The Pseudomonas aeruginosa tonB gene encodes a novel TonB protein.
The Pseudomonas aeruginosa tonB gene was cloned by complementation of the tonB mutation of Pseudomonas putida strain TE516 (W. Bitter, J. Tommassen & P.J. Weisbeek, 1993, Mol Microbiol 7, 117-130). The gene was 1025 bp in length, capable of encoding a protein of 36860 Da. As with previously described TonB proteins, the P. aeruginosa TonB (TonBp.a.) was rich in Pro residues (18.1%) and contained Glu-Pro/Lys-Pro repeats. Unlike previously described TonB proteins, however, TonBp.a. lacked an N-terminal membrane anchor (signal) sequence and contained, instead, a predicted internal signal/anchor sequence, expected to yield an atypical N-terminal cytoplasmic domain in this protein. TonB proteins are essential components in iron-siderophore uptake in bacteria, apparently functioning as energy transducers in coupling the energized state of the cytoplasmic membrane to outer-membrane receptor function. As expected, tonB derivatives of P. aeruginosa were defective in siderophore-mediated iron acquisition. tonB gene expression was inducible by iron-limitation, consistent with the identification of a Fur consensus binding sequence upstream of the gene. TonBp.a. showed substantially greater similarity to the Escherichia coli TonB protein than the Pseudomonas putida protein (31% identity vs. 20% identity) and tonBp.a. was able to complement deficiencies in the acquisition of ferric enterobactin and vitamin B12, and sensitivity to phage phi 80 of an E. coli tonB strain. The larger size of TonBp.a. and its ability to function in both E. coli and P. putida make it a unique TonB protein whose characterization should enhance our understanding of TonB function in bacteria.


Visceral analgesia induced by acute and repeated water avoidance stress in rats: sex difference in opioid involvement.
Chronic psychological stress-induced alterations in visceral sensitivity have been predominantly assessed in male rodents. We investigated the effect of acute and repeated water avoidance stress (WAS) on the visceromotor response (VMR) to colorectal distension (CRD) and the role of opioids in male and cycling female Wistar rats using a novel non-invasive manometric technique. After a baseline VMR (1st CRD, day 0), rats were exposed to WAS (1 h day(-1) ) either once or for four consecutive days, without injection or with naloxone (1 mg kg(-1) ) or saline injected subcutaneously before each WAS session. The VMR to CRD recorded on day 1 or 4 immediately after the last WAS was reduced in both females and males. The visceral analgesia was mainly naloxone-dependent in females, but naloxone-independent in males. In non-injected animals, on days 2 and 5, VMR was not significantly different from baseline in males whereas females exhibited a significant VMR increase at 60 mmHg on day 5. Basal CRD and CRD on days 1, 2, and 5 in both sexes without WAS induced similar VMR. When monitored non-invasively, psychological stress induces an immediate poststress visceral analgesia mediated by an opiate signaling system in females while naloxone-independent in males, and hyperalgesia at 24 h after repeated stress only in females. These data highlight the importance of sex-specific interventions to modulate visceral pain response to stress.


Ex-vivo Expansion of Adult Human Pancreatic Beta-Cells.
Ex-vivo generation of human insulin-producing cells is considered a promising approach to providing an abundant source of cells for beta-cell replacement therapy in diabetes. Expansion of adult beta-cells from the limited number of islet donors is an attractive prospect. However, while evidence supports the replicative capacity of both rodent and human beta-cells in vivo, attempts at expanding these cells in tissue culture result in loss of beta-cell phenotype, making it difficult to track their fate during continuous propagation and raising doubts about their therapeutic potential. Recent lineage-tracing studies demonstrate the ability of human beta-cells to survive and replicate to a significant degree in vitro. Beta-cell delamination out of the normal epithelial structure, a process that results in dedifferentiation, seems to be required for significant in-vitro proliferation. Therefore, ways must be found of inducing redifferentiation of the expanded cells ex vivo, or of restoring their function upon transplantation. Elucidation of the signaling pathways altered during beta-cell adaptation to growth in culture may provide clues to cell redifferentiation. In a recent study, we found that human beta-cell dedifferentiation and entrance into the cell cycle in vitro correlated with activation of the Notch pathway and downregulation of the cell cycle inhibitor p57. Inhibition of the Notch downstream target HES1 using short hairpin RNA reduced beta-cell dedifferentiation and replication, suggesting a potential target for inducing cell redifferentiation following expansion in culture. This review critically discusses the potential for using ex-vivo beta-cell replication and redifferentiation in cell replacement therapy in diabetes.


The role of glandular kallikrein in the formation of a salivary proline-rich protein A by cleavage of a single bond in salivary protein C.
An enzyme was purified from human parotid saliva that can cleave a single arginine-glycine peptide bond between residues 106 and 107 in human salivary proline-rich protein C, hereby giving rise to another proline-rich protein A, which is also found in saliva. The enzyme was purified 2400-fold. It cleaved salivary protein C at the rate of 59 micrograms of protein/h per microgram of enzyme and had amino acid composition, molecular weight and inhibition characteristics similar to those reported for human salivary kallikrein. Confirmation that the enzyme was kallikrein was demonstrated by its kinin-generating ability. Histochemical evidence indicates that a post-synthetic cleavage of protein C by kallikrein would have to take place during passage of saliva through the secretory ducts. In secreted saliva, cleavage of salivary protein C can only be observed after 72 h incubation. In addition, there is no effect of salivary flow rate on the relative amounts of proteins A and C in saliva. On the basis of the experimental observations, it is proposed that in vivo it is unlikely that kallikrein secreted from ductal cells plays a significant role in converting protein C into protein A.


Analysis of maternal microchimerism in rhesus monkeys (Macaca mulatta) using real-time quantitative PCR amplification of MHC polymorphisms.
Although pregnancy-associated microchimerism is known to exist in humans, its clinical significance remains unclear. Fetal microchimerism has been documented in rhesus monkeys, but the trafficking and persistence of maternal cells in the monkey fetus and infant have not been fully explored. To investigate the frequency of maternal microchimerism in the rhesus monkey (Macaca mulatta), a real-time polymerase chain reaction (PCR) strategy was developed and validated to target polymorphic major histocompatibility complex (MHC) gene sequences. Informative PCR assays were identified for 19 of 25 dams and their respective offspring. Analyses were performed on tissues (thymus, liver, spleen, lymph nodes, and bone marrow) and peripheral blood mononuclear cells (PBMCs) collected prenatally and postnatally in a subset of animals. Seven of 19 monkeys had detectable maternal microchimerism in at least one compartment (range: 0.001-1.9% chimeric cells). In tissues, maternal microchimerism was found in 2 of 7 fetuses and 3 of 12 juveniles (1-1.5 years of age), and most of the animals that were positive had microchimeric cells in more than one tissue. Maternal microchimerism was detected in PBMCs from all (4 of 4) fetuses. These observations suggest that maternal microchimerism occurs in the rhesus monkey fetus and can be detected in tissues in a subset of offspring after birth.


E-cigarette Use Among Youth: Results from the 2019 South Dakota Youth Tobacco Survey.
The prevalence of e-cigarette use among youth is increasing in an alarming rate nationwide with over 5 million students currently using e-cigarettes in 2019. To better understand e-cigarette use in South Dakota, we examined the prevalence, access, reasons, and harm perception of e-cigarette among South Dakota youth. We analyzed a representative sample of sixth to eighth grade students from the 2019 South Dakota Youth Tobacco Survey (YTS). Weighted estimates were computed to account for the complex sampling design and estimates were analyzed by geographic location and race/ethnicity. Among the 2,346 students, the prevalence of ever use of e-cigarettes among middle school students was 16.0 percent, a nearly 100 percent increase from 2017 YTS findings (8.2 percent). Approximately, seven percent (6.7 percent) reported use of an e-cigarette in the past 30 days, a nearly threefold increase from 2017 results (2.5 percent). Prevalence of current e-cigarette use was significantly higher among American Indian (12.7 percent) students and rural (8.3 percent) school students. The most common reported reasons for e-cigarette use was having friends/ family members that use them (49.6 percent) but also availability of flavors (18.6 percent). E-cigarette users obtained e-cigarettes predominantly through social sources (e.g., friends and peers). Nearly 30 percent (27.3 percent) reported not having enough information about e-cigarette's harm. In 2019, e-cigarettes were the most commonly used tobacco product among South Dakota middle school students. We found geographic and racial/ethnic disparity in e-cigarette use. Social influence and availability of flavors were important factors in promoting e-cigarette use. Continued efforts to implement population-level strategies to reduce e-cigarette use among youth are needed.


Concussion Incidence and Impact on Player Performance in Major League Baseball Players Before and After a Standardized Concussion Protocol.
Sports-related concussions have garnered significant attention in recent years because of the negative effects they can have on a player's cognitive health and performance. In response to this growing concern, Major League Baseball (MLB) introduced a standardized concussion protocol during the 2011-2012 season. The purpose of this study was to compare the reported incidence of concussions and the subsequent performance of MLB players before and after the introduction of the standardized concussion protocol. We hypothesized that the introduction of the standardized concussion protocol would not have an impact on player performance postconcussion. Cohort study; Level of evidence, 3. Players who suffered a concussion between 2001 and 2018 were identified from the MLB transactions page. Incidence and player performance were compared before and after the introduction of the standardized concussion protocol. Player performance was evaluated using traditional data and sabermetric data, which are advanced statistics used in conjunction with standard statistics to better compare players and teams. Player averages were calculated and compared using paired t tests for 30 days before and after concussion, 1 year before and after concussion, and career before and after concussion. Averages were also compared before and after the institution of the standardized concussion protocol using independent-measures t tests. There were a total of 114 players who suffered 142 concussions, with 77% of those occurring after the introduction of the concussion protocol (32 before, 110 after). The average time missed because of concussion significantly decreased from 33.7 days (range, 10-122 days) to 18.9 days (range, 6-111 days) after the concussion protocol (P = .0005). There was no difference in player performance (including batting average, on-base percentage, and slugging for batters; earned run average, fielding-independent pitching, and walks plus hits per inning pitched for pitchers) after concussion at any time point (30 days, 1 year, or career) when comparing these statistics before versus after the MLB concussion protocol. However, batter performance was significantly poorer 1 year after concussion and over the remainder of the players' careers (P < .05). The number of reported concussions increased after the introduction of the MLB concussion protocol. However, players spent significantly less time on the disabled list without any adverse effect on player performance. Despite these changes, long-term batting performance was significantly poorer after concussion.


