Structure and mutational analysis of the archaeal GTP:AdoCbi-P guanylyltransferase (CobY) from Methanocaldococcus jannaschii: insights into GTP binding and dimerization.
In archaea and bacteria, the late steps in adenosylcobalamin (AdoCbl) biosynthesis are collectively known as the nucleotide loop assembly (NLA) pathway. In the archaeal and bacterial NLA pathways, two different guanylyltransferases catalyze the activation of the corrinoid. Structural and functional studies of the bifunctional bacterial guanylyltransferase that catalyze both ATP-dependent corrinoid phosphorylation and GTP-dependent guanylylation are available, but similar studies of the monofunctional archaeal enzyme that catalyzes only GTP-dependent guanylylation are not. Herein, the three-dimensional crystal structure of the guanylyltransferase (CobY) enzyme from the archaeon Methanocaldococcus jannaschii (MjCobY) in complex with GTP is reported. The model identifies the location of the active site. An extensive mutational analysis was performed, and the functionality of the variant proteins was assessed in vivo and in vitro. Substitutions of residues Gly8, Gly153, or Asn177 resulted in ≥94% loss of catalytic activity; thus, variant proteins failed to support AdoCbl synthesis in vivo. Results from isothermal titration calorimetry experiments showed that MjCobY(G153D) had 10-fold higher affinity for GTP than MjCobY(WT) but failed to bind the corrinoid substrate. Results from Western blot analyses suggested that the above-mentioned substitutions render the protein unstable and prone to degradation; possible explanations for the observed instability of the variants are discussed within the framework of the three-dimensional crystal structure of MjCobY(G153D) in complex with GTP. The fold of MjCobY is strikingly similar to that of the N-terminal domain of Mycobacterium tuberculosis GlmU (MtbGlmU), a bifunctional acetyltransferase/uridyltransferase that catalyzes the formation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc).


Complications of invasive subdural electrode monitoring at St. Louis Children's Hospital, 1994-2005.
The purpose of this study was to better define the incidence of complications associated with placement of subdural electrodes for localization of seizure foci and functional mapping in children. The authors retrospectively reviewed the records of 112 consecutive patients (53 boys, 59 girls; mean age 10.9 years, range 10 months-21.7 years) with medically intractable epilepsy who underwent invasive monitoring at the Pediatric Epilepsy Center at St. Louis Children's Hospital between January 1994 and July 2005. There were 122 implantation procedures (85 grids and strips, 32 strips only, five grids only, four with additional depth electrodes), with a mean monitoring period of 7.1 days (range 2-21 days). Operative complications included the need for repeated surgery for additional electrode placement (5.7%); wound infection (2.4%); cerebrospinal fluid leak (1.6%); and subdural hematoma, symptomatic pneumocephalus, bone flap osteomyelitis, and strip electrode fracture requiring operative retrieval (one patient [0.8%] each). There were four cases of transient neurological deficit (3.3%) and no permanent deficit or death associated with invasive monitoring. Placement of subdural grid and strip electrodes for invasive video electroencephalographic monitoring is generally well tolerated in the pediatric population. The authors found that aggressive initial electrode coverage was not associated with higher rates of blood transfusion or perioperative complications, and reduced the frequency of repeated operations for placement of supplemental electrodes.


The RNA polymerase II C-terminal domain promotes splicing activation through recruitment of a U2AF65-Prp19 complex.
Pre-mRNA splicing is frequently coupled to transcription by RNA polymerase II (RNAPII). This coupling requires the C-terminal domain of the RNAPII largest subunit (CTD), although the underlying mechanism is poorly understood. Using a biochemical complementation assay, we previously identified an activity that stimulates CTD-dependent splicing in vitro. We purified this activity and found that it consists of a complex of two well-known splicing factors: U2AF65 and the Prp19 complex (PRP19C). We provide evidence that both U2AF65 and PRP19C are required for CTD-dependent splicing activation, that U2AF65 and PRP19C interact both in vitro and in vivo, and that this interaction is required for activation of splicing. Providing the link to the CTD, we show that U2AF65 binds directly to the phosphorylated CTD, and that this interaction results in increased recruitment of U2AF65 and PRP19C to the pre-mRNA. Our results not only provide a mechanism by which the CTD enhances splicing, but also describe unexpected interactions important for splicing and its coupling to transcription.


Comparative study of the chemotactic responses of periodontal ligament cells and gingival fibroblasts to polypeptide growth factors.
Selective recruitment of periodontal ligament cells to a previously exposed root surface is believed to enhance periodontal regeneration. It has been hypothesized that competition from gingival fibroblasts may reduce the potential of periodontal regeneration. We compared the migratory responses of PDL cells and gingival fibroblasts to a variety of biologicals. Parallel experiments designed to examine the directed migration responses of both periodontal ligament cells (PDL cells) and gingival fibroblasts (GF) isolated from the same donors were conducted using Platelet Derived Growth Factor (PDGF), Insulin Like Growth Factor-I, -II (IGF-I, -II), Epidermal Growth Factor (EGF), Transforming Growth Factor-beta (TGF-beta), and the chemotactic factor derived from the conditioned culture media of PDL cells (termed PDL-CTX) as attractants. Both PDL cells and GF exhibited dose-dependent migratory responses when challenged with PDGF, IGF-I, IGF-II, EGF, and TGF-beta. However, when these cells were challenged with PDL-CTX, only PDL cells migrated in a specific dose-dependent manner, while GF were refractive to PDL-CTX stimulation. Additionally, concentrated conditioned culture media from cultures of gingival fibroblasts did not stimulate PDL cell migratory responses. In other experiments, antibody directed against PDGF, FGF, TGF-beta, IGF-I, IGF-II, NGF, and EGF did not inhibit the PDL-CTX-elicited response in PDL cells. Previous studies have suggested that success of periodontal therapy depends on the specific attachment, migration, and proliferation of selected periodontal ligament cells. The data presented in this manuscript suggest that both PDL cells and gingival fibroblasts respond to a multitude of growth factors. PDL-CTX was found to be PDL-cell-specific for directed migration. Thus, we conclude that any biological therapeutic regime for periodontal regeneration should include PDL-cell-specific agents.


Evolution of enzymatic activity in the enolase superfamily: structural and mutagenic studies of the mechanism of the reaction catalyzed by o-succinylbenzoate synthase from Escherichia coli.
o-Succinylbenzoate synthase (OSBS) from Escherichia coli, a member of the enolase superfamily, catalyzes an exergonic dehydration reaction in the menaquinone biosynthetic pathway in which 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) is converted to 4-(2'-carboxyphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB). Our previous structural studies of the Mg(2+).OSB complex established that OSBS is a member of the muconate lactonizing enzyme subgroup of the superfamily: the essential Mg(2+) is coordinated to carboxylate ligands at the ends of the third, fourth, and fifth beta-strands of the (beta/alpha)(7)beta-barrel catalytic domain, and the OSB product is located between the Lys 133 at the end of the second beta-strand and the Lys 235 at the end of the sixth beta-strand [Thompson, T. B., Garrett, J. B., Taylor, E. A, Meganathan, R., Gerlt, J. A., and Rayment, I. (2000) Biochemistry 39, 10662-76]. Both Lys 133 and Lys 235 were separately replaced with Ala, Ser, and Arg residues; all six mutants displayed no detectable catalytic activity. The structure of the Mg(2+).SHCHC complex of the K133R mutant has been solved at 1.62 A resolution by molecular replacement starting from the structure of the Mg(2+).OSB complex. This establishes the absolute configuration of SHCHC: the C1-carboxylate and the C6-OH leaving group are in a trans orientation, requiring that the dehydration proceed via a syn stereochemical course. The side chain of Arg 133 is pointed out of the active site so that it cannot function as a general base, whereas in the wild-type enzyme complexed with Mg(2+).OSB, the side chain of Lys 133 is appropriately positioned to function as the only acid/base catalyst in the syn dehydration. The epsilon-ammonium group of Lys 235 forms a cation-pi interaction with the cyclohexadienyl moiety of SHCHC, suggesting that Lys 235 also stabilizes the enediolate anion intermediate in the syn dehydration via a similar interaction.


