (TAG). Ricinoleate is synthesized from oleate (18:1n-9) esterified to the sn-2 position of phosphatidylcholine (PtdCho) catalyzed by oleoyl-12-hydroxylase. PtdCho-derived diacylglycerol (DAG) is an important substrate pool for TAG synthesis, and the interconversion between PtdCho and DAG has been shown to play a critical role in channeling hydroxy fatty acids (HFA) to TAG. Although phospholipase D (PLD) has been reported to catalyze the hydrolysis of PtdCho to produce phosphatidic acid which can then be converted to DAG, its potential functions in the channeling of RA from PtdCho to DAG and the assembly of RA on TAG is largely unknown. In the present study, 11 PLD genes were identified from the Castor Bean Genome Database. Gene expression analysis indicated that RcPLD9 is expressed at relatively high levels in developing seeds compared to other plant tissues. Sequence and phylogenetic analyses revealed that RcPLD9 is a homolog of Arabidopsis PLDζ2. Overexpression of RcPLD9 in the Arabidopsis CL7 line producing C18-HFA resulted in RA content reductions in the polar lipid fraction (mainly PtdCho) and mono-HFA-TAG, but increased RA content in di-HFA-TAG. Since part of RA in di-HFA-TAG is derived from HFA-DAG, the results indicated that RcPLD9 facilitates the channeling of RA from PtdCho to DAG for its assembly on TAG in developing seeds.


Male and female rate differences in carpal tunnel syndrome injuries: personal attributes or job tasks?
Carpal tunnel syndrome (CTS) exacts a significant toll on the health and productivity of American workers. In 1996, 29,937 workers lost time from work due to CTS. Half of these were out for more than 25 days, compared to a median of 5 days for all injuries/illnesses. There are striking gender disparities in CTS rates. Overall, three times more women suffer from CTS than men. While some authors have emphasized the role of gender attributes in this risk disparity, the multifactoral causes of CTS, and the sex segregation of women into jobs with high-risk tasks, may be obscuring the work-related contributions to CTS risk. We argue here that men and women doing the same work tasks will have similar rates of CTS. To examine this premise, we used Bureau of Labor Statistics injury counts (numerator) and census data from the Current Population Survey (denominator) to determine injury rates of CTS for both men and women in six high-risk occupations: (1) assembler, (2) laborer-nonconstruction, (3) packaging and filling machine operators, (4) janitors and cleaners, (5) butchers and meat cutters, and (6) data entry keyers. Variable job tasks exist within five of the six high-risk occupational titles. Among those five, the male to female (M:F) risk rate ratio ranged from 0.29 to 0.50. However, the sixth occupational title, data entry keyers, which requires a single physical task, had a risk rate ratio of 1.06. This suggests that an equal risk between genders exists when the occupational tasks (exposure) are truly similar. Job task analysis unmasks potential biases that may wrongly attribute disproportionate CTS rates to gender attributes. This focus on gender attributes fails all workers because preventive interventions in the workplace are deferred. It fails women disproportionately because they are overrepresented in jobs at high risk for CTS.


Melatonin and its agonists in pain modulation and its clinical application.
Melatonin, the hormone of darkness has many physiological functions in the body and also exerts a number of pharmacological effects. Most of these actions of melatonin are mediated through melatonin membrane receptors like MT1/MT2 receptors or through nuclear orphan receptors like RZR/ROR receptors or through calcium binding proteins in the cytosol. The finding that pain perception is circadian in nature has prompted many to suggest that "pain modulation" is one of the most important physiological functions of melatonin. By using a number of animal models of pain perception, it has been found that melatonin exerts antinociceptive and antiallodynic effects. Number of studies has shown that melatonin modulates pain perception by acting through opioid receptors, NMDA receptors and G-protein, and they have been analyzed using specific antagonists like naloxone or NMDA-G protein receptor antagonists. Recently it has been shown that melatonin exerts its antinociceptive effects through MT1 and MT2 melatonergic receptors located in the dorsal region of the spinal cord as well as in various parts of the brain concerned with pain modulation. Evidences for this have been obtained by using common melatonergic receptor antagonist like luzindole or specific MT2 receptor antagonist like 4P-PDOT or K-185. In a few clinical studies undertaken during surgery, melatonin has been shown to have analgesic effects. Melatonin is emerging as a new analgesic drug with a novel mechanism of actions and has the potential to be used as a natural pain killer in inflammatory, neuropathic pain conditions and also during surgical procedures.