Airborne pollen survey for Lincoln, Nebraska. III. Weeds.
Pollen counts in 1988, 1989, and 1990 revealed 16 weed pollen types. The weed pollination season in Lincoln extends from March through October. Very low amounts of pigweed-type and nettle pollen appear in April. Dock pollination begins in May. June has the greatest diversity of weed types and moderately high total counts (highest total June counts ca. 300 grains/m3). July is low in both diversity and absolute numbers of pollen grains (highest total July counts ca. 35 grains/m3). In early August pollen counts begin to rise with the primary pigweed-type pollination season and the first ragweed pollen. Ragweed pollen peaks in the last week of August and first week of September with more than 460 ragweed grains/m3 and total counts of more than 780 gr/m3.


Setbacks to shoot growth are common in woody plants, so how are shoots of some species safer than others?
Tissue turnover is a critical facet of plant life history variation. This study quantifies losses from setbacks to growth of terminal woody shoots 1.2m long, across 83 species and seven sites in eastern Australia. Setbacks, where the leading meristem had been removed or died and a new leader had emerged, were common (median three per shoot). Shoots had lost an average of 0.25 m of lead-stem length for 1.2 m net shoot-length gain. Insects like girdlers and borers were prominent causes of large setbacks. The sites spanned tropical to temperate and humid to semiarid climates, but variation in stem loss was much greater across species than across sites. We measured 17 plant functional traits related to growth form, mechanics, hydraulics, and economics. Only four traits were correlated with variation across species in stem losses: stem diameter, stem nitrogen content, bark thickness, and maximum photosynthetic rate. The correlations were weak. Stem specific gravity (wood density) showed no correlation with risk. Our results suggest a pattern similar to the growth risk trade-off known for herbaceous plants, where traits associated with fast growth increase tissue turnover and herbivory, but the weak correlations leave ample scope for other influences that remain to be identified.


Characterization of Oryzias latipes glucocorticoid receptors and their unique response to progestins.
Various receptor bioassays, including estrogens, androgens and thyroid hormones, have been developed and applied successfully for assessing hormone function in a wide range of animal species, including fish. In fish, corticosteroids play a pivotal role in physiology as they do in mammals, but far less is known about the corticosteroid receptor system in fish compared with in mammals. Here we established a transient transactivation assay using the Japanese medaka, Oryzias latipes, glucocorticoid receptors (olGRs) and mineralocorticoid receptor to analyse their functional properties in a fish. We found that olGR2 was highly responsive to glucocorticoids, similar to the human GR, whereas the olGR1 subtype was minimally responsive. Thus, olGR2 most likely mediates glucocorticoid signaling in medaka. We further tested crosstalk between GRs and other steroid hormones, and found that progestins could activate or inactivate olGR2-mediating transcription, depending on the presence or absence of cortisol. The transactivation assays developed for medaka GRs provide tools to gain useful insights into corticosteroid signaling in fish and for in vitro screening of environmental substances activating GRs.


Alkaline peroxidase activity of cupric oxide nanoparticles and its modulation by ammonia.
We herein report the intrinsic alkaline peroxidase-like activity exhibited by CuO nanoparticles when 3-(4-hydroxyphenyl)propionic acid was employed as a substrate. Based on this observation, a fluorometric assay method with a low detection limit of 0.81 μM was established for H2O2 determination under alkaline conditions. Notably, ammonia was found to inhibit the alkaline peroxidase-like activity of the CuO nanoparticles. Thus, a sensing platform for the determination of urea and urease was successfully constructed, with the limits of detection for urea and urease being 27 μM and 2.6 U L-1, respectively. This platform was then applied for the detection of urea in human urine and urease in soil, which yielded satisfactory results. These results suggest that it is possible to extend the catalytic potential of peroxidase and its mimetics from acidic and neutral conditions to include activity in alkaline media as well. Furthermore, this strategy is a novel method for the analysis of urea and urease. The assay developed in this work is facile, inexpensive, convenient, and highly selective and sensitive. Therefore, it is expected that this system can serve as a template for the development of similar enzyme nano-mimics.


The long and winding road to physical activity maintenance.
To build upon state-of-the-art theory and empirical data to estimate the strength of multiple mediators of the efficacious Keep Active Minnesota (KAM) physical activity (PA) maintenance intervention. The total, direct, and indirect effects through which KAM helped randomized participants (KAM n = 523; UC n = 526) maintain moderate or vigorous PA (MVPA) for up to 2 years were estimated using structural equation modeling. Multiple mediators explained half (beta = .052, P = .13) of the effect of KAM on MVPA (beta = .105, P = .004). Self-efficacy was the upstream variable in 2 endogenously mediated effects, and the self-concept mediator emerged as the strongest predictor of MVPA. KAM positively impacted self-efficacy, which was associated with PA enjoyment, integration into the self-concept, and PA maintenance. Successful long-term PA maintenance appears to be influenced by multiple small interrelated mediational pathways. Future research evaluating maintenance models should specify recursive relationships among mediators and outcomes.


Tracking and perceiving diverse motion signals: Directional biases in human smooth pursuit and perception.
Human smooth pursuit eye movements and motion perception behave similarly when observers track and judge the motion of simple objects, such as dots. But moving objects in our natural environment are complex and contain internal motion. We ask how pursuit and perception integrate the motion of objects with motion that is internal to the object. Observers (n = 20) tracked a moving random-dot kinematogram with their eyes and reported the object's perceived direction. Objects moved horizontally with vertical shifts of 0, ±3, ±6, or ±9° and contained internal dots that were static or moved ±90° up/down. Results show that whereas pursuit direction was consistently biased in the direction of the internal dot motion, perceptual biases differed between observers. Interestingly, the perceptual bias was related to the magnitude of the pursuit bias (r = 0.75): perceptual and pursuit biases were directionally aligned in observers that showed a large pursuit bias, but went in opposite directions in observers with a smaller pursuit bias. Dissociations between perception and pursuit might reflect different functional demands of the two systems. Pursuit integrates all available motion signals in order to maximize the ability to monitor and collect information from the whole scene. Perception needs to recognize and classify visual information, thus segregating the target from its context. Ambiguity in whether internal motion is part of the scene or contributes to object motion might have resulted in individual differences in perception. The perception-pursuit correlation suggests shared early-stage motion processing or perception-pursuit interactions.


Impact of 21-Gene Breast Cancer Assay on Treatment Decision for Patients with T1-T3, N0-N1, Estrogen Receptor-Positive/Human Epidermal Growth Receptor 2-Negative Breast Cancer: Final Results of the Prospective Multicenter ROXANE Study.
The ROXANE Italian prospective study evaluated the impact of the 21-gene Recurrence Score (RS) results on adjuvant treatment decision for patients with early breast cancer. Nine centers participated. Physicians used the RS test whenever unsure about adjuvant treatment recommendation for patients with estrogen receptor-positive/human epidermal growth receptor 2-negative, T1-T3, N0-N1 early breast cancer. Pre-RS and post-RS treatment recommendations were collected. A total of 251 patients were included. N0 patients (61%) showed higher grade (p < .001) and higher Ki67 (p = .001) and were more frequently progesterone receptor negative (p = .012) as compared with N1 patients. RS results were as follows: <11, n = 63 (25.1%); 11-25, n = 143 (57%); and ≥26, n = 45 (17.9%). Higher RS was found in N0 vs. N1 patients (p = .001) and in cases of G3 (p < .001) and higher Ki67 (p < .001). The rate of change in treatment decision was 30% (n = 75), mostly from chemotherapy (CT) plus hormone therapy (CT + HT) to hormone therapy (HT; 76%, n = 57/75). The proportion of patients recommended to CT + HT was significantly reduced from pre-RS to post-RS (52% to 36%, p < .0001). CT use reduction was more evident for N1 patients (55% to 27%) than for N0 patients (50% to 42%) and was observed only in cases of RS ≤17. Physicians predominantly used the 21-gene assay in N0 patients with a more aggressive biology or in N1 patients showing more indolent biology. In this selected patient population, the use of RS testing led to a 30% rate of change in treatment decision. In the N1 patient subgroup, the use of RS testing contributed to reduce CT use by more than half. This study shows that, even in a context in which physicians recommend a high proportion of patients to endocrine treatment alone before knowing the results of the Recurrence Score (RS) assay, the use of the RS test, whenever uncertainty regarding adjuvant treatment recommendation is present, significantly contributes in further reducing the use of chemotherapy, especially for N1 patients.


The Meaning of Social Support in Nature-Based Services for Young Adults with Mental Health Problems.
In previous studies, social context and social support have been found to be important in nature-based services. However, no studies have previously focused on the meaning of different dimensions of social support in these contexts. The aim of this study is therefore to uncover dimensions of social support in relation to mental health among young adults with mental health problems participating in nature-based services in Norway. This study applies data from a survey of 93 young adults participating in nature-based services; in addition, qualitative interview data from 20 interviews are also used. The data are analysed using qualitative content analysis, descriptive statistics, and correlation analysis. The results indicate that participants in nature-based services experience emotional, esteem, informational, and instrumental support in addition to social integration and opportunities for nurturance in these services. The service leader, other participants, and the animals are important contributors to these dimensions of social support. Nature-based services may be a helpful intervention for young adults with mental health problems. The unique context of these services, including nature and animals, adds special qualities to mental health and social work practices.