Machine learning models to predict surgical case duration compared to current industry standards: scoping review.
Surgical waiting lists have risen dramatically across the UK as a result of the COVID-19 pandemic. The effective use of operating theatres by optimal scheduling could help mitigate this, but this requires accurate case duration predictions. Current standards for predicting the duration of surgery are inaccurate. Artificial intelligence (AI) offers the potential for greater accuracy in predicting surgical case duration. This study aimed to investigate whether there is evidence to support that AI is more accurate than current industry standards at predicting surgical case duration, with a secondary aim of analysing whether the implementation of the models used produced efficiency savings. PubMed, Embase, and MEDLINE libraries were searched through to July 2023 to identify appropriate articles. PRISMA extension for scoping reviews and the Arksey and O'Malley framework were followed. Study quality was assessed using a modified version of the reporting guidelines for surgical AI papers by Farrow et al. Algorithm performance was reported using evaluation metrics. The search identified 2593 articles: 14 were suitable for inclusion and 13 reported on the accuracy of AI algorithms against industry standards, with seven demonstrating a statistically significant improvement in prediction accuracy (P < 0.05). The larger studies demonstrated the superiority of neural networks over other machine learning techniques. Efficiency savings were identified in a RCT. Significant methodological limitations were identified across most studies. The studies suggest that machine learning and deep learning models are more accurate at predicting the duration of surgery; however, further research is required to determine the best way to implement this technology.


The importance of 8993C>T (Thr399Ile) TLR4 polymorphism in etiology of osteoporosis in postmenopausal women.
Toll-like receptors (TLR) may play a key role in initiating cellular signaling pathways by increasing the levels of inflammatory cytokines which, cooperating with osteoclasts, influence bone turnover Numerous research articles focused on the genetic background of this condition, among others on polymorphic variants in TLR genes. The aim of the study was to examine the role of 20877G>A (Arg753GIn) in TLR2 gene and 8993C>T (Thr399lle) in TLR4 gene in the etiopathogenesis of postmenopausal osteoporosis in Polish women. This study included 180 postmenopausal women (t-score < or = -2.5), 153 postmenopausal women with osteopenia (t-score between -2.5 and -1), and 91 postmenopausal healthy women with correct t-score (t-score >-1). The 20877G>A TLR2 and 8993C>T TLR4 polymorphisms were determined by PCR/RFLP analysis. The analysis did not reveal statistically significant differences in the distribution of genetic variants of 20877G>A TLR2 polymorphism between the investigated groups of women. The most interesting results were connected with 8993C>T TLR4 polymorphism. Comparison of the group with osteoporosis and controls revealed overrepresentation of heterozygous 8993CT genotype (13.3 vs. 5.5%, OR=2.65, p=0.03). Also, mutated 8993T allele was overrepresented in the group with osteoporosis (6.7 vs. 2.7%, OR=2.52, p=0.04). Higher frequency of heterozygous 8993CT genotype (13.3 vs. 4.6%, OR=3.21, p=0.004) and mutated 8993T allele (6.7 vs. 2.3%, OR=3.05, p=0. 005) was noted in osteoporotic women as compared to the group with osteopenia. Higher frequency of heterozygous 8993CT genotype (13.3% vs. 5.3%, OR=2.73, p=0.003) and mutated 8993T allele (6.7 vs. 2.7%, OR=2.67, p=0.004) was observed in the group with osteoporosis as compared to women with osteopenia and with correct t-score. Results of our study suggest an important role of mutated 8993T allele of 8993C>T TLR4 polymorphisms in the etiology of postmenopausal osteoporosis. Nevertheless, this observation requires further investigation with larger sample size comprised of Polish women.


[CMV retinitis in AIDS--a pre-final complication?].
The appearance of cytomegalovirus (CMV) retinitis in AIDS is regarded as an unfavourable sign, it was even considered in the first years of the HIV-epidemic to be a pre-final complication. The survival period after diagnosis of the retinitis is under virostatic therapy generally given as several months, only exceptionally as more than a year. We report here 3 cases of CMV retinitis in AIDS having an unusually long duration of 14-24 months. The clinical and histological results with the pecularities (resistance to therapy, optic atrophy, retinal atrophy with detachment, atypical peripheral fundus lesions) are presented. The importance of ophthalmological care of HIV-patients is indicated and a screening procedure is suggested.


Repeat confirmatory testing for persons with discordant whole blood and oral fluid rapid HIV test results: findings from post marketing surveillance.
Reactive oral fluid and whole blood rapid HIV tests must be followed with a confirmatory test (Western blot (WB), immunofluorescent assay (IFA) or approved nucleic acid amplification test (NAAT)). When the confirmatory result is negative or indeterminate (i.e. discordant with rapid result), repeat confirmatory testing should be conducted using a follow-up specimen. Previous reports have not described whether repeat testing adequately resolves the HIV-infection status of persons with discordant results. Post-marketing surveillance was conducted in 368 testing sites affiliated with 14 state and 2 city health departments from August 11, 2004 to June 30, 2005 and one health department through December 31, 2005. For persons with discordant results, data were collected on demographics, risk behaviors, HIV test results and specimen types. Persons with repeat confirmatory results were classified as HIV-infected or uninfected. Regression models were created to assess risk factors for not having repeat testing. Of 167,371 rapid tests conducted, 2589 (1.6%) were reactive: of these, 2417 (93%) had positive WB/IFA, 172 (7%) had negative or indeterminate WB/IFA. Of 89/172 (52%) persons with a repeat confirmatory test: 17 (19%) were HIV-infected, including 3 with indeterminate WB and positive NAAT; 72 (81%) were uninfected, including 12 with repeat indeterminate WB. Factors associated with HIV-infection included having an initial indeterminate WB/IFA (vs. negative) (p<0.001) and having an initial oral fluid WB (vs. serum) (p<0.001). Persons who had male-female sex (vs. male-male sex) were at increased risk for not having a repeat test [adjusted OR 2.6, 95% CI (1.3, 4.9)]. Though only half of persons with discordant results had repeat confirmatory testing, of those who did, nearly one in five were HIV-infected. These findings underscore the need for rapid HIV testing programs to increase repeat confirmatory testing for persons with discordant results. Because of the lower sensitivity of oral fluid WBs, confirmatory testing following a reactive rapid test should be conducted using serum or plasma, when possible.