The Impact of Dobbs on US Graduate Medical Education.
The Dobbs decision will directly affect patients and reproductive rights; it will also impact patients indirectly in many ways, one of which will be changes in the physician workforce through its impact on graduate medical education. Current residency accreditation standards require training in all forms of contraception in addition to training in the provision of abortion. State bans on abortions may diminish access to training as approximately half of obstetrics and gynecology residency programs are in states with significant abortion restrictions. The Dobbs decision creates numerous hurdles for trainees and their programs. Trainees in restrictive states will have to travel to learn in a different program in a protective state. As training opportunities diminish, potentially leading to a decline in clinical skills, knowledge, and experience in the provision of abortion, the rate of complications and maternal mortality are likely to rise. This will likely have a disproportionately negative effect on preexisting disparities in reproductive health fueled by a longstanding history of systemic racism and inequities. This work aims to both define the looming problem in abortion training created by Dobbs and propose solutions to ensure that an adequate workforce is available in the future to serve patient needs.


Gene transfer in cereals.
Until recently, gene transfer in plants was achieved only by sexual hybridization. Now, in addition, plant genetic manipulation, with the use of both recombinant DNA and protoplast fusion technology, is being applied to an increasing range of plants. The soil bacterium Agrobacterium tumefaciens, with its associated plasmid, is used as a vector for introducing DNA into the genomes of dicotyledonous plants, but it has not proved suitable for cereals. Instead, the direct uptake of plasmid DNA into cereal protoplasts is being used for the transformation of cells in rice, wheat, and maize. Transformation efficiencies, in some cases, are becoming comparable to those obtained in dicotyledons with Agrobacterium. In rice it is now possible to regenerate efficiently whole plants from protoplasts, and this capability may soon be extended to the other cereals. By means of direct interaction of cereal protoplasts with plasmids, coupled with improved procedures for the regeneration of plants from their protoplasts, gene transfer in the cereals is becoming established at the frontiers of recombinant DNA technology.


Homing and reparative effect of intra-articular injection of autologus mesenchymal stem cells in osteoarthritic animal model.
This work aimed to study the homing evidence and the reparative effect of mesenchymal stem cells (MSCs) in the healing process of induced osteoarthritis in experimental animal model (donkeys). Twenty-seven donkeys were equally divided into 3 groups based on the observation period after induction of arthritis (3, 6 and 9 weeks) to achieve different degrees of osteoarthritis. Each group was subdivided into three subgroups of three animals each based on the follow-up period (1, 2 and 6 months) after treatment. The induction was done through intra-articular (IA) injection of 2 ml of Amphotericin-B in both carpal joints. MSCs were harvested in a separate procedure, labeled with green fluorescent protein (GFP) using monster GFP vector and suspended in hyaluronic acid for IA injection. Treatment approaches consisted of cell-treatment using MSCs suspended in 3 ml of hyaluronic acid (HA) for the right carpal joint; and using the same amount of (HA) but without MSCs for the left contralateral carpal joint to serve as a control. Animals were assessed clinically and radiologically before and after treatment. Synovial fluid was also evaluated. Histopathologically; articular cartilage structural changes, reduction of articular cartilage matrix staining, osteophyte formation, and subchondral bone plate thickening were graded. Data was summarized using median and percentile for scores of histopathologic grading. Comparison between groups was done using non-parametric Mann Whitney test. The reparative effect of MSCs was significant both clinically and radiologically in all treated groups (P < 0.05) compared to the control groups. Fluorescence microscopy of sections of the cell-treated joints of all animals indicated that the GFP-transduced injected cells have participated effectively in the reparative process of the damaged articular surface and have integrated within the existing articular cartilage. The cells were associated with the surface of the cartilage and, were also detected in the interior. Homing was confirmed by the incorporation of injected GFP-labeled MSCs within the repaired newly formed cartilage. Significant recovery proves that the use of IA injection of autologous MSCs is a viable and a practical option for treating different degrees of osteoarthritis.