Fluorescence dequenching makes haem-free soluble guanylate cyclase detectable in living cells.
In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e.g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions.


Globally weaker and topologically different: resting-state connectivity in youth with autism.
There is a lack of agreement about functional connectivity differences in individuals with autism spectrum disorder (ASD). Studies using absolute strength have found reduced connectivity, while those using relative strength--a measure of system topology--reveal mostly enhanced connectivity. We hypothesized that mixed findings may be driven by the metric of functional connectivity. Resting-state echo planar 3 T functional magnetic resonance imaging scans were acquired on a Siemens Verio Scanner from 6 to 17-year-old youth with ASD (n = 81) and a matched typically developing control group (n = 82). All functional time series data were preprocessed using a confound regression procedure that has been previously validated in large-scale developmental datasets. It has also been shown to be highly effective at reducing the influence of motion artifact on connectivity data. We extracted time series data from a 333-node parcellation scheme, which was previously mapped to 13 functional systems. A Pearson's correlation was calculated and transformed to Fisher's z between every pair of nodes to create a weighted 333 × 333 adjacency matrix. Mean absolute functional connectivity strength was the mean Fisher's z of the matrix. Relative functional connectivity was corrected for individual differences in mean absolute functional connectivity (i.e., each connection in the matrix was divided by their mean z), and functional connectivity was evaluated within and across each of the functional networks in the parcellation scheme. Absolute functional connectivity strength was lower in ASD, and lower functional connectivity was correlated with greater ASD symptom severity. Relative functional connectivity was higher for the ASD group in the ventral attention and retrosplenial-temporal systems, with lower cross-system functional connectivity between the ventral attention and somatomotor-mouth systems. Functional connectivity within the ventral attention and retro-splenial systems correlated significantly with ASD symptom severity. Within a context of globally weaker functional connectivity, youth with ASD have an atypical topology of brain systems that support social perception and communication. This study clarifies the mixed results reported previously and demonstrates that the functional connectivity metric influences the observed direction of functional connectivity differences for individuals with ASD.


Purification of phosphatidylinositol kinase from bovine brain myelin.
A membrane-bound phosphatidylinositol (PI) kinase (EC 126.96.36.199) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.


Treatment of contaminated soil with phosphorus and manganese oxide reduces lead absorption by Sprague-Dawley rats.
This study was conducted to determine the extent of Pb absorption into young rats (Rattus norvegicus var. Sprague-Dawley) fed untreated Pb-contaminated soil or Pb-contaminated soil treated with two different sources of P and P + Mn oxide. Data were compared from an in vitro, physiologically based extraction test (PBET) with the animal data to support the validity of the in vitro test to assess bioavailable Pb from a treated Pb-contaminated soil. Soil with a total Pb concentration of 2290 mg kg(-1) was used. Rats were fed 19 different test diets for 21 consecutive days. The test diets represented 95 g AIN93G rat meal kg(-1) diet with varying proportions of silica sand or soil to provide low, medium, or high doses of Pb from either Pb acetate, treated, or untreated soil. Blood, liver, kidney, and bone Pb concentrations were examined. For all four tissues, Pb concentrations for the Pb acetate groups were significantly higher than concentrations for all the soil groups. In general, either triple superphosphate (TSP) or phosphate rock (PR) treatments resulted in significant reductions in tissue Pb concentrations compared with untreated soil. Blood and kidney Pb concentrations for the PR + Mn oxide group were significantly lower than those of the PR group at the low and high doses. Relative bioavailability of Pb, as measured in all tissues, was significantly reduced when comparing untreated with amended soil. Correlation between the in vitro and in vivo tests, based on bone and liver tissue, showed that the in vitro test is successful at predicting Pb bioavailability.


Induction of an epithelial to mesenchymal transition in human immortal and malignant keratinocytes by TGF-beta1 involves MAPK, Smad and AP-1 signalling pathways.
Recent data indicate that transforming growth factor-beta1 (TGF-beta1) can act to promote tumour progression in the late stages of carcinogenesis. The mechanism by which this occurs is unknown although a ligand-induced epithelial-mesenchymal transition (EMT) is thought to be important. In this study, we demonstrate that active Ras is required for TGF-beta1-induced EMT in human keratinocytes and that epidermal growth factor (EGF) can substitute for mutant Ras. EMT was reversed by the removal of TGF-beta1. Under conditions of TGF-beta1-induced EMT, cells were growth inhibited by the ligand resulting in G1 arrest. In cells containing normal Ras, TGF-beta1-activated ERK and p38 mitogen-activated protein kinases (MAPKs), and levels of activation were further increased by co-treatment with EGF. Inhibition of MAPK pathways and Smad2/3 signalling blocked the induction of EMT by TGF-beta1. Further, inhibition of the AP-1 transcriptional complex by [6]-Gingerol, or by the ectopic expression of JDP2, blocked TGF-beta1-induced EMT and conversely, stimulation of AP-1 by 12-O-tetradecanoylphorbol 13-acetate (TPA) substituted for EGF in the induction of EMT by TGF-beta1 in cells containing normal Ras. The presence of oncogenic Ras, the treatment of cells with EGF, or the treatment of cells with TPA to activate AP-1, potentiated TGF-beta1-induced Smad-dependent transcription, an effect that was attenuated by the inhibition of MAPKs and AP-1. The results demonstrate that active Ras and TGF-beta1 co-operate to reversibly induce EMT in human keratinocytes by mechanisms that involve MAPKs, Smad2/3 and AP-1. Further we demonstrate that MAPK/AP-1 signalling enhances Smad transcriptional activity under conditions associated with TGF-beta1-induced EMT.


Estimating the cost and efficiency gain of rolling out a multi-month dispensing programme for antiretroviral treatment in Tanzania.
Background: Globally, efforts to curtail the HIV pandemic are growing. The Joint United Nations Programme on HIV and AIDS (UNAIDS) and partners set the 95-95-95 targets to be achieved by 2025. Tanzania's ongoing transition from single-month ARV to longer multi-month dispensing (MMD) involves significant planning and shifts in existing resources, including health commodities, clinical staff and storage space. This study aimed at evaluating the costs and efficiency gains of rolling out MMD compared to the prior monthly dispending (MD) standard of care before the new guidelines.Methods: The analysis employed a health provider perspective utilising prior costing data collected to estimate cost of treatment for HIV/AIDS, including salaries, laboratory costs, antiretroviral drugs, other supplies and overhead costs. The projections were run from 2018 to 2030 using the Spectrum package for Tanzania.Results: Our model estimated that total treatment cost without MMD (including salaries, laboratory costs, antiretroviral drugs, other supplies, and overhead costs) is estimated to rise from USD 189 million in 2018 to USD 244 million in 2030. The introduction of a six-month MMD would lead to the total annual facility-based treatment costs being reduced to USD 205 million in 2030. When comparing MD to a six-month MMD, the total savings over the 13-year period would be USD 425 million. The introduction of six-month MMD for stable patients would reduce the average cost from USD 180 to USD 156 per patient per year if stable patients were only required to make six-monthly visit.Conclusions: The introduction of differentiated service delivery models (DSDMs) and MMD is already contributing to significant cost savings for Tanzania and will continue to do so as the country puts more stable patients on MMD. The potential gains from MMD implantation could further be harnessed if retention of treatment and viral suppression monitoring are prioritised.


Isolation of naturally occurring novel isoflavonoids: an update.
Covering: Jan. 2012 to Dec. 2017 This review describes the 391 new isoflavonoids isolated and identified from natural sources between January 2012 and December 2017, commenting on their sources, identification and biological activities. Due to the length of this review, the synthesis of isoflavonoids is not included. Furthermore, new applications of analytical techniques for the isolation and identification of isoflavonoids are also reviewed.


Persistently higher serum sCD40L levels are associated with outcome in septic patients.
Soluble CD40 ligand (sCD40L) exhibits proinflammatory and procoagulant effects. Recent data indicated that sCD40L plays a significant role in septic patients. The aim of the present study was to determine sCD40L changes in surgical patients without sepsis (SWS) and surgical sepsis patients (SS) during the first 3 days after intensive care unit (ICU) admission and to observe the association between sCD40L and mortality. Time changes in sCD40L levels were assessed for 3 days after ICU admission in 49 patients with SS and compared with those in 19 SWS patients. Serum sCD40L concentration was detected by ELISA. Survival at 28 days served as the endpoint. SS had significantly higher sCD40L levels than SWS and control patients. We observed an association between sCD40L levels ≥1028.75 pg/mL at day 2 and 28-day mortality (odds ratio = 7.888; 95% confidence interval = 1.758 to 35.395; P = 0.007). We could not discover any significant differences in sex, presence of septic shock, site of infection, length of stay in the ICU, PaO2/FiO2 ratio, incidence of AKI, ARDS, or type of surgery between nonsurvivors and survivors. Septic patients show persistently higher circulating sCD40L levels in the first 3 days after ICU admission, and serum sCD40L levels are associated with the mortality of patients with sepsis. Thus, serum sCD40L may be used as a reliable biomarker and therapeutic target in sepsis.


Evaluation of tablet computers for visual function assessment.
Recent advances in technology and the increased use of tablet computers for mobile health applications such as vision testing necessitate an understanding of the behavior of the displays of such devices, to facilitate the reproduction of existing or the development of new vision assessment tests. The purpose of this study was to investigate the physical characteristics of one model of tablet computer (iPad mini Retina display) with regard to display consistency across a set of devices (15) and their potential application as clinical vision assessment tools. Once the tablet computer was switched on, it required about 13 min to reach luminance stability, while chromaticity remained constant. The luminance output of the device remained stable until a battery level of 5%. Luminance varied from center to peripheral locations of the display and with viewing angle, whereas the chromaticity did not vary. A minimal (1%) variation in luminance was observed due to temperature, and once again chromaticity remained constant. Also, these devices showed good temporal stability of luminance and chromaticity. All 15 tablet computers showed gamma functions approximating the standard gamma (2.20) and showed similar color gamut sizes, except for the blue primary, which displayed minimal variations. The physical characteristics across the 15 devices were similar and are known, thereby facilitating the use of this model of tablet computer as visual stimulus displays.