Beyond nutrition and agriculture policy: collaborating for a food policy.
Global interest in food policy is emerging in parallel with mounting challenges to the food supply and the rising prevalence of diet-related chronic health conditions. Some of the foundational elements of food policies are agricultural practices, finite resources, as well as economic burdens associated with a growing and ageing population. At the intersection of these interests is the need for policy synchronisation and a better understanding of the dynamics within local, regional and national government decision-making that ultimately affect the wellness of the populous and the safety, quality, affordability and quantity of the food supply. Policies, synchronised or not, need to be implemented and, for the food industry, this has seen a myriad of approaches with respect to condensing complex nutritional information and health claims. These include front and/or back of pack labelling, traffic light systems, etc. but in general there is little uniformity at the more regional and global scales. This translation of the nutritional and health-beneficial messages accompanying specific products to the consumer will undoubtedly be an area of intense activity, and hopefully interaction with policy makers, as the food industry continues to become a more global industry.


Overlooked Short Toxin-Like Proteins: A Shortcut to Drug Design.
Short stable peptides have huge potential for novel therapies and biosimilars. Cysteine-rich short proteins are characterized by multiple disulfide bridges in a compact structure. Many of these metazoan proteins are processed, folded, and secreted as soluble stable folds. These properties are shared by both marine and terrestrial animal toxins. These stable short proteins are promising sources for new drug development. We developed ClanTox (classifier of animal toxins) to identify toxin-like proteins (TOLIPs) using machine learning models trained on a large-scale proteomic database. Insects proteomes provide a rich source for protein innovations. Therefore, we seek overlooked toxin-like proteins from insects (coined iTOLIPs). Out of 4180 short (<75 amino acids) secreted proteins, 379 were predicted as iTOLIPs with high confidence, with as many as 30% of the genes marked as uncharacterized. Based on bioinformatics, structure modeling, and data-mining methods, we found that the most significant group of predicted iTOLIPs carry antimicrobial activity. Among the top predicted sequences were 120 termicin genes from termites with antifungal properties. Structural variations of insect antimicrobial peptides illustrate the similarity to a short version of the defensin fold with antifungal specificity. We also identified 9 proteins that strongly resemble ion channel inhibitors from scorpion and conus toxins. Furthermore, we assigned functional fold to numerous uncharacterized iTOLIPs. We conclude that a systematic approach for finding iTOLIPs provides a rich source of peptides for drug design and innovative therapeutic discoveries.


Gold Nanoparticles for Monitoring of Mesenchymal Stem-Cell-Loaded Bioresorbable Polymeric Wraps for Arteriovenous Fistula Maturation.
Mesenchymal stem cell (MSC)-seeded polymeric perivascular wraps have been shown to enhance arteriovenous fistula (AVF) maturation. However, the wraps' radiolucency makes their placement and integrity difficult to monitor. Through electrospinning, we infused gold nanoparticles (AuNPs) into polycaprolactone (PCL) wraps to improve their radiopacity and tested whether infusion affects the previously reported beneficial effects of the wraps on the AVF's outflow vein. Sprague Dawley rat MSCs were seeded on the surface of the wraps. We then compared the effects of five AVF treatments-no perivascular wrap (i.e., control), PCL wrap, PCL + MSC wrap, PCL-Au wrap, and PCL-Au + MSC wrap-on AVF maturation in a Sprague Dawley rat model of chronic kidney disease (n = 3 per group). Via micro-CT, AuNP-infused wraps demonstrated a significantly higher radiopacity compared to that of the wraps without AuNPs. Wraps with and without AuNPs equally reduced vascular stenoses, as seen via ultrasonography and histomorphometry. In the immunofluorescence analysis, representative MSC-seeded wraps demonstrated reduced neointimal staining for markers of infiltration with smooth muscle cells (α-SMA), inflammatory cells (CD45), and fibroblasts (vimentin) compared to that of the control and wraps without MSCs. In conclusion, AuNP infusion allows in vivo monitoring via micro-CT of MSC-seeded polymeric wraps over time, without compromising the benefits of the wrap for AVF maturation.


Intragroup and intragenomic conflict over chemical defense against predators.
Insects are often chemically defended against predators. There is considerable evidence for a group-beneficial element to their defenses, and an associated potential for individuals to curtail their own investment in costly defense while benefitting from the investments of others, termed "automimicry." Although females in chemically defended taxa often lay their eggs in clusters, leading to siblings living in close proximity, current models of automimicry have neglected kin-selection effects, which may be expected to curb the evolution of such selfishness. Here, we develop a general theory of automimicry that explicitly incorporates kin selection. We investigate how female promiscuity modulates intragroup and intragenomic conflicts overinvestment into chemical defense, finding that individuals are favored to invest less than is optimal for their group, and that maternal-origin genes favor greater investment than do paternal-origin genes. We translate these conflicts into readily testable predictions concerning gene expression patterns and the phenotypic consequences of genomic perturbations, and discuss how our results may inform gene discovery in relation to economically important agricultural products.


Localization and Speciation of Iron Impurities within a Fluid Catalytic Cracking Catalyst.
Fluid catalytic cracking is a chemical conversion process of industrial scale. This process, utilizing porous catalysts composed of clay and zeolite, converts heavy crude-oil fractions into transportation fuel and petrochemical feedstocks. Among other factors iron-rich reactor and feedstream impurities cause these catalyst particles to permanently deactivate. Herein, we report tomographic X-ray absorption spectroscopy measurements that reveal the presence of dissimilar iron impurities of specific localization within a single deactivated particle. Whereas the iron natural to clay in the composite seems to be unaffected by operation, exterior-facing and feedstream-introduced iron was found in two forms. Those being minute quantities of ferrous oxide, located near regions of increased porosity, and impurities rich in Fe3+ , preferentially located in the outer dense part of the particle and suggested to contribute to the formation of an isolating amorphous silica alumina envelope.


Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development.
Embryonic stem (ES) cells are immortal and pluripotent cells derived from early mammalian embryos. Transcription factor Oct3/4 is essential for self-renewal of ES cells and early mouse development. However, only a few Oct3/4 target genes have been identified. In this study, we found that F-box-containing protein Fbx15 was expressed predominantly in mouse undifferentiated ES cells. Inactivation of Oct3/4 in ES cells led to rapid extinction of Fbx15 expression. Reporter gene analyses demonstrated that this ES cell-specific expression required an 18-bp enhancer element located approximately 500 nucleotides upstream from the transcription initiation site. The enhancer contained an octamer-like motif and an adjacent Sox-binding motif. Deletion or point mutation of either motif abolished the enhancer activity. The 18-bp fragment became active in NIH 3T3 cells when Oct3/4 and Sox2 were coexpressed. A gel mobility shift assay demonstrated cooperative binding of Oct3/4 and Sox2 to the enhancer sequence. In mice having a beta-galactosidase gene knocked into the Fbx15 locus, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining was detected in ES cells, early embryos (two-cell to blastocyst stages), and testis tissue. Despite such specific expression of Fbx15, homozygous mutant mice showed no gross developmental defects and were fertile. Fbx15-null ES cells were normal in morphology, proliferation, and differentiation. These data demonstrate that Fbx15 is a novel target of Oct3/4 but is dispensable for ES cell self-renewal, development, and fertility.