Identification of a minimal promoter sequence for the human N-acetyltransferase Type I gene that binds AP-1 (activator protein 1) and YY-1 (Yin and Yang 1).
Human N -acetyltransferase Type I (NAT1) catalyses the acetylation of many aromatic amine and hydrazine compounds and it has been implicated in the catabolism of folic acid. The enzyme is widely expressed in the body, although there are considerable differences in the level of activity between tissues. A search of the mRNA databases revealed the presence of several NAT1 transcripts in human tissue that appear to be derived from different promoters. Because little is known about NAT1 gene regulation, the present study was undertaken to characterize one of the putative promoter sequences of the NAT1 gene located just upstream of the coding region. We show with reverse-transcriptase PCR that mRNA transcribed from this promoter (Promoter I) is present in a variety of human cell-lines, but not in quiescent peripheral blood mononuclear cells. Using deletion mutant constructs, we identified a 20 bp sequence located 245 bases upstream of the translation start site which was sufficient for basal NAT1 expression. It comprised an AP-1 (activator protein 1)-binding site, flanked on either side by a TCATT motif. Mutational analysis showed that the AP-1 site and the 3' TCATT sequence were necessary for gene expression, whereas the 5' TCATT appeared to attenuate promoter activity. Electromobility shift assays revealed two specific bands made up by complexes of c-Fos/Fra, c-Jun, YY-1 (Yin and Yang 1) and possibly Oct-1. PMA treatment enhanced expression from the NAT1 promoter via the AP-1-binding site. Furthermore, in peripheral blood mononuclear cells, PMA increased endogenous NAT1 activity and induced mRNA expression from Promoter I, suggesting that it is functional in vivo.


The experience with 113 patients with Kawasaki disease in Fars Province, Iran.
This study was conducted to determine the epidemiology, clinical manifestations, cardiac involvements and laboratory findings of Kawasaki disease (KD) in Fars Province, Iran from January 1991 to December 2002. One hundred and thirteen patients with KD were confirmed. This syndrome occurred mostly in males (ratio=2.1:1). The most commonly affected age group was one to five years old. There was no secondary case in families, none of the patients were related and recurrence of disease was seen in only one patient. Prolonged fever and thrombocytosis were significant risk factors for developing coronary artery disease (p<0.05, respectively). Among the 10 patients with cardiac involvement, seven patients had dilated coronary arteries (>4 mm) and three patients had aneurysm of coronary artery in their first echocardiography. It has been suggested that a high index of clinical suspicion of KD could improve diagnosis and implementation of preventive treatment.


[Environmental factors and human reproductive system].
Problems concerning regularities and mechanisms of the environmental factor influence on human reproductive system are discussed. It is indicated that the response of reproductive system to external challenges is based on the evolutionary formed links between the structures responsible for reproductive organ function (epiphysis, hypothalamus, pituitary), the nature of environmental factors and the regularities of their changes depending on the season and geographical location. Damaging external challenges caused by biosphere pollution by waste products of human economic activity and extreme ecological conditions are realized both by direct influence on target tissues (ionizing radiation) and indirectly through neuroendocrine mechanisms regulating reproductive organs function. Factors and regularities of the development of diseases and pathological conditions of human reproductive system, caused by external challenge including the injurious industrial factor influence were analyzed.


From best evidence to best practice: effective implementation of change in patients' care.
Major difficulties arise when introducing evidence and clinical guidelines into routine daily practice. Data show that many patients do not receive appropriate care, or receive unnecessary or harmful care. Many approaches claim to offer solutions to this problem; which ones are as yet the most effective and efficient is unclear. We aim to provide an overview of present knowledge about initiatives to changing medical practice. Substantial evidence suggests that to change behaviour is possible, but this change generally requires comprehensive approaches at different levels (doctor, team practice, hospital, wider environment), tailored to specific settings and target groups. Plans for change should be based on characteristics of the evidence or guideline itself and barriers and facilitators to change. In general, evidence shows that none of the approaches for transferring evidence to practice is superior to all changes in all situations.


Application of an Ecological Momentary Assessment Protocol in a Workplace Intervention: Assessing Compliance, Criterion Validity, and Reactivity.
Ecological momentary assessment (EMA) is a method of collecting behavioral data in real time. The purpose of this study was to examine EMA compliance, identify factors predicting compliance, assess criterion validity of, and reactivity to, using EMA in a workplace intervention study. Forty-five adults (91.1% female, 39.7 [9.6] y) were recruited for a workplace standing desk intervention. Participants received 5 surveys each day for 5 workdays via smartphone application. EMA items assessed current position (sitting/standing/stepping). EMA responses were time matched to objectively measured time in each position before and after each prompt. Multilevel logistic regression models estimated factors influencing EMA response. Cohen kappa measured interrater agreement between EMA-reported and device-measured position. Reactivity was assessed by comparing objectively measured sitting/standing/stepping in the 15 minutes before and after each EMA prompt using multilevel repeated-measures models. Participants answered 81.4% of EMA prompts. Differences in compliance differed by position. There was substantial agreement between EMA-reported and device-measured position (κ = .713; P < .001). Following the EMA prompt, participants sat 0.87 minutes more than before the prompt (P < .01). The use of EMA is a valid assessment of position when used in an intervention to reduce occupational sitting and did not appear to disrupt sitting in favor of the targeted outcome.