[Pulmonary arterial hypertension. Part I: pathobiologic, pathophysiologic, clinical and diagnostic aspects].
Pulmonary hypertension is a pathophysiologic condition characterized by the increase of mean pulmonary arterial pressure > or =25 mmHg. A concomitant increase of pulmonary wedge pressure >15 mmHg may be present (post-capillary pulmonary hypertension) or not (precapillary pulmonary hypertension). The increase of pulmonary arterial pressure and of pulmonary vascular resistance and consequent elevation of the right ventricular afterload lead to right ventricular failure after variable periods of time. Pulmonary hypertension is present in multiple clinical conditions which have been classified in five groups. Pulmonary arterial hypertension (group 1) includes the familial and the idiopathic form and the forms associated with anorexigen drug use, connective tissue diseases, congenital heart diseases, HIV infection and portal hypertension. Group 2 includes all left heart diseases characterized by the increase of left atrial pressure and pulmonary wedge pressure (post-capillary pulmonary hypertension). Group 3 includes parenchymal lung diseases (chronic obstructive lung disease, lung fibrosis, ecc). Chronic thromboembolic pulmonary hypertension (group 4) is characterized by the obstruction of elastic pulmonary arteries at different levels by organized thromboembolism. Group 5 includes heterogeneous conditions such as sarcoidosis and histiocytosis X. These clinical groups are characterized by different pathobiologic and pathophysiologic mechanisms and therapeutic strategies. The exact pathobiologic mechanisms leading to pulmonary arterial hypertension (group 1) are unknown. Genetic factors (inheritable forms), predisposing factors (female gender) and exogenous factors (drugs, antibodies, viruses, congenital heart disease, etc). Endothelial dysfunction of lung microcirculation is invariably present and is characterized by the reduction of vasodilator and antiproliferative substances (prostacyclin, nitric oxide) and by the increase of vasoconstrictor and mitogenic factors (endothelin, thromboxane A2). Current approved therapies are targeted to the correction of this imbalance, which leads to the progressive increase of pulmonary vascular resistance. Different therapeutic strategies that are effective in diverse groups require an appropriate diagnostic algorithm in order to identify the precise group and specific conditions within the group. Evaluation of vasoreactivity and assessment of the severity of functional and hemodynamic changes are also required in pulmonary arterial hypertension for an appropriate therapeutic decision-making and estimate of results.


Differences between the time constant of sensory and motor peripheral nerve fibers: further studies and considerations.
Using a method of latent addition, we previously demonstrated that sensory fibers had time constants that were about three times longer than those of motor fibers. The aim of the present work was to confirm this difference by determining the time constants for single sensory axons by using microneurography and for single motor axons by recording single motor units with bipolar concentric needle electrodes. To determine the influence of the conditioning pulse on the neural time constant, we used both depolarizing and hyperpolarizing conditioning pulses. When hyperpolarizing conditioning pulses at comparable intensity were applied, the tendency was to find shorter time constants than when depolarizing pulses were applied, although still with the motor time constant being slightly shorter. Although the absolute values varied with the different methods, the sensory time constant was generally three times the motor time constant for depolarizing conditioning stimuli, whereas for hyperpolarizing conditioning stimuli the difference dropped to about one and a half. These characteristics improve understanding of the behavior of sensory and motor axons, and, in particular, explain the differential excitability. Determination of neural time constants might prove valuable for clinical use.


A three-dimensional micro-electrode array for in-vitro neuronal interfacing.
In this paper, we report on the development of an easy-to-fabricate three-dimensional Micro-Electrode Array (3D-MEA) specifically designed for brain-on-a-dish applications. The proposed device consists of pillar-shaped gold microelectrodes realized by electroplating directly on top of a standard MEA, making this approach highly versatile and convenient for batch fabrication. Moreover, with this simple technique, it is possible to obtain electrodes with a height of more than 100 µm onto different kind of substrates, ranging from glass to flexible plastic ones. This novel 3D-MEA structure has been validated with acute brain slices, successfully recording both epileptiform-like discharges (upon the administration of 4-AP), and electrically-evoked neuronal activity. The preliminary validation showed a substantial improvement in the signals amplitude with respect to both commercial and custom planar electrodes thanks to a better coupling offered by the peculiar shape of the three-dimensional electrodes. Beside the versatility of the fabrication approach, which allows to obtain 3D MEA devices onto both rigid and flexible substrates, the reported validation showed how the pillar approach can outperform standard planar MEA recordings in terms of signal amplitude. Moreover, thanks to the possibility of obtaining multi-level 3D structures within the same device, the proposed fabrication technique offers an interesting and flexible approach for the development of a new family of electrophysiological tools for 3D in vitro electrophysiology, in particular for acute brain slices and 3D neuronal cultures for brain-on-a-dish applications.


Ranolazine for the treatment of refractory angina in a veterans population.
Pivotal ranolazine trials did not require optimization of conventional medical therapy including coronary revascularization and antianginal drug therapy prior to ranolazine use. This case series describes the use of ranolazine for the treatment of chronic stable angina refractory to maximal medical treatment in a veterans population. A total of 18 patients with a median age of 66 years were identified. All patients had prior percutaneous coronary intervention and/or coronary artery bypass graft surgery; 83% had three-vessel coronary artery disease, with left main disease present in 39% of patients. Prior to initiating ranolazine, antianginal use consisted of beta blockers (94%), long-acting nitrates (83%) and calcium channel blockers (61%). Median blood pressure (116.2/61.8 mmHg) and pulse (65 beats per min) were controlled. Median preranolazine angina episodes and sublingual nitroglycerin (SLNTG) doses per week were 14 and 10, respectively, with a Canadian Cardiovascular Society (CCS) angina grade of III-IV in 67% of patients. After initiation of ranolazine, median angina episodes per week and SLNTG doses used per week decreased to 0.7 and 0, respectively, with CCS grade of III-IV declining to 17%. Of the 18 subjects enrolled, 44% had complete resolution of angina episodes. The addition of ranolazine to maximally tolerated conventional antianginal drug therapy post coronary revascularization was associated with decreases in angina episodes and SLNTG utilization and improvement in CCS angina grades. Ranolazine may provide an effective treatment option for revascularized patients with refractory angina.


Construction and validation of risk prediction model for gastrointestinal bleeding in patients after coronary artery bypass grafting.
This study aimed to develop a risk prediction model for gastrointestinal bleeding in patients after coronary artery bypass grafting (CABG) and assessed its accuracy. A retrospective analysis was conducted on 232 patients who underwent CABG under general anesthesia in our hospital between January 2022 and December 2022. The patients were divided into gastrointestinal bleeding (GIB) group (n = 52) and group without gastrointestinal bleeding (non-GIB) (n = 180). The independent risk factors for gastrointestinal bleeding in post-CABG patients were analyzed using χ2 test, t test and logistic multivariate regression analysis. A prediction model was established based on the identified risk factors. To verify the accuracy of the prediction model, a verification group of 161 patients who met the criteria was selected between January to June 2023, and the Bootstrap method was used for internal validation. The discrimination of the prediction model was evaluated using the area under the curve (AUC), where a higher AUC indicates a stronger discrimination effect of the model. The study developed a risk prediction model for gastrointestinal bleeding after CABG surgery. The model identified four independent risk factors: duration of stay in the intensive care unit (ICU) (OR 0.761), cardiopulmonary bypass time (OR 1.019), prolonged aortic occlusion time (OR 0.981) and re-operation for bleeding (OR 0.180). Based on these factors, an individualized risk prediction model was constructed. The C-index values of the modeling group and the verification group were 0.805 [95% CI (0.7303-0.8793)] and 0.785 [95% CI (0.6932-0.8766)], respectively, which indicated a good accuracy and discrimination of this model. The calibration and standard curves showed similar results, which further supported the accuracy of the risk prediction model. In conclusion, ICU time, cardiopulmonary bypass time, aortic occlusion time and re-operation for bleeding are identified as independent risk factors for gastrointestinal bleeding in patients after CABG. The risk prediction model developed in this study demonstrates strong predictive performance and provides valuable insights for clinical medical professionals in evaluating gastrointestinal complications in CABG patients.


[Scale of Concern about COVID-19 Infection in Peruvian Health Care Workers].
The global SARS-CoV-2 pandemic has generated concern about contagion, especially among healthcare workers who are on the front line. To design and analyse the evidence of content validity, internal structure and reliability of a measure of concern about the spread of COVID-19 among Peruvian healthcare workers. Quantitative study and instrumental design. The scale was administered to 321 health science professionals (78 males and 243 females), whose ages ranged from 22 to 64 years (38.12 ± 9.61). Aiken's V-coefficient values were statistically significant. An exploratory factor analysis was performed which indicated the existence of a single factor, and confirmatory factor analysis (CFA) confirmed a satisfactory six-factor model. The CFA solution obtained adequate fit indices (RMSEA = 0.079; p = 0.05; TLI = 0.967; IFC = 0.980; GFI = 0.971, and AGFI = 0.931), and showed good internal consistency based on Cronbach's alpha coefficient (α = 0.865; CI 95%, 0.83-0.89). The scale of concern for COVID-19 infection is a valid and reliable brief measure that can be used for research and professional purposes.