Biotransformation of organic nitrates to nitric oxide by vascular smooth muscle and endothelial cells.
The vasodilator action of organic nitrates is thought to be mediated by an increase in the level of cGMP following stimulation of the cytosolic enzyme guanylate cyclase in the vascular smooth muscle cell. However, direct evidence for the formation of the putative active metabolite, nitric oxide (NO) within the different compartments of the vascular wall is still missing. We here demonstrate for the first time that cultured vascular smooth muscle cells as well as endothelial cells from different species actively metabolize organic nitrates to NO. We furthermore present evidence for an outward transport of cGMP from both cell types following stimulation of soluble guanylate cyclase. The rate of NO release closely correlated with the rate of cGMP egression. Biotransformation of organic nitrates to NO appeared to comprise at least two different components, a heat-sensitive enzymatic pathway which is short-lived and prone to rapid desensitization and a second non-enzymatic component which is apparently unsaturable and longer lasting. The marked decrease in the release of NO and cGMP upon the repeated administration of organic nitrates suggests that the phenomenon of "nitrate tolerance" is mainly due to an impaired biotransformation. We propose that the metabolism of nitrates to NO may have important implications for the prevention of atherosclerosis and the therapeutic modulation of blood cell function.


Plasmid-mediated resistance in Enterobacteriaceae: changing landscape and implications for therapy.
Antimicrobial resistance is increasing worldwide, and pathogenic microorganisms that are resistant to all available antimicrobial agents are increasingly reported. Emerging plasmid-encoded extended-spectrum β-lactamases (ESBLs) and carbapenemases are increasingly reported worldwide. Carbapenemase production encoded by genes located on mobile genetic elements is typically accompanied by genes encoding resistance to other drug classes, often but not necessarily located on the same mobile element. Multiple plasmid-mediated mechanisms of resistance against the fluoroquinolones and aminoglycosides have been described, and the combination of plasmid-mediated resistance with chromosomally encoded resistance mechanisms of multiple drug classes now results in strains that are resistant to all of the main classes of commonly used antimicrobial drugs. Clinical studies of antimicrobial therapy and outcome of patients infected with ESBL- or carbapenemase-producing strains of Enterobacteriaceae compared with patients infected with susceptible strains are limited in their design but suggest a worse outcome after infection with resistant strains. Alternative options for the treatment of infections caused by carbapenem-resistant strains of Enterobacteriaceae are limited. Current strategies include colistin, fosfomycin, tigecycline and temocillin. Although in vitro testing suggests strong activity for each of these drugs against a large proportion of carbapenem-resistant strains of Enterobacteriaceae, clinical evaluations do not provide strong evidence for equivalent or improved outcome. Oral treatment with fosfomycin tromethamine is effective against lower urinary tract infections (UTIs) caused by ESBL-producing Escherichia coli. Intravenous fosfomycin may be beneficial and safe for patients when used as part of a combination therapy in the management of severe infections caused by carbapenem-resistant Klebsiella pneumoniae. Tigecycline is only indicated for the treatment of complicated skin and skin structure infections and complicated intra-abdominal infections in Europe, and is also approved for treatment of community-acquired pneumonia in the US. Clearly, further research on the clinical and safety outcomes in the treatment of multidrug-resistant Enterobacteriaceae with these existing alternative drugs, and the development of new and unrelated drugs, are urgently warranted.


Lacrimal gland development and Fgf10-Fgfr2b signaling are controlled by 2-O- and 6-O-sulfated heparan sulfate.
Heparan sulfate, an extensively sulfated glycosaminoglycan abundant on cell surface proteoglycans, regulates intercellular signaling through its binding to various growth factors and receptors. In the lacrimal gland, branching morphogenesis depends on the interaction of heparan sulfate with Fgf10-Fgfr2b. To address if lacrimal gland development and FGF signaling depends on 2-O-sulfation of uronic acids and 6-O-sulfation of glucosamine residues, we genetically ablated heparan sulfate 2-O and 6-O sulfotransferases (Hs2st, Hs6st1, and Hs6st2) in developing lacrimal gland. Using a panel of phage display antibodies, we confirmed that these mutations disrupted 2-O and/or 6-O but not N-sulfation of heparan sulfate. The Hs6st mutants exhibited significant lacrimal gland hypoplasia and a strong genetic interaction with Fgf10, demonstrating the importance of heparan sulfate 6-O sulfation in lacrimal gland FGF signaling. Altering Hs2st caused a much less severe phenotype, but the Hs2st;Hs6st double mutants completely abolished lacrimal gland development, suggesting that both 2-O and 6-O sulfation of heparan sulfate contribute to FGF signaling. Combined Hs2st;Hs6st deficiency synergistically disrupted the formation of Fgf10-Fgfr2b-heparan sulfate complex on the cell surface and prevented lacrimal gland induction by Fgf10 in explant cultures. Importantly, the Hs2st;Hs6st double mutants abrogated FGF downstream ERK signaling. Therefore, Fgf10-Fgfr2b signaling during lacrimal gland development is sensitive to the content or arrangement of O-sulfate groups in heparan sulfate. To our knowledge, this is the first study to show that simultaneous deletion of Hs2st and Hs6st exhibits profound FGF signaling defects in mammalian development.


Combinatorial screening of biochemical and physical signals for phenotypic regulation of stem cell-based cartilage tissue engineering.
Despite great progress in biomaterial design strategies for replacing damaged articular cartilage, prevention of stem cell-derived chondrocyte hypertrophy and resulting inferior tissue formation is still a critical challenge. Here, by using engineered biomaterials and a high-throughput system for screening of combinatorial cues in cartilage microenvironments, we demonstrate that biomaterial cross-linking density that regulates matrix degradation and stiffness-together with defined presentation of growth factors, mechanical stimulation, and arginine-glycine-aspartic acid (RGD) peptides-can guide human mesenchymal stem cell (hMSC) differentiation into articular or hypertrophic cartilage phenotypes. Faster-degrading, soft matrices promoted articular cartilage tissue formation of hMSCs by inducing their proliferation and maturation, while slower-degrading, stiff matrices promoted cells to differentiate into hypertrophic chondrocytes through Yes-associated protein (YAP)-dependent mechanotransduction. in vitro and in vivo chondrogenesis studies also suggest that down-regulation of the Wingless and INT-1 (WNT) signaling pathway is required for better quality articular cartilage-like tissue production.


Advancing Italian biomedical information extraction with transformers-based models: Methodological insights and multicenter practical application.
The introduction of computerized medical records in hospitals has reduced burdensome activities like manual writing and information fetching. However, the data contained in medical records are still far underutilized, primarily because extracting data from unstructured textual medical records takes time and effort. Information Extraction, a subfield of Natural Language Processing, can help clinical practitioners overcome this limitation by using automated text-mining pipelines. In this work, we created the first Italian neuropsychiatric Named Entity Recognition dataset, PsyNIT, and used it to develop a Transformers-based model. Moreover, we collected and leveraged three external independent datasets to implement an effective multicenter model, with overall F1-score 84.77 %, Precision 83.16 %, Recall 86.44 %. The lessons learned are: (i) the crucial role of a consistent annotation process and (ii) a fine-tuning strategy that combines classical methods with a "low-resource" approach. This allowed us to establish methodological guidelines that pave the way for Natural Language Processing studies in less-resourced languages.