Altered Mucins (MUC) trafficking in benign and malignant conditions.
Mucins are high molecular weight O-glycoproteins that are predominantly expressed at the apical surface of epithelial cells and have wide range of functions. The functional diversity is attributed to their structure that comprises of a peptide chain with unique domains and multiple carbohydrate moieties added during posttranslational modifications. Tumor cells aberrantly overexpress mucins, and thereby promote proliferation, differentiation, motility, invasion and metastasis. Along with their aberrant expression, accumulating evidence suggest the critical role of altered subcellular localization of mucins under pathological conditions due to altered endocytic processes. The mislocalization of mucins and their interactions result in change in the density and activity of important cell membrane proteins (like, receptor tyrosine kinases) to facilitate various signaling, which help cancer cells to proliferate, survive and progress to more aggressive phenotype. In this review article, we summarize studies on mucins trafficking and provide a perspective on its importance to pathological conditions and to answer critical questions including its use for therapeutic interventions.


SYCP3 regulates strand invasion activities of RAD51 and DMC1.
The synaptonemal complex is a higher-ordered proteinaceous architecture formed between homologous chromosomes. SYCP3 is a major component of the lateral/axial elements in the synaptonemal complex and is essential for meiotic recombination. Previous genetic studies showed that SYCP3 functions in meiotic homologous recombination biased to interhomologous chromosomes, by regulating the strand invasion activities of the RAD51 and DMC1 recombinases. However, the mechanism by which SYCP3 regulates RAD51- and DMC1-mediated strand invasion remains elusive. In this study, we found that SYCP3 significantly suppresses the RAD51-mediated, but not the DMC1-mediated, strand invasion reaction by competing with HOP2-MND1, which is an activator for both RAD51 and DMC1. A SYCP3 mutant with defective RAD51 binding does not inhibit the RAD51-mediated homologous recombination in human cells. Therefore, SYCP3 may promote the DMC1-driven homologous recombination by attenuating the RAD51 activity during meiosis.


A comparison study of hair examination methodologies.
A study was conducted to investigate the accuracy between two methods of hair analysis: PCR-STR DNA analysis and microscopic comparison analysis. Standard sets of pubic hairs were collected from volunteers, and unknown sets were generated from these samples. Three out of five (60%) of the hairs analyzed produced full DNA profiles that were correctly matched to the standard sets. DNA analysis was inconclusive (partial or no DNA profile) for two out of five (40%) of the samples. In contrast, the microscopic comparison analysis correctly matched four out of five (80%) of the samples to the standard sets but mis-identified one out of five (20%) of the samples. These results reinforce the practice of preliminary microscopic hair examination in narrowing down a set of hairs for DNA analysis. Microscopic comparison analysis is sufficiently reliable to remain a rapid and inexpensive method for forensic hair analysis.


Harnessing the influence of reactive edges and defects of graphene substrates for achieving complete cycle of room-temperature molecular sensing.
Molecular doping and detection are at the forefront of graphene research, a topic of great interest in physical and materials science. Molecules adsorb strongly on graphene, leading to a change in electrical conductivity at room temperature. However, a common impediment for practical applications reported by all studies to date is the excessively slow rate of desorption of important reactive gases such as ammonia and nitrogen dioxide. Annealing at high temperatures, or exposure to strong ultraviolet light under vacuum, is employed to facilitate desorption of these gases. In this article, the molecules adsorbed on graphene nanoflakes and on chemically derived graphene-nanomesh flakes are displaced rapidly at room temperature in air by the use of gaseous polar molecules such as water and ethanol. The mechanism for desorption is proposed to arise from the electrostatic forces exerted by the polar molecules, which decouples the overlap between substrate defect states, molecule states, and graphene states near the Fermi level. Using chemiresistors prepared from water-based dispersions of single-layer graphene on mesoporous alumina membranes, the study further shows that the edges of the graphene flakes (showing p-type responses to NO₂ and NH₃) and the edges of graphene nanomesh structures (showing n-type responses to NO₂ and NH₃) have enhanced sensitivity. The measured responses towards gases are comparable to or better than those which have been obtained using devices that are more sophisticated. The higher sensitivity and rapid regeneration of the sensor at room temperature provides a clear advancement towards practical molecule detection using graphene-based materials.