Expression and function of Nkx6.3 in vertebrate hindbrain.
Homeodomain transcription factors serve important functions in organogenesis and tissue differentiation, particularly with respect to the positional identity of individual cells. The Nkx6 subfamily controls tissue differentiation in the developing central nervous system where they function as transcriptional repressor proteins. Recent work indicates that Nkx6.3 is expressed in hindbrain V2 interneurons that co-express Nkx6.1, suggesting the possibility of functional redundancy. Here, we report that Nkx6.3 expression is specific to Chx10+ V2a interneurons but not to Gata3+ V2b interneurons of the hindbrain, and that Nkx6.3 expression appears to mark cells of the prospective medullary reticular formation. Molecular analysis of Nkx6.3 null embryonic mouse hindbrain did not reveal detectable defects in progenitor markers, motor neuron or V2 interneuron sub-types. Forced expression of Nkx6.3 and Nkx6.1 promote V2 interneuron differentiation in the developing chick hindbrain. These findings indicate Nkx6.3 function is dispensable for CNS development and lead to the proposal that absence of overt defects is due to functional compensation from a related homeodomain transcription factor.


An NAD(P)H oxidase regulates growth and transcription in melanoma cells.
Malignant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-kappaB activation and suppress cell proliferation (11), the nature of the enzyme responsible for ROS production in melanoma cells has not been determined. To address this issue, we now have characterized the source of ROS production in melanoma cells. We report that ROS are generated by isolated, cytosol-free melanoma plasma membranes, with inhibition by NAD(P)H oxidase inhibitors. The p22(phox), gp91(phox), and p67(phox) components of the human phagocyte NAD(P)H oxidase and the gp91(phox) homolog NOX4 were demonstrated in melanomas by RT-PCR and sequencing, and protein product for both p22(phox) and gp91(phox) was detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only p22(phox) and NOX4. Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for p22(phox) and NOX4. Also, the flavoprotein inhibitor diphenylene iodonium inhibited constitutive DNA binding of nuclear protein to the NF-kappaB and cAMP-response element consensus oligonucleotides, without affecting DNA binding activity to activator protein-1 or OCT-1. This suggests that ROS generated in autocrine fashion by an NAD(P)H oxidase may play a role in signaling malignant melanoma growth.


Albumin adsorption to contact lens materials: a review.
During contact lens wear, tear film components such as lipids, mucins and proteins tend to deposit on and within the lens material and may cause discomfort, reduced vision and inflammatory reactions. The tear film protein that has attracted most interest when studying contact lens deposition is the small (14 kDa), positively charged protein lysozyme. Albumin, which is a much larger protein (66 kDa) with an overall net negative charge is also of interest, and shows very different adsorption patterns to lysozyme. The concentration of albumin in the tear film is relatively low compared to the concentration in blood serum, but this value increases markedly under various conditions, including when the eye is closed, during contact lens wear and in various dry eye states. Gaining an understanding of the manner in which albumin deposits on biomaterials is of importance for contact lens wear, as well as for other medical applications where HEMA-based materials are used for implants, artificial blood vessels or drug delivery devices. This review paper summarizes the impact of individual material compositions, water content, hydrophobicity and electrostatic attraction on the adsorption behavior of the protein albumin.


A cell-free, biomimetic hydrogel based on probiotic membrane vesicles ameliorates wound healing.
Probiotic bacteria, such as Lactobacilli, have been shown to elicit beneficial effects in various tissue regeneration applications. However, their formulation as living bacteria is challenging, and their therapeutic use as proliferating microorganisms is especially limited in immunocompromised patients. Here, we propose a new therapeutic avenue to circumvent these shortcomings by developing a bacteriomimetic hydrogel based on membrane vesicles (MVs) produced by Lactobacilli. We coupled MVs from Lactobacillus plantarum and Lactobacillus casei, respectively, to the surface of synthetic microparticles, and embedded those bacteriomimetics into a pharmaceutically applicable hydrogel matrix. The wound microenvironment changes during the wound healing process, including adaptions of the pH and changes of the oxygen supply. We thus performed proteomic characterization of the MVs harvested under different culture conditions and identified characteristic proteins related to the biological effect of the probiotics in every culture state. In addition, we highlight a number of unique proteins expressed and sorted into the MVs for every culture condition. Using different in vitro models, we demonstrated that increased cell migration and anti-inflammatory effects of the bacteriomimetic microparticles were dependent on the culture condition of the secreting bacteria. Finally, we demonstrated the bacteriomimetic hydrogel's ability to improve healing in an in vivo mouse full-thickness wound model. Our results create a solid basis for the future application of probiotic-derived vesicles in the treatment of inflammatory dispositions and stimulates the initiation of further preclinical trials.


Home-Based Rehabilitation of the Shoulder Using Auxiliary Systems and Artificial Intelligence: An Overview.
Advancements in modern medicine have bolstered the usage of home-based rehabilitation services for patients, particularly those recovering from diseases or conditions that necessitate a structured rehabilitation process. Understanding the technological factors that can influence the efficacy of home-based rehabilitation is crucial for optimizing patient outcomes. As technologies continue to evolve rapidly, it is imperative to document the current state of the art and elucidate the key features of the hardware and software employed in these rehabilitation systems. This narrative review aims to provide a summary of the modern technological trends and advancements in home-based shoulder rehabilitation scenarios. It specifically focuses on wearable devices, robots, exoskeletons, machine learning, virtual and augmented reality, and serious games. Through an in-depth analysis of existing literature and research, this review presents the state of the art in home-based rehabilitation systems, highlighting their strengths and limitations. Furthermore, this review proposes hypotheses and potential directions for future upgrades and enhancements in these technologies. By exploring the integration of these technologies into home-based rehabilitation, this review aims to shed light on the current landscape and offer insights into the future possibilities for improving patient outcomes and optimizing the effectiveness of home-based rehabilitation programs.


Weight-loss-associated changes in bone mineral density and bone turnover after partial weight regain with or without aerobic exercise in obese women.
Moderate, long-term weight loss results in the loss of bone mass in overweight or obese premenopausal women. However, whether these changes persist during weight maintenance or regain remains to be determined. Overweight or obese (body mass index: 25.8-42.5 kg/m(2)) women (n=40) with at least two risk factors for the metabolic syndrome participated in this 12-month study that examined the effects of prescribed weight loss and regain, with or without exercise, on bone turnover and on bone mineral density (BMD) in a subset of participants (n=24). During the first 6 month, participants lost ≈ 10% of their initial body weight via energy restriction and supervised aerobic exercise. Following weight loss, participants were randomly assigned to either an exercise or a no exercise treatment for the regain (+50% of weight lost) phase. A one-way (time) repeated measures one-factor analysis of variance (RMANOVA) tested the effects of weight loss on BMD and bone turnover, and a two-way RMANOVA (time, exercise) was used to examine the effects of exercise during weight regain. Hip (P=0.007) and lumbar spine (P=0.05) BMD decreased with weight loss, and remained reduced after weight regain with or without exercise. Likewise, the weight-loss-associated increases in osteocalcin (P<0.001) and C-terminal peptide of type I collagen (P<0.001) persisted following weight regain, independent of exercise. The results of the present study, which is the first to examine changes in bone mass and turnover during carefully controlled weight regain, suggest that weight-loss-induced perturbations in bone mass and turnover persist after partial weight regain, regardless of whether regular weight-bearing aerobic exercise was continued.


Teratogenic effects of alcohol in humans and laboratory animals.
The teratogenicity of alcohol has been demonstrated in humans through clinical studies, behavioral studies, and epidemiologic studies, and in animals through controlled laboratory experiments. In humans exposed to alcohol during gestation the effects can range from fetal alcohol syndrome in some offspring of chronic alcoholic women to reduced average birth weight in offspring of women reporting an average consumption of two to three drinks or more per day. The behavioral effects of such exposure may range from mental retardation in children with fetal alcohol syndrome to milder developmental and behavioral effects in infants born to social drinkers. In animals, exposure to alcohol in utero may result in death, malformation, and growth deficiency as well as behavioral and developmental abnormalities. The mechanisms of impairment and related risk factors are yet to be elucidated.


[Autonomous pharmacodynamics of organic nitrates in patients with unstable angina].
The pharmacological test with the use of high-resolution rhythmocardiography for the analysis of cardiac rhythm variability associated with peripheral vegetative regulation of the synoatrial node was applied to study effects of a single dose of organic nitrates (nitroglycerin and mononitrate isosorbide) in 41 patients with unstable angina. Rhythmocardiograms were obtained before and after a morning sublingual dose of nitrates preceding intake of basal drugs. The results suggest negative action of nitrates on peripheral vegetative regulation of pacemaker activity of the synoatrial node.


Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes.
We have developed a primary cell culture system derived from embryonic and larval stages of Drosophila. This allows for high-resolution imaging and genetic analyses of endocytic processes. Here, we have investigated endocytic pathways of three types of molecules: an endogenous receptor that binds anionic ligands (ALs), glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP), and markers of the fluid phase in primary hemocytes. We find that the endogenous AL-binding receptor (ALBR) is internalized into Rab5-positive endosomes, whereas the major portion of the fluid phase is taken up into Rab5-negative endosomes; GPI-APs are endocytosed into both classes of endosomes. ALBR and fluid-phase-containing early endosomes subsequently fuse to yield a population of Rab7-positive late endosomes. In primary culture, the endocytic phenotype of ALBR internalization in cells carrying mutations in Drosophila Dynamin (dDyn) at the shibire locus (shits) parallels the temperature-sensitive behavior of shits animals. At the restrictive temperature in shits cells, receptor-bound ALs remain completely surface accessible, localized to clathrin and alpha-adaptin-positive structures. On lowering the temperature, ALs are rapidly sequestered, suggesting a reversible block at a late step in dDyn-dependent endocytosis. By contrast, GPI-AP and fluid-phase endocytosis are quantitatively unaffected at the restrictive temperature in shits hemocytes, demonstrating a constitutive dDyn and Rab5-independent endocytic pathway in Drosophila.