High-dose corticotropin (ACTH) versus prednisone for infantile spasms: a prospective, randomized, blinded study.
To compare the efficacy of corticotropin (ACTH) (150 U/m2/day) and prednosone (2 mg/kg/day) given for 2 weeks, in suppressing clinical spasms and hypsarrhythmic electroencephalogram (EEG) in infantile spasms (IS). AACTH and prednisone are standard treatments for IS. ACTH at high doses causes severe dose- and duration-dependent side effects, but may be superior to prednisone, based on retrospective or uncontrolled studies. Blinded prospecive studies have shown equal efficacy of prednisone and low-dose ACTH, and low versus high-dose ACTH. A prospective, randomized, single-blinded study. Patient population consisted of consecutive infants fulfilling entry criteria, including the presence of clinical spasms, hypsarrhythmia (or variants) during a full sleep cycle video-EEG, and no prior steroid/ACTH treatment. Response required both cessation of spasms and elimination of hypsarrhythmia by the end of the 2-week treatment period, as determined by an investigator "blinded" to treatment. Treatment of responders was tapered off over 12 days; those failing one hormone were crossed-over to the other. OF 34 eligible infants, 29 were enrolled. Median age of patients was 6 months. Twenty-two infants were "symptomatic" with known or suspected cause, and seven were cryptogenic (two normal). Of 15 infants randomized to ACTH, 13 responded by EEG and clinical criteria (86.6%); Seizures stopped in an additional infant, but EEG remained hypsarrhythmic (considered a failure). Four of the 14 patients given prednisone responded (28.6%,, with complete clinical-EEG correlation), significantly less than with ACTH, (chi2 test). Using a prospective, randomized approach, a 2-week course of high-dose ACTH is superior to 2 weeks of prednsone for treatment of IS, as assessed by both clinical and EEG criteria.


Imagined positive emotions and inhibitory control: the differentiated effect of pride versus happiness.
Inhibitory control is a cognitive mechanism that contributes to successful self-control (i.e., adherence to a long-term goal in the face of an interfering short-term goal). This research explored the effect of imagined positive emotional events on inhibition. The authors proposed that the influence of imagined emotions on inhibition depends on whether the considered emotion corresponds to the attainment of a long-term goal (i.e., pride) or a short-term goal (i.e., happiness). The authors predicted that in an antisaccade task that requires inhibition of a distractor, imagining a happiness-eliciting event is likely to harm inhibitory processes compared with imagining a pride-eliciting event, because the former but not the latter primes interfering short-term goals. The results showed that imagining a happiness-eliciting event decreased inhibition relative to imagining a pride-eliciting event. The results suggest a possible mechanism underlying the role of imagined positive emotions in pursuit of goals that require self-control.


Yarning as a method for building sexual wellbeing among urban Aboriginal young people in Australia.
This paper describes the strategies used by Aboriginal young people to build positive relationships and sexual wellbeing. It does so to counter the risk-focussed narratives present in much existing research and to showcase the resourcefulness of Aboriginal young people. We used peer-interview methods to collect qualitative data from 52 Aboriginal young people living in western Sydney, Australia. Participants reported a strong desire to stay safe and healthy in their sexual relationships and to achieve this they relied heavily on oral communication and yarning strategies. Participants viewed communication as a way to gain or give advice (about bodies, infections, pregnancy, relationships); to assess the acceptability and safety of potential partners; to negotiate consent with partners; to build positive relationships; and to get themselves out of unhealthy relationships. Participants also discussed 'self-talk' as a strategy for building sexual wellbeing, referring to narratives of self-respect and pride in culture as important in establishing Aboriginal young people's positive views of self and as deserving of respectful and safe sexual relationships. These findings suggest that future programmes and interventions based on yarning could be well-regarded, given it is a cultural form of pedagogy and a strategy Aboriginal young people already use to build positive relationships and identities.


Optimizing pain treatment interventions (OPTI): A pilot randomized controlled trial of collaborative care to improve chronic pain management and opioid safety-Rationale, methods, and lessons learned.
Veterans seeking care in VA medical facilities have high rates of chronic pain, which often co-occur with mental health and substance use disorders, including prescription opioid misuse. The overall goal of the Optimizing Pain Treatment Interventions (OPTI) study was to pilot a 12-week Collaborative Care intervention to improve opioid safety, chronic pain disability, and use of non-pharmacological pain management strategies in veterans in VA primary care. Between November 2014 and January 2017, 100 veteran patients with chronic pain and high-risk prescription opioid use (e.g., high-dose therapy, early refills, etc.) were enrolled and completed an initial one-hour study visit with a primary care provider (PCP) within 4 weeks of enrollment. Study PCPs were guided by a web-based opioid management decision support program and templated notes in the VA electronic medical record. After assessment and education, study PCPs used Shared Decision-Making to formulate a Pain Care Plan aligned with a participant's personal values and goals. After the initial visit, patients randomized to Collaborative Care received one Motivational Interviewing (MI) session with a Care Manager followed by 3 Care Manager-delivered brief telephone MI sessions at 6, 8, and 12 weeks to reinforce Pain Care Plans; patients randomized to an Attention Control condition met with a Care Manager briefly, followed by 3 brief scripted telephone psychoeducation sessions at 6, 8, and 12 weeks. Masked evaluators assessed outcomes at baseline, end of intervention (12 weeks), and after eight weeks of no contact (20 weeks). We present study rationale, detailed methods, preliminary results and lessons learned.


A behavioral design approach to improving a Chagas disease vector control campaign in Peru.
Individual behavior change is a critical ingredient in efforts to improve global health. Central to the focus on behavior has been a growing understanding of how the human brain makes decisions, from motivations and mindsets to unconscious biases and cognitive shortcuts. Recent work in the field of behavioral economics and related fields has contributed to a rich menu of insights and principles that can be engineered into global health programs to increase impact and reach. However, there is little research on the process of designing and testing interventions informed by behavioral insights. In a study focused on increasing household participation in a Chagas disease vector control campaign in Arequipa, Peru, we applied Datta and Mullainathan's "behavioral design" approach to formulate and test specific interventions. In this Technical Advance article we describe the behavioral design approach in detail, including the Define, Diagnosis, Design, and Test phases. We also show how the interventions designed through the behavioral design process were adapted for a pragmatic randomized controlled field trial. The behavioral design framework provided a systematic methodology for defining the behavior of interest, diagnosing reasons for household reluctance or refusal to participate, designing interventions to address actionable bottlenecks, and then testing those interventions in a rigorous counterfactual context. Behavioral design offered us a broader range of strategies and approaches than are typically used in vector control campaigns. Careful attention to how behavioral design may affect internal and external validity of evaluations and the scalability of interventions is needed going forward. We recommend behavioral design as a useful complement to other intervention design and evaluation approaches in global health programs.