Role of fibronectin in topographical guidance of neurite extension on electrospun fibers.
Bridging of long peripheral nerve gaps remains a significant clinical challenge. Electrospun nanofibers have been used to direct and enhance neurite extension in vitro and in vivo. While it is well established that oriented fibers influence neurite outgrowth and Schwann cell migration, the mechanisms by which they influence these cells are still unclear. In this study, thin films consisting of aligned poly-acrylonitrile methylacrylate (PAN-MA) fibers or solvent casted smooth, PAN-MA films were fabricated to investigate the potential role of differential protein adsorption on topography-dependent neural cell responses. Aligned nanofiber films promoted enhanced adsorption of fibronectin compared to smooth films. Studies employing function-blocking antibodies against cell adhesion motifs suggest that fibronectin plays an important role in modulating Schwann cell migration and neurite outgrowth from dorsal root ganglion (DRG) cultures. Atomic Force Microscopy demonstrated that aligned PAN-MA fibers influenced fibronectin distribution, and promoted aligned fibronectin network formation compared to smooth PAN-MA films. In the presence of topographical cues, Schwann cell-generated fibronectin matrix was also organized in a topographically sensitive manner. Together these results suggest that fibronectin adsorption mediated the ability of topographical cues to influence Schwann cell migration and neurite outgrowth. These insights are significant to the development of rational approaches to scaffold designs to bridge long peripheral nerve gaps.


Sensory anatomy of the most aquatic of carnivorans: the Antarctic Ross seal, and convergences with other mammals.
Transitions to and from aquatic life involve transformations in sensory systems. The Ross seal, Ommatophoca rossii, offers the chance to investigate the cranio-sensory anatomy in the most aquatic of all seals. The use of non-invasive computed tomography on specimens of this rare animal reveals, relative to other species of phocids, a reduction in the diameters of the semicircular canals and the parafloccular volume. These features are independent of size effects. These transformations parallel those recorded in cetaceans, but these do not extend to other morphological features such as the reduction in eye muscles and the length of the neck, emphasizing the independence of some traits in convergent evolution to aquatic life.


TMARKER: A free software toolkit for histopathological cell counting and staining estimation.
Histological tissue analysis often involves manual cell counting and staining estimation of cancerous cells. These assessments are extremely time consuming, highly subjective and prone to error, since immunohistochemically stained cancer tissues usually show high variability in cell sizes, morphological structures and staining quality. To facilitate reproducible analysis in clinical practice as well as for cancer research, objective computer assisted staining estimation is highly desirable. We employ machine learning algorithms as randomized decision trees and support vector machines for nucleus detection and classification. Superpixels as segmentation over the tissue image are classified into foreground and background and thereafter into malignant and benign, learning from the user's feedback. As a fast alternative without nucleus classification, the existing color deconvolution method is incorporated. Our program TMARKER connects already available workflows for computational pathology and immunohistochemical tissue rating with modern active learning algorithms from machine learning and computer vision. On a test dataset of human renal clear cell carcinoma and prostate carcinoma, the performance of the used algorithms is equivalent to two independent pathologists for nucleus detection and classification. We present a novel, free and operating system independent software package for computational cell counting and staining estimation, supporting IHC stained tissue analysis in clinic and for research. Proprietary toolboxes for similar tasks are expensive, bound to specific commercial hardware (e.g. a microscope) and mostly not quantitatively validated in terms of performance and reproducibility. We are confident that the presented software package will proof valuable for the scientific community and we anticipate a broader application domain due to the possibility to interactively learn models for new image types.


Validating Domains of Patient Contextual Factors Essential to Preventing Contextual Errors: A Qualitative Study Conducted at Chicago Area Veterans Health Administration Sites.
"Patient context" indicates patient circumstances and characteristics or states that are essential to address when planning patient care. Specific patient "contextual factors," if overlooked, result in an inappropriate plan of care, a medical error termed a "contextual error." The myriad contextual factors that constitute patient context have been grouped into broad domains to create a taxonomy of challenges to consider when planning care. This study sought to validate a previously identified list of contextual domains. This qualitative study used directed content analysis. In 2014, 19 Department of Veterans Affairs (VA) providers (84% female) and 49 patients (86% male) from two VA medical centers and four outpatient clinics in the Chicago area participated in semistructured interviews and focus groups. Topics included patient-specific, community, and resource-related factors that affect patients' abilities to manage their care. Transcripts were analyzed with a previously identified list of contextual domains as a framework. Analysis of responses revealed that patients and providers identified the same 10 domains previously published, plus 3 additional ones. Based on comments made by patients and providers, the authors created a revised list of 12 domains from themes that emerged. Six pertain to patient circumstances such as access to care and financial situation, and 6 to patient characteristics/states including skills, abilities, and knowledge. Contextual factors in patients' lives may be essential to address for effective care planning. The rubric developed can serve as a "contextual differential" for clinicians to consider when addressing challenges patients face when planning their care.