Using domiciliary non-invasive ventilator data downloads to inform clinical decision-making to optimise ventilation delivery and patient compliance.
Ventilation parameter data from patients receiving home mechanical ventilation can be collected via secure data cards and modem technology. This can then be reviewed by clinicians and ventilator prescriptions adjusted. Typically available measures include tidal volume (VT), leak, respiratory rate, minute ventilation, patient triggered breaths, achieved pressures and patient compliance. This study aimed to assess the potential impact of ventilator data downloads on management of patients requiring home non-invasive ventilation (NIV). A longitudinal within-group design with repeated measurements was used. Baseline ventilator data were downloaded, reviewed and adjustments made to optimise ventilation. Leak, VT and compliance data were collected for comparison at the first review and 3-7 weeks later. Ventilator data were monitored and amended remotely via a modem by a consultant physiotherapist between the first review and second appointment. Analysis of data from 52 patients showed increased patient compliance (% days used >4 hours) from 90% to 96% (p=0.007), increased usage from 6.53 to 6.94 hours (p=0.211) and a change in VT(9.4 vs 8.7 mL/kg/ideal body weight, p=0.022). There was no change in leak following review of NIV prescriptions (mean (SD): 43 (23.4) L/min vs 45 (19.9)L/min, p=0.272). Ventilator data downloads, via early remote assessment, can help optimise patient ventilation through identification of modifiable factors, in particular interface leak and ventilator prescriptions. However, a prospective study is required to assess whether using ventilator data downloads provides value in terms of patient outcomes and cost-effectiveness. The presented data will help to inform the design of such a study.


Muscle Na-K-pump and fatigue responses to progressive exercise in normoxia and hypoxia.
To investigate the effects of hypoxia and incremental exercise on muscle contractility, membrane excitability, and maximal Na(+)-K(+)-ATPase activity, 10 untrained volunteers (age = 20 +/- 0.37 yr and weight = 80.0 +/- 3.54 kg; +/- SE) performed progressive cycle exercise to fatigue on two occasions: while breathing normal room air (Norm; Fi(O(2)) = 0.21) and while breathing a normobaric hypoxic gas mixture (Hypox; Fi(O(2)) = 0.14). Muscle samples extracted from the vastus lateralis before exercise and at fatigue were analyzed for maximal Na(+)-K(+)-ATPase (K(+)-stimulated 3-O-methylfluorescein phosphatase) activity in homogenates. A 32% reduction (P < 0.05) in Na(+)-K(+)-ATPase activity was observed (90.9 +/- 7.6 vs. 62.1 +/- 6.4 nmol.mg protein(-1).h(-1)) in Norm. At fatigue, the reductions in Hypox were not different (81 +/- 5.6 vs. 57.2 +/- 7.5 nmol.mg protein(-1).h(-1)) from Norm. Measurement of quadriceps neuromuscular function, assessed before and after exercise, indicated a generalized reduction (P < 0.05) in maximal voluntary contractile force (MVC) and in force elicited at all frequencies of stimulation (10, 20, 30, 50, and 100 Hz). In general, no differences were observed between Norm and Hypox. The properties of the compound action potential, amplitude, duration, and area, which represent the electromyographic response to a single, supramaximal stimulus, were not altered by exercise or oxygen condition when assessed both during and after the progressive cycle task. Progressive exercise, conducted in Hypox, results in an inhibition of Na(+)-K(+)-ATPase activity and reductions in MVC and force at different frequencies of stimulation; these results are not different from those observed with Norm. These changes occur in the absence of reductions in neuromuscular excitability.


Potent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA.
Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA.


Plasma Leptin, Adiponectin, Neuropeptide Y Levels in Drug Naive Children With ADHD.
ADHD is one of the most common childhood psychiatric disorders. Research indicates that there is some link between obesity/overweight and ADHD, though the mechanism of this association remains uncertain. It is the aim of the present study to explore the association between ADHD, obesity, and plasma leptin, neuropeptide Y (NPY), and adiponectin levels. Thirty-six patients diagnosed with ADHD were included in the study. The control group consisted of 40 healthy children and adolescents who had similar age and gender features with the patient group. Plasma leptin, adiponectin, NPY levels were measured, and body mass index (BMI), weight for height, and standard deviation scores (SDS) of height, weight, and BMI were calculated. No significant difference was found between patients and healthy children in terms of BMI and BMI percentile. Participants were classified into three groups according to their weight to height values. There was no significant difference between the two groups, but 10% of the control group and 30.6% of the ADHD group were classified as overweight, which was 3 times higher than the control group. The adiponectin plasma level was significantly lower and leptin/adiponectin (L/A) ratio was significantly higher in the ADHD group. There was no significant difference between serum NPY levels. In the ADHD group, the mean leptin plasma level was high, but was not statistically significant. We think that a low adiponectin level and high L/A ratio may be the underlying mechanism of the obesity in ADHD patients.


Surgical intervention versus expectant management for endometrial polyps in subfertile women.
Endometrial polyps, which are benign growths of the endometrium, may be a factor in female subfertility. Possible mechanisms include physical interference with gamete transport, alteration of the endometrial milieu and unresponsiveness to the cyclical global endometrial changes. As such polyps remain mostly asymptomatic, their diagnosis is often incidental during routine investigations prior to embarking on assisted reproductive treatment. Transvaginal sonography, hysterosalpingography and saline infusion sonography are the diagnostic tools most commonly employed. However, hysteroscopy remains the gold standard for diagnosis, as well as for treatment. Due to the possible effect of endometrial polyps on fertility, their removal prior to any subfertility treatment is widely practiced. To determine the effectiveness and safety of removal of endometrial polyps in subfertile women. Electronic databases were searched, including the Cochrane Menstrual Disorders and Subfertility Group Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, PsycINFO, CINAHL and trial registers. The reference lists of identified articles were checked. The last search was performed on 30 July 2014. Only randomised controlled trials, reporting pregnancy or live birth rates and complication rates as primary or secondary outcomes, in which polyps were removed surgically prior to treatment of subfertility were eligible for inclusion. The diagnosis of endometrial polyps was required to be made by transvaginal ultrasound, hysterosalpingography, saline infusion, sono-hysterography or hysteroscopy. Any surgical technique of polyp removal was acceptable, with no intervention in the control groups. Two review authors independently screened the titles, abstracts and full articles to assess their suitability for inclusion in this review. Quality assessment was attempted independently by two authors with discrepancies being settled by consensus or consultation with a third review author.No data extraction was performed due to the absence of useable data in the one eligible study. If there had been data to include, two review authors would have independently extracted the data from the studies using a data extraction form designed and pilot tested by the authors. Any disagreements would have been resolved by discussion or by a third review author. Only one randomised controlled trial of endometrial polypectomy was identified for inclusion. However, a single set of data could not be extracted from this study due to internal inconsistencies of the results reported. Attempts to contact the authors to resolve the issue were unsuccessful, by phone, post and e-mail. Removal of endometrial polyps in subfertile women is commonly being performed in many countries with an aim to improve the reproductive outcome. We did not identify any analysable randomised trials which would allow us to reach any sound scientific conclusions on the efficacy of endometrial polypectomy in subfertile women. Well designed, methodologically sound, randomised controlled trials are urgently needed.


Reliability and stability of the thymidine labeling index of breast carcinoma.
We have approached the question of the reliability of the thymidine labeling index (TLI) of breast carcinoma by comparing paired samples. Because the distribution of the TLI is approximately lognormal, comparisons were made after transformation to the natural logarithm. For 38 paired samples from the primary carcinoma (biopsy versus residual tumor in mastectomy specimen), r, 0.89, p less than 0.0001. For 68 primary carcinomas versus their axillary metastases, r, 0.90, p less than 0.0001. Substantial correlation was also observed between the TLIs of 18 primary breast carcinomas and their recurrent or metastatic lesions studied after relapse (r, 0.75, p less than 0.0003), and between 14 pairs of first and second primary carcinomas recovered from the same breast (r, 0.72, p less than 0.003). Good agreement was achieved among three observers in measuring the TLI from a set of breast carcinoma autoradiographs. We conclude that the TLI is sufficiently reliable for semiquantitative use as a means of separating breast carcinomas into groups with slow, moderate, and rapid rates of proliferation. The substantial similarities between the TLIs of breast carcinoma samples from different sites of the primary tumor, between primary tumor and axillary metastases, and between primary tumor and subsequent samples of relapsed tumor suggest that clonal evolution either is infrequent or does not usually affect the cell kinetics of the carcinoma. The similarity of the TLIs of synchronous dual primary carcinomas is an additional interesting finding.


Morphological and functional characterization of predifferentiation of myelinating glia-like cells from human bone marrow stromal cells through activation of F3/Notch signaling in mouse retina.
Recently, we have demonstrated that F3/contactin and NB-3 are trans-acting extracellular ligands of Notch that promote differentiation of neural stem cells and oligodendrocyte precursor cells into mature oligodendrocytes (OLs). Here, we demonstrate that human bone marrow stromal cells (hBMSCs) can be induced to differentiate into cells with myelinating glial cell characteristics in mouse retina after predifferentiation in vitro. Isolated CD90(+) hBMSCs treated with beta-mercaptoethanol for 1 day and retinoic acid for 3 days in culture changed into myelinating glia-like cells (MGLCs). More cells expressed NG2, an early OL marker, after treatment, but expression of O4, a mature OL marker, was negligible. Subsequently, the population of O4(+) cells was significantly increased after the MGLCs were predifferentiated in culture in the presence of either F3/contactin or multiple factors, including forskolin, basic fibroblast growth factor, platelet-derived growth factor, and heregulin, in vitro for another 3 days. Notably, 2 months after transplantation into mouse retina, the predifferentiated cells changed morphologically into cells resembling mature MGLCs and expressing O4 and myelin basic protein, two mature myelinating glial cell markers. The cells sent out processes to contact and wrap axons, an event that normally occurs during early stages of myelination, in the retina. The results suggest that CD90(+) hBMSCs are capable of morphological and functional differentiation into MGLCs in vivo through predifferentiation by triggering F3/Notch signaling in vitro.