Immunosuppression in mice induced by dioxin (TCDD) in feed.
Juvenile and adult mice (4 and 7 weeks old, respectively) were fed various levels of 2, 3, 7, 8-tetracholorodibenzo-p-dioxin (TCDD) incorporated in mouse feed for five weeks or more. Animal parameters monitored included body weight, organ weights, white blood counts, hematocrits, certain serum protein levels (see below), symptoms of overt toxicity and mortality. High exposure levels (100 ppb) produced marked suppression in total serum protein, gamma globulin and albumin, but an increase in the beta-globulins. Feeding levels of 10 ppb TCDD or more reduced the primary and secondary antibody response to both tetanus toxoid and sheep erythrocytes. The amount of suppression appeared to be dose related, with juvenile animals showing greater suppression than adults. Antibody suppression from the 10 ppb feed level was roughly equivalent to that observed from a single high dose (200 mg/kg) of cyclophosphamide (CY). No evidence of enhanced IgE synthesis was obtained from TCDD exposed animals. TCDD feeding also lowered contact sensitization to dinitrofluorobenzene and resistance to challenge with either Salmonella typhimurium or Listeria monocytogenes.


A novel endoscopic ablation of gastric antral vascular ectasia.
An 80-year-old woman was admitted to our hospital because of tarry stool with iron deficiency anemia. Her past history included autoimmune hepatitis. Esophagogastroduodenal endoscopy was performed to investigate the bleeding source and revealed multiple linear gastric vascular malformations in the antrum and cardia, compatible with Gastric antral vascular ectasia (GAVE). Endoscopic ablation was carried out with the tip of the hot biopsy forceps without opening at soft coagulation mode of 80W. The patient tolerated the procedure well and there were no complications associated with endoscopic therapies. After two sessions of endoscopic ablation her anemia improved to around 10 g/dL, an increase of 3.6 g/dL. Various endoscopic treatments have been described to manage GAVE. The most popular is argon plasma coagulation (APC), although APC is associated with over-distension induced by the argon plasma gas. To avoid over-distension and to reduce the abdominal discomfort/pain of this patient, we have used hot biopsy forceps instead of APC. Our case suggests that this procedure is effective, easy and convenient, as no special equipment or skill is necessary.


Integrated tumor and germline whole-exome sequencing identifies mutations in MAPK and PI3K pathway genes in an adolescent with rosette-forming glioneuronal tumor of the fourth ventricle.
The integration of genome-scale studies such as whole-exome sequencing (WES) into the clinical care of children with cancer has the potential to provide insight into the genetic basis of an individual's cancer with implications for clinical management. This report describes the results of clinical tumor and germline WES for a patient with a rare tumor diagnosis, rosette-forming glioneuronal tumor of the fourth ventricle (RGNT). Three pathogenic gene alterations with implications for clinical care were identified: somatic activating hotspot mutations in FGFR1 (p.N546K) and PIK3CA (p.H1047R) and a germline pathogenic variant in PTPN11 (p.N308S) diagnostic for Noonan syndrome. The molecular landscape of RGNT is not well-described, but these data are consistent with prior observations regarding the importance of the interconnected MAPK and PI3K/AKT/mTOR signaling pathways in this rare tumor. The co-occurrence of FGFR1, PIK3CA, and PTPN11 alterations provides further evidence for consideration of RGNT as a distinct molecular entity from pediatric low-grade gliomas and suggests potential therapeutic strategies for this patient in the event of tumor recurrence as novel agents targeting these pathways enter pediatric clinical trials. Although RGNT has not been definitively linked with cancer predisposition syndromes, two prior cases have been reported in patients with RASopathies (Noonan syndrome and neurofibromatosis type 1 [NF1]), providing an additional link between these tumors and the mitogen-activated protein kinase (MAPK) signaling pathway. In summary, this case provides an example of the potential for genome-scale sequencing technologies to provide insight into the biology of rare tumors and yield both tumor and germline results of potential relevance to patient care.


Cyclotides are a component of the innate defense of Oldenlandia affinis.
Cyclotides are small cysteine-rich plant peptides similar in size and processing to the defensins. Long-term growth of the Rubiaceae family plant Oldenlandia affinis under different conditions reveals a diverse cyclotide gene and peptide expression profile, including tissue specificity, suggesting that different cyclotides are regulated differently both spatially and in response to the environment. To determine whether cyclotide precursor gene regulation was dynamic we exposed O. affinis to a range of abiotic, biotic, and hormonal stimuli and monitored Oak1-4 expression over a 48-h period. Unlike some defensins, the genes for cyclotide precursor proteins Oak1-4 did not display dynamic change, indicating that they contribute to the basal defense of O. affinis. Despite this lack of dynamism, the cyclotide profile of plants grown on plates differed markedly from field-grown plants and so prompted attempts to discover novel cyclotides and precursor genes. The two most abundant cyclotides from plate-grown O. affinis were sequenced and one was found to be an unusual linear cyclotide derivative, kalata B20-lin. Degenerate PCR of plate-grown O. affinis obtained five novel cyclotide genes including Oak9 which encodes for kalata B20-lin and appears to have arisen by the presence of a premature stop codon.


Effects of therapeutic ultrasound on range of motion and stretch pain.
[Purpose] This study aimed to clarify the effects of therapeutic ultrasound on range of motion and stretch pain and the relationships between the effects. [Subjects] The subjects were 15 healthy males. [Methods] Subjects performed all three interventions: (1) ultrasound (US group), (2) without powered ultrasound (placebo group), and (3) rest (control group). Ultrasound was applied at 3 MHz with an intensity of 1.0 W/cm(2) and a 100% duty cycle for 10 minutes. The evaluation indices were active and passive range of motion (ROM), stretch pain (visual analog scale; VAS), and skin surface temperature (SST). The experimental protocol lasted a total of 40 minutes; this was comprised of 10 minutes before the intervention, 10 minutes during the intervention (US, placebo, and control), and 20 minutes after the intervention. [Results] ROM and SST were significantly higher in the US group than in the placebo and control groups for the 20 minutes after ultrasound, though there was no change in stretch pain. [Conclusion] The effects of ultrasound on ROM and SST were maintained for 20 minutes after the intervention. The SST increased with ultrasound and decreased afterwards. Additionally, the SST tended to return to baseline levels within 20 minutes after ultrasound exposure. Therefore, these effects were caused by a combination of thermal and mechanical effects of the ultrasound.


Immediate changes in anticipatory activity of caudate neurons associated with reversal of position-reward contingency.
The primate caudate nucleus plays a crucial role in transforming cognitive/motivational information into eye movement signals. A subset of caudate projection neurons fire before a visual target's onset. This anticipatory activity is sensitive to position-reward contingencies and correlates with saccade latency, which is shorter toward a rewarded position. We recorded single-unit activity of caudate projection neurons to examine the dynamics of change in anticipatory activity immediately after switches of the position-reward contingency. Two monkeys performed a visually guided saccade task where only one position was associated with reward. The position-reward mapping remained constant within a block, but was reversed frequently between blocks without any indication to the monkey. Therefore the switch could be detected only by unexpected reward delivery or unexpected lack of reward. After the switch, both saccade latency and anticipatory activity showed reliable changes already in the second trial, whether or not the first trial was rewarded. However, anticipatory activity in the second trial was generally higher if the first trial was rewarded, and the measured saccade latencies could be better explained by the difference in anticipatory activity between the two caudate nuclei. We suggest that anticipatory activity of caudate neurons reflects the reversal set of reward-position contingency.