Intake of trans fatty acids from partially hydrogenated vegetable and fish oils and ruminant fat in relation to cancer risk.
Intake of trans fatty acids (TFA) may influence systemic inflammation, insulin resistance and adiposity, but whether TFA intake influences cancer risk is insufficiently studied. We examined the association between TFA intake from partially hydrogenated vegetable oils (PHVO-TFA), partially hydrogenated fish oils (PHFO-TFA), and ruminant fat (rTFA) and cancer risk in the Norwegian counties study, a large cohort study with a participation rate >80%. TFA intake was assessed three times in 1974-1988 by questionnaire. A total of 77,568 men and women were followed up through 2007, during which time 12,004 cancer cases occurred. Hazard ratios (HRs) and confidence intervals (CIs) were estimated with Cox regression for cancer sites with ≥150 cases during follow-up. Significantly increased or decreased risks were found when comparing the highest and lowest intake categories (HRs, 95% CIs) for PHVO-TFA and pancreatic cancer in men (0.52, 0.31-0.87) and non-Hodgkin lymphoma (NHL) in both genders (0.70, 0.50-0.98); PHFO-TFA and rectal cancer (1.43, 1.09-1.88), prostate cancer (0.82, 0.69-0.96), and multiple myeloma (2.02, 1.24-3.28); and rTFA and all cancers (1.09, 1.02-1.16), cancer of the mouth/pharynx (1.59, 1.08-2.35), NHL (1.47, 1.06-2.04) and multiple myeloma (0.45, 0.24-0.84). Furthermore, positive trends were found for PHFO-TFA and stomach cancer (p(trend) = 0.01) and rTFA and postmenopausal breast cancer (p(trend) = 0.03). Inverse trends were found for PHVO-TFA and all cancers (p(trend) = 0.006) and cancer of the central nervous system in women (p(trend) = 0.005). PHFO-TFA, but not PHVO-TFA, seemed to increase cancer risk. The increased risks observed for rTFA may be linked to saturated fat.


Effect of lysozyme on Mycobacterium leprae.
Because large amounts of lysozyme are present in phagocytes, it was selected, as one of the bactericidal substances, to study the in vitro effects on M. leprae. The results obtained led the author to conclude that M. leprae is not susceptible to lysozyme as many other atypical as well as attenuated pathogenic mycobacteria. This resistance of M. leprae to lysozyme was seen up to a level of 100-500 micrograms per ml, while levels of 1000 micrograms and more had very significant effect; the ATP content of such treated M. leprae declined to 30% of the original levels in 5 days of incubation at 37 degrees C, with optimum pH of 6.0, and these M. leprae after such exposure to lysozyme failed to multiply in the foot pads of mice. The interpretation of these results, though not definitive, have been discussed.


Breast cancer cells induce stromal fibroblasts to express MMP-9 via secretion of TNF-alpha and TGF-beta.
We used 2D-cocultures employing fibroblasts of different genetic backgrounds and MCF10A-derived human breast epithelial cells of increasingly malignant potential to investigate tumor-stroma interactions in breast cancer and to identify possible signaling pathways involved. Tumor cells induced expression of matrix-metalloproteinase 9 (MMP-9) in fibroblasts in a pattern dependent on the degree of their malignancy. In-situ zymography localized the main gelatinolytic activity around stromal cells in cocultures and xenografted tumors. Use of Smad3 knockout fibroblasts, small molecule inhibitors, and neutralizing antibodies showed that MMP-9 expression was induced by tumor cell-derived TNF-alpha and TGF-beta, dependent on Smad-, Ras-, and PI3-kinase-signaling, and likewise modulated by subsequent HGF- and EGF-signaling. Together, our results indicate that MMP-9 levels in tumor fibroblasts are regulated by a complex tumor-stroma cross-talk, involving multiple ligands and cellular signaling pathways.