Mechanisms of Human Immunodeficiency Virus-Associated Lymphocyte Regulated Cell Death.
Human immunodeficiency virus-1 (HIV-1) causes CD4 T cell depletion through a number of mechanisms, including programmed cell death pathways (both apoptotic and nonapoptotic). In the setting of HIV-1 infection, the enhanced lymphocyte cell death occurs as a consequence of complex interactions between the host immune system and viral factors, which are reviewed herein. On the other hand, the main challenge to HIV-1 eradication is the development of latent infection in a subset of long lived cells, including CD4+ T cells and macrophages, which resist HIV-induced cell death. Understanding the potential mechanisms of how HIV-1 induces lymphocyte cell death is critical to the "kick and kill" cure strategy, which relies on the effective killing of reactivated, HIV-1-infected cells.


An evaluation of residues at an oil refinery site following fires.
Soil pollution at the oil refinery at Novi Sad following destruction of crude oil and its products in storage tanks during the Kosovo conflict was investigated. More than 100,000 t of crude oil and its products were destroyed, and about 90% of these were burnt off, 10% leached and 130 t recovered. The acute injection of polycyclic aromatic hydrocarbons (PAHs) to the air of the town was widespread depending on the weather conditions and ranged from 1-431,000 ng/m3. The presence of PAHs, polychlorinated biphenyls (PCBs), and elements in the surface zone and soil core samples taken from various sites were determined up to a depth of 100 cm. Concentrations of PAHs were in the range between 0.75 and 86.19 microg/g dry soil. The contaminated soil can be expected to act as a permanent pollution source, while the mobile constituents are likely to cause groundwater pollution.


Evolution and development of the dentition.
The genetic code that produces human teeth began to develop in primitive vertebrates around 500 million years ago. Some parts of the information appear to have been very stable, particularly the mineralized matrices, while others have evolved. The development of tooth shape and tooth number are very rigidly controlled by genes in each species and are responsive to relatively rapid genetic selection by the environment, as are bone shape and associated soft tissues. Their developmental independence is reflected in the ability of the embryonic tooth bud to develop in vitro. Part of the genetic control of tooth size and shape is correlated with the genetic control of size and shape of the jaws, but the jaws are more responsive to environmental variables modifying individual development than are the teeth. There are recognized mutations producing changes in teeth which act at all levels of control, the development of the embryonic bud, the morphogenesis of the bell stage, the production of enamel and dentin and the formation of the roots. The mechanisms of this genetic control are at the molecular and submolecular levels which are just beginning to be examined. Tooth germs are a good system for study of these processes, and changes in our knowledge will lead to increased understanding of the variation of teeth and its relation to the structure of other tissues both normal and abnormal.


Underactivation of the adiponectin-adiponectin receptor 1 axis in clear cell renal cell carcinoma: implications for progression.
Energy-sensing pathways, normally coordinated by 5' AMP-activated protein kinase (AMPK), are dysregulated in renal cell carcinoma (RCC). Obesity can accentuate the pre-existing pro-tumorigenic metabolic machinery in RCC cells through its associated obesogenic hormonal milieu, characterized by lower circulating levels of adiponectin. In RCC patients, low adiponectin levels associate clinically with more aggressive disease. We investigated the adiponectin signaling pathway in RCC, focusing on adiponectin receptor 1 (AdipoR1) and associated activation of AMPK. AdipoR1 protein in RCC and normal surrounding renal tissues was determined by Western blot analysis and immunohistochemistry. Anti-tumorigenic effects of adiponectin in RCC cells in vitro were investigated via VEGF and MMP ELISA and invasion assays. Using in vivo models of RCC, the effect of AdipoR1-knockdown (shRNA) on tumor latency, growth and dissemination were determined. AdipoR1 protein was significantly reduced in clear cell RCC specimens. Adiponectin treatment inhibited VEGF, MMP-2 and MMP-9 secretion and activity and invasive and migratory capacities of RCC cells. AMPKα1-knockdown (shRNA) attenuated adiponectin's effects. In cells stably expressing AdipoR1-specific shRNA, AMPK activation by adiponectin was significantly reduced compared to cells expressing control shRNA. In vivo, AdipoR1 knockdown increased the growth, dissemination and angiogenesis of RCC. These findings suggest that deficiencies in the entire adiponectin hormonal axis (the hormone and its receptor) result in underactivation of AMPK leading to increased angiogenic and invasive capacities of RCC. The established link between obesity and RCC can therefore be further explained by the adiponectin deficiency in obese individuals together with reduced AdipoR1 protein in RCC.


A bioflocculant-supported dissolved air flotation system for the removal of suspended solids, lipids and protein matter from poultry slaughterhouse wastewater.
In this study, two previously identified isolates, i.e. Comamonas aquatica (BF-3) and Bacillus sp. BF-2, were determined to be suitable candidates to utilise in a bioflocculant-supported dissolved air flotation (Bio-DAF) system as a pretreatment system for poultry slaughterhouse wastewater (PSW). A 2% (v/v) (bioflocculant:PSW) strategy was used for the DAF to reduce total suspended solids (TSS), lipids and proteins in the PSW, by supplementing the bioflocculants produced and the co-culture (C. aquatica BF-3 and Bacillus sp. BF-2) directly into the DAF. The Bio-DAF was able to reduce 91% TSS, 79% proteins and 93% lipids when the DAF system was operating at steady state, in comparison with a chemical DAF operated using 2% (v/v) alum that was able to only reduce 84% TSS, 71% proteins and 92% lipids. It was concluded that the Bio-DAF system worked efficiently for the removal of suspended solids, lipids and proteins, achieving better results than when alum was used.


Revisiting Pentacam Parameters in the Diagnosis of Subclinical and Mild Keratoconus Based on Different Grading System Definitions.
To retest the performance of Pentacam parameters in the detection of eyes with subclinical keratoconus (KC) and mild KC based on different definitions from the Amsler-Krumeich (AK), Collaborative Longitudinal Evaluation of Keratoconus (CLEK), and ABCD systems. This cross-sectional university-based study comprised 24 eyes with subclinical KC, 144 eyes with mild KC (based on AK in 101 eyes, CLEK in 28 eyes, and ABCD in 15 eyes), and 70 controls. Diagnostic ability of the thinnest point (TP) pachymetry, KISA% index, inferior-superior asymmetry, corneal aberrations, Pentacam indices, front/ back elevations, pachymetric progression index, Ambrósio-Relational Thickness (ARTmax), and Belin/Ambrósio Enhanced Ectasia Display scores (Df, Db, Dp, Dt, Da, and D-final) were evaluated. ARTmax (83.3% sensitivity/74.3% specificity) had the highest ability in distinguishing subclinical KC from normal, followed by TP pachymetry, Dt, and Da. D-final showed excellent sensitivity/specificity in mild KC diagnosis based on AK (98%/100%) and CLEK (97.4%/100%) descriptions. In the mild KC-ABCD group, index of vertical asymmetry accurately detected all eyes with mild KC and 97.1% of the controls. Conclusion: This study points out the gray zone in the detection of eyes with subclinical and mild KC due to overlapping terminology and grading criteria. Pentacam parameters seem to have modest capability in subclinical KC detection, indicating the necessity for additional diagnostic modalities. However, eyes with mild KC can be diagnosed with high accuracy using Pentacam parameters, although the strongest parameters may vary according to the definition of "mild KC." Nevertheless, uniform and definitive criteria for subclinical and clinical KC classification are required for a diagnostic and therapeutic consensus in KC.


Inhibition of RON kinase potentiates anti-CTLA-4 immunotherapy to shrink breast tumors and prevent metastatic outgrowth.
The advent of immune checkpoint blockade as a new strategy for immunotherapy has changed the outlook for many aggressive cancers. Although complete tumor eradication is attainable in some cases, durable clinical responses are observed only in a small fraction of patients, underlining urgent need for improvement. We previously showed that RON, a receptor tyrosine kinase expressed in macrophages, suppresses antitumor immune responses, and facilitates progression and metastasis of breast cancer. Here, we investigated the molecular changes that occur downstream of RON activation in macrophages, and whether inhibition of RON can cooperate with checkpoint immunotherapy to eradicate tumors. Activation of RON by its ligand, MSP, altered the gene expression profile of macrophages drastically and upregulated surface levels of CD80 and PD-L1, ligands for T-cell checkpoint receptors CTLA-4 and PD-1. Genetic deletion or pharmacological inhibition of RON in combination with anti-CTLA-4, but not with anti-PD-1, resulted in improved clinical responses against orthotopically transplanted tumors compared to single-agent treatment groups, resulting in complete tumor eradication in 46% of the animals. Positive responses to therapy were associated with higher levels of T-cell activation markers and tumor-infiltrating lymphocytes. Importantly, co-inhibition of RON and anti-CTLA-4 was also effective in clearing metastatic breast cancer cells in lungs, resulting in clinical responses in nearly 60% of the mice. These findings suggest that RON inhibition can be a novel approach to potentiate responses to checkpoint immunotherapy in breast cancer.