Mechanism of miR-181a-5p in Regulatory T/T-Helper 17 Immune Imbalance and Asthma Development in Mice with Allergic Rhinitis.
Allergic rhinitis (AR) is an immune disorder and also a risk factor of asthma. microRNAs (miRNAs) are implicated in autoimmune diseases, including RA. This study investigated effect of miR-181a-5p on regulatory T (Treg)/ T-helper (Th) 17 immune imbalance in AR. A murine model of AR was established and treated with lentivirus modified miR-181a-5p. The allergic symptoms of mice were examined. The contents of Th17-related cytokines (interferon [IFN]-γ and interleukin [IL]-6), Treg-related cytokine (IL-10), and Treg-specific nuclear transcription factor (Foxp3) in nasal mucosa and lung tissues were determined. The proportion of Treg and Th17 cells was analyzed by flow cytometry. The level of ovalbumin-specific immunoglobulin E in the serum, and the contents of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid and IFN-γ, IL-6, and IL-10 in nasal lavage fluid were measured. The targeting relationship between miR-181a-5p and high mobility group box chromosomal protein 1 (HMGB1) was verified. HMGB1 and receptor for advanced glycation end products (RAGE) expression in RA were determined, and the interaction between HMGB1 and RAGE was detected. miR-181a-5p expression was reduced in AR mice. miR-181a-5p overexpression attenuated allergic behaviors, alleviated Treg/Th17 imbalance, and delayed asthma development. HMGB1 and RAGE were elevated in AR mice. miR-181a-5p targeted HMGB1, and HMGB1 bound to RAGE, while miR-181a-5p overexpression reduced the binding between them. Activating HMGB1/RAGE reversed the protective effect of miR-181a-5p overexpression on AR and induced the development of asthma. miR-181a-5p overexpression reduced the binding of HMGB1 and RAGE by inhibiting HMGB1, thus alleviating Treg/Th17 immune imbalance and blocking AR from developing into asthma.


Efficacy of pitfall trapping, Winkler and Berlese extraction methods for measuring ground-dwelling arthropods in moist-deciduous forests in the Western Ghats.
The present study provides data to decide on the most appropriate method for sampling of ground-dwelling arthropods measured in a moist-deciduous forest in the Western Ghats in South India. The abundance of ground-dwelling arthropods was compared among large numbers of samples obtained using pitfall trapping, Berlese and Winkler extraction methods. Highest abundance and frequency of most of the represented taxa indicated pitfall trapping as the ideal method for sampling of ground-dwelling arthropods. However, with possible bias towards surface-active taxa, pitfall-trapping data is inappropriate for quantitative studies, and Berlese extraction is the better alternative. Berlese extraction is the better method for quantitative measurements than the other two methods, whereas pitfall trapping would be appropriate for qualitative measurements. A comparison of the Berlese and Winkler extraction data shows that in a quantitative multigroup approach, Winkler extraction was inferior to Berlese extraction because the total number of arthropods caught was the lowest; and many of the taxa that were caught from an identical sample via Berlese extraction method were not caught. Significantly a greater frequency and higher abundance of arthropods belonging to Orthoptera, Blattaria, and Diptera occurred in pitfall-trapped samples and Psocoptera and Acariformes in Berlese-extracted samples than that were obtained in the other two methods, indicating that both methods are useful, one complementing the other, eliminating a chance for possible under-representation of taxa in quantitative studies.


Vertical smooth pursuit as a diagnostic marker of traumatic brain injury.
Neural deficits were measured via the eye tracking of vertical smooth pursuit (VSP) as markers of traumatic brain injury (TBI). The present study evaluated the ability of the eye tracking tests to differentiate between different levels of TBI severity and healthy controls. Ninety-two individuals divided into four groups (those with mild, moderate or severe TBI and healthy controls) participated in a computerized test of VSP eye movement using a remote eye tracker. The VSP eye tracking test was able to distinguish between severe and moderate levels of TBI but unable to detect differences in the performance of participants with mild TBI and healthy controls. The eye-tracking technology used to measure VSP eye movements is able to provide a timely and objective method of differentiating between individuals with moderate and severe levels of TBI.


Anomalous Scaling of Dynamical Large Deviations.
The typical values and fluctuations of time-integrated observables of nonequilibrium processes driven in steady states are known to be characterized by large deviation functions, generalizing the entropy and free energy to nonequilibrium systems. The definition of these functions involves a scaling limit, similar to the thermodynamic limit, in which the integration time τ appears linearly, unless the process considered has long-range correlations, in which case τ is generally replaced by τ^{ξ} with ξ≠1. Here, we show that such an anomalous power-law scaling in time of large deviations can also arise without long-range correlations in Markovian processes as simple as the Langevin equation. We describe the mechanism underlying this scaling using path integrals and discuss its physical consequences for more general processes.


Surface microdialysis measures local tissue metabolism after Ivor Lewis esophagectomy; an attempt to predict anastomotic defect.
Anastomotic defect (AD) after esophagectomy can lead to severe complications with need for surgical or endoscopic intervention. Early detection enables early treatment and can limit the consequences of the AD. As of today, there are limited methods to predict AD. In this study, we have used microdialysis (MD) to measure local metabolism at the intrathoracic anastomosis. Feasibility and possible diagnostic use were investigated. Sixty patients planned for Ivor Lewis esophagectomy were enrolled. After construction of the anastomosis, surface MD (S-MD) probes were attached to the outer surface of the esophageal remnant and the gastric conduit in close vicinity of the anastomosis and left in place for 7 postoperative days (PODs). Continuous sampling of local tissue concentrations of metabolic substances (glucose, lactate, and pyruvate) was performed postoperatively. Outcome, defined as AD or not according to Esophagectomy Complications Consensus Group definitions, was recorded at discharge or at first postoperative follow up. Difference in concentrations of metabolic substances was analyzed retrospectively between the two groups by means of artificial neural network technique. S-MD probes can be attached and removed from the gastric tube reconstruction without any adverse events. Deviating metabolite concentrations on POD 1 were associated with later development of AD. In subjects who developed AD, no difference in metabolic concentrations between the esophageal and the gastric probe was recorded. The technical failure rate of the MD probes/procedure was high. S-MD can be used in a clinical setting after Ivor Lewis esophagectomy. Deviation in local tissue metabolism on POD 1 seems to be associated with development of AD. Further development of MD probes and procedure is required to reduce technical failure.


Estimation of patient's inspiratory effort from the electrical activity of the diaphragm.
To calculate an index (termed Pmusc/Eadi index) relating the pressure generated by the respiratory muscles (Pmusc) to the electrical activity of the diaphragm (Eadi), during assisted mechanical ventilation and to assess if the Pmusc/Eadi index is affected by the type and level of ventilator assistance. The Pmusc/Eadi index was also used to measure the patient's inspiratory effort from Eadi without esophageal pressure. Crossover study. One general ICU. Ten patients undergoing assisted ventilation. Pressure support and neurally adjusted ventilator assist delivered, each, at three levels of ventilatory assistance. Airways flow and pressure, esophageal pressure, and Eadi were continuously recorded. Sixty tidal volumes for each ventilator settings were analyzed off-line, at three time points during inspiration. For each time point, Pmusc/Eadi index was calculated. Pmusc/Eadi index was also calculated from airway pressure drop during end-expiratory occlusions. Pmusc/Eadi index was very variable among patients, but within one patient it was not affected by type and level of ventilator assistance. Pmusc/Eadi index decreased during the inspiration. Pmusc/Eadi index obtained during an occlusion from airway pressure swing was tightly correlated with that derived from esophageal pressure during tidal ventilation and allowed to estimate pressure time product. Pmusc is tightly related to Eadi, by a proportionality coefficient that we termed Pmusc/Eadi index, stable within each patient under different conditions of ventilator assistance. The derivation of the Pmusc/Eadi index from Eadi and airway pressure during an expiratory occlusion enables a continuous estimate of patient's inspiratory effort.


PRPF mutations are associated with generalized defects in spliceosome formation and pre-mRNA splicing in patients with retinitis pigmentosa.
Proteins PRPF31, PRPF3 and PRPF8 (RP-PRPFs) are ubiquitously expressed components of the spliceosome, a macromolecular complex that processes nearly all pre-mRNAs. Although these spliceosomal proteins are conserved in eukaryotes and are essential for survival, heterozygous mutations in human RP-PRPF genes lead to retinitis pigmentosa, a hereditary disease restricted to the eye. Using cells from patients with 10 different mutations, we show that all clinically relevant RP-PRPF defects affect the stoichiometry of spliceosomal small nuclear RNAs (snRNAs), the protein composition of tri-small nuclear ribonucleoproteins and the kinetics of spliceosome assembly. These mutations cause inefficient splicing in vitro and affect constitutive splicing ex-vivo by impairing the removal of at least 9% of endogenously expressed introns. Alternative splicing choices are also affected when RP-PRPF defects are present. Furthermore, we show that the steady-state levels of snRNAs and processed pre-mRNAs are highest in the retina, indicating a particularly elevated splicing activity. Our results suggest a role for PRPFs defects in the etiology of PRPF-linked retinitis pigmentosa, which appears to be a truly systemic splicing disease. Although these mutations cause widespread and important splicing defects, they are likely tolerated by the majority of human tissues but are critical for retinal cell survival.


Agreement between visual and goniometric assessments of adductor and popliteal angles in infants.
Amiel-Tison method is a commonly used technique for assessing tone and neurological status of infants. There is a paucity of data on the reliability of visual assessment of angles, a component of this method. We compared the visual and the goniometric assessment of adductor and popliteal angles in infants with hypertonia and neurologically normal controls. A total of 16 infants with hypertonia and 15 normal infants underwent blinded assessment of the adductor and popliteal angles. The mean and standard deviation for the difference between visual and goniometric measurements were calculated for popliteal and adductor angles. The mean differences between visual and goniometric measurements for the popliteal angle were 4.94 (SD3.40) and 8.73 (SD6.10) degrees for the cases and controls respectively. Similarly, the values for adductor angle measurements were 8.94 (SD8.23) and 14.47 (SD8.47) degrees respectively. The deviation of visual assessment from goniometric measurement was found to be less for popliteal angle measurement as compared to adductor angle measurements. It was note-worthy that the difference was less for the measurements of children with spasticity.


Predicting Endocrine Disruption Using Conformal Prediction - A Prioritization Strategy to Identify Hazardous Chemicals with Confidence.
Receptor-mediated molecular initiating events (MIEs) and their relevance in endocrine activity (EA) have been highlighted in literature. More than 15 receptors have been associated with neurodevelopmental adversity and metabolic disruption. MIEs describe chemical interactions with defined biological outcomes, a relationship that could be described with quantitative structure-activity relationship (QSAR) models. QSAR uncertainty can be assessed using the conformal prediction (CP) framework, which provides similarity (i.e., nonconformity) scores relative to the defined classes per prediction. CP calibration can indirectly mitigate data imbalance during model development, and the nonconformity scores serve as intrinsic measures of chemical applicability domain assessment during screening. The focus of this work was to propose an in silico predictive strategy for EA. First, 23 QSAR models for MIEs associated with EA were developed using high-throughput data for 14 receptors. To handle the data imbalance, five protocols were compared, and CP provided the most balanced class definition. Second, the developed QSAR models were applied to a large data set (∼55,000 chemicals), comprising chemicals representative of potential risk for human exposure. Using CP, it was possible to assess the uncertainty of the screening results and identify model strengths and out of domain chemicals. Last, two clustering methods, t-distributed stochastic neighbor embedding and Tanimoto similarity, were used to identify compounds with potential EA using known endocrine disruptors as reference. The cluster overlap between methods produced 23 chemicals with suspected or demonstrated EA potential. The presented models could be utilized for first-tier screening and identification of compounds with potential biological activity across the studied MIEs.


Spawning season, recruitment and early life distribution of Anchoa tricolor (Spix and Agassiz, 1829) in a tropical bay in southeastern Brazil.
The pattern of the use of the Sepetiba Bay, a semi-closed 450 km(2) area in southeastern Brazil, by Anchoa tricolor was investigated to assess spawning period, recruitment and to detect spatial-temporal patterns of this major fishery resource. Fishes were sampled by seine nets, from spring, 1998 to winter, 1999 and by beach seine, from spring, 1998 to winter, 2000. Reproductive season, measured in terms of GSI, gonad development and appearance of recruits, indicate that reproduction occurs from August to March, when they reach the best condition. Recruitment peaks in winter/spring at sandy beaches where they stay until late summer, moving toward deeper bay areas during autumn. After that, they join adults and perform movements between the bay and the adjacent continental shelf to reproduce.


Sofosbuvir and ribavirin in adolescents 12-17 years old with hepatitis C virus genotype 2 or 3 infection.
Children with chronic hepatitis C virus infection have limited treatment options. We evaluated the all-oral combination of sofosbuvir and ribavirin in adolescents aged 12-17 with hepatitis C virus genotype 2 or 3 (ClinicalTrials.gov NCT02175758). Fifty-two patients received sofosbuvir 400 mg once daily and weight-based ribavirin twice daily for 12 (genotype 2) or 24 (genotype 3) weeks. The pharmacokinetics of sofosbuvir and its metabolite GS-331007 were evaluated by intensive plasma sampling at day 7 in the first 10 patients enrolled and by sparse sampling in all patients throughout treatment. The primary efficacy endpoint was the percentage of patients with a sustained virologic response 12 weeks after treatment (SVR12). The median age of patients was 15 years, and 75% had genotype 3. Eighty-three percent of patients were treatment-naive, and 73% were infected by vertical transmission. Forty percent were assessed as not having cirrhosis; the remainder did not have a cirrhosis determination. Overall, SVR12 was achieved by 98% of patients (51/52; 95% confidence interval, 90%-100%). SVR12 rates were 100% (13/13) for patients with genotype 2 and 97% (38/39) for those with genotype 3. The single patient who did not achieve SVR12