Examining the relationship between health-related quality of life and increasing numbers of diagnoses.
Little is known about estimating utilities for comorbid (or 'joint') health states. Several joint health state prediction models have been suggested (for example, additive, multiplicative, best-of-pair, worst-of-pair, etc.), but no general consensus has been reached. The purpose of the study is to explore the relationship between health-related quality of life (HRQoL) and increasing numbers of diagnoses. We analyzed a large dataset containing respondents' ICD-9 diagnoses and preference-based HRQoL (EQ-5D and SF-6D). Data were stratified by the number of diagnoses, and mean HRQoL values were estimated. Several adjustments, accounting for the respondents' age, sex, and the severity of the diagnoses, were carried out. Our analysis fitted additive and multiplicative models to the data and assessed model fit using multiple standard model selection methods. A total of 39,817 respondents were included in the analyses. Average HRQoL values were represented well by both linear and multiplicative models. Although results across all analyses were similar, adjusting for severity of diagnoses, age, and sex strengthened the linear model's performance measures relative to the multiplicative model. Adjusted R (2) values were above 0.99 for all analyses (i.e., all adjusted analyses, for both HRQoL instruments), indicating a robust result. Additive and multiplicative models perform equally well within our analyses. A practical implication of our findings, based on the presumption that a linear model is simpler than an additive model, is that an additive model should be preferred unless there is compelling evidence to the contrary.


Complications associated with eye blocks and peripheral nerve blocks: an american society of anesthesiologists closed claims analysis.
Concern for block-related injury and liability has dissuaded many anesthesiologists from using regional anesthesia for eye and extremity surgery, despite many studies demonstrating the benefits of regional over general anesthesia. To determine injury patterns and liability associated with eye and peripheral nerve blocks, we re-examined the American Society of Anesthesiologists Closed Claims Database as part of the American Society of Regional Anesthesia and Pain Medicine's Practice Advisory on Neurologic Complications of Regional Anesthesia and Pain Medicine. Claims with eye or peripheral nerve blocks performed perioperatively from 1980 through 2000 were analyzed. The liability profile of anesthesiologists who provided both the eye block and sedation for eye surgery was compared with the profile of anesthesiologists who provided sedation only. The injury patterns associated with peripheral nerve blocks and payment factors were analyzed. Anesthesiologists who provided both the eye block and sedation for eye surgery (n = 59) had more injuries associated with block placement (P < .001), a higher proportion of claims with permanent injury (P < .05), and a higher proportion of claims with plaintiff payment (P < .05), compared with anesthesiologists who provided sedation only (n = 38). Peripheral nerve blocks (n = 159) were primarily associated with temporary injuries (56%). Local anesthetic toxicity was associated with 7 of 19 claims with death or brain damage. Performance of eye blocks by anesthesiologists significantly alters their liability profile, primarily related to permanent eye damage from block needle trauma. Though most peripheral nerve block claims are associated with temporary injuries, local anesthetic toxicity is a major cause of death or brain damage in these claims.


Infiltrated photonic crystal fiber: experiments and liquid crystal scattering model.
Experimental results obtained by means of a cut-back technique indicate low attenuations (< 1 dB x cm(-1)) for a solid core photonic crystal fiber filled with the nematic liquid crystal E7. These results observed in the visible wavelength range are compared with electromagnetic field simulations. The latter are carried out with a full vectorial finite element algorithm. Based on the modal properties under the condition of perpendicular anchoring of the liquid crystal molecules, the wavelength dependent attenuation is estimated using a power loss model considering the turbidity of the nematic liquid crystal. The results indicate that the scattering properties of this type of materials make them extremely interesting for fiber optical filters in the visible wavelength range and that filling materials with a relatively high turbidity are in general potentially useful as filling materials for solid core photonic crystal fibers.


Global proteomic analysis of the oocyst/sporozoite of Toxoplasma gondii reveals commitment to a host-independent lifestyle.
Toxoplasmosis is caused by the apicomplexan parasite Toxoplasma gondii and can be acquired either congenitally or via the oral route. In the latter case, transmission is mediated by two distinct invasive stages, i.e., bradyzoites residing in tissue cysts or sporozoites contained in environmentally resistant oocysts shed by felids in their feces. The oocyst plays a central epidemiological role, yet this stage has been scarcely investigated at the molecular level and the knowledge of its expressed proteome is very limited. Using one-dimensional gel electrophoresis coupled to liquid chromatography-linked tandem mass spectrometry, we analysed total or fractionated protein extracts of partially sporulated T. gondii oocysts, producing a dataset of 1304 non reduntant proteins (~18% of the total predicted proteome), ~59% of which were classified according to the MIPS functional catalogue database. Notably, the comparison of the oocyst dataset with the extensively covered proteome of T. gondii tachyzoite, the invasive stage responsible for the clinical signs of toxoplasmosis, identified 154 putative oocyst/sporozoite-specific proteins, some of which were validated by Western blot. The analysis of this protein subset showed that, compared to tachyzoites, oocysts have a greater capability of de novo amino acid biosynthesis and are well equipped to fuel the Krebs cycle with the acetyl-CoA generated through fatty acid β-oxidation and the degradation of branched amino acids. The study reported herein significantly expanded our knowledge of the proteome expressed by the oocyst/sporozoite of T. gondii, shedding light on a stage-specifc subset of proteins whose functional profile is consistent with the adaptation of T. gondii oocysts to the nutrient-poor and stressing extracellular environment.


[Effect of tiapride on headache and various other types of pain].
The effects of tiapride were studied in 180 patients, including 165 with cephalalgia originating in various causes and 15 with other types of pain. 110 of the 165 patients with cephalalgia completed the study; results were good or excellent in 78 (71%), with no differences related the the cause of the headache. 13 of the 15 patients with other types of pain completed the study, with good or excellent results in 10. Tiapride was given in a daily dose of three tablets a day in outpatients, and two daily intramuscular injections in hospitalized patients. Tolerance was excellent in 109 of the 123 patients (88%). Recorded side-effects were drowsiness in 4 patients, asthenia in 3, ebrious manifestations in 3, amenorrhea-galactorrhea in 2 and constipation in 2.


A Custom-Made Device for Reproducibly Depositing Pre-metered Doses of Nebulized Drugs on Pulmonary Cells in vitro.
The deposition of pre-metered doses (i.e., defined before and not after exposition) at the air-liquid interface of viable pulmonary epithelial cells remains an important but challenging task for developing aerosol medicines. While some devices allow quantification of the deposited dose after or during the experiment, e.g., gravimetrically, there is still no generally accepted way to deposit small pre-metered doses of aerosolized drugs or pharmaceutical formulations, e.g., nanomedicines. Here, we describe a straightforward custom-made device, allowing connection to commercially available nebulizers with standard cell culture plates. Designed to tightly fit into the approximately 12-mm opening of either a 12-well Transwell® insert or a single 24-well plate, a defined dose of an aerosolized liquid can be directly deposited precisely and reproducibly (4.8% deviation) at the air-liquid interface (ALI) of pulmonary cell cultures. The deposited dose can be controlled by the volume of the nebulized solution, which may vary in a range from 20 to 200 μl. The entire nebulization-deposition maneuver is completed after 30 s and is spatially homogenous. After phosphate-buffered saline (PBS) deposition, the viability and barrier properties transepithelial electrical resistance (TEER) of human bronchial epithelial Calu-3 cells were not negatively affected. Straightforward in manufacture and use, the device enables reproducible deposition of metered doses of aerosolized drugs to study the interactions with pulmonary cell cultures grown at ALI conditions.


Glucose-induced changes in pancreatic islet blood flow mediated by central nervous system.
Earlier experiments with the microsphere technique suggested that a heightened serum glucose concentration consistently leads to an increase in islet blood flow (IBF). Several lines of evidence suggest that this glucose-sensitive control mechanism is located at an extrapancreatic site. The purpose of this study was to define the possible role of the central nervous system in such a mechanism. D-glucose, L-glucose, 3-O-methylglucose, or saline were therefore infused into the carotid artery, each at a dose of 1 mg X kg body wt-1 X min-1 for 3 min, and the pancreatic and islet blood flows were measured. None of these substances affected the systemic serum glucose level. The intracarotid infusion of D-glucose, however, caused a rapid increase in both the serum insulin concentration and IBF. The blood flow to the whole pancreas nevertheless remained unchanged, indicating a redistribution of flow within the gland. Carotid infusion of the other test substances or a similar amount of D-glucose given in a femoral vein did not affect these parameters. Both the increase in serum insulin concentration and the increase in IBF caused by D-glucose could be abolished by vagotomy or administration of atropine. When the systemic blood glucose concentration was increased by intraperitoneal glucose administration (2 g/kg body wt), vagotomy blocked the increase in islet blood flow but not the concomitant insulin release. These observations suggest that the glucose-induced increase in IBF is mediated by vagal cholinergic influences.


Integrating Augmented In Situ Measurements and a Spatiotemporal Machine Learning Model To Back Extrapolate Historical Particulate Matter Pollution over the United Kingdom: 1980-2019.
Historical PM2.5 data are essential for assessing the health effects of air pollution exposure across the life course or early life. However, a lack of high-quality data sources, such as satellite-based aerosol optical depth before 2000, has resulted in a gap in spatiotemporally resolved PM2.5 data for historical periods. Taking the United Kingdom as an example, we leveraged the light gradient boosting model to capture the spatiotemporal association between PM2.5 concentrations and multi-source geospatial predictors. Augmented PM2.5 from PM10 measurements expanded the spatiotemporal representativeness of the ground measurements. Observations before and after 2009 were used to train and test the models, respectively. Our model showed fair prediction accuracy from 2010 to 2019 [the ranges of coefficients of determination (R2) for the grid-based cross-validation are 0.71-0.85] and commendable back extrapolation performance from 1998 to 2009 (the ranges of R2 for the independent external testing are 0.32-0.65) at the daily level. The pollution episodes in the 1980s and pollution levels in the 1990s were also reproduced by our model. The 4-decade PM2.5 estimates demonstrated that most regions in England witnessed significant downward trends in PM2.5 pollution. The methods developed in this study are generalizable to other data-rich regions for historical air pollution exposure assessment.


Nutritional Care and Rehabilitation for Frailty, Sarcopenia, and Malnutrition.
Frailty, sarcopenia, and malnutrition are highly prevalent among the older population [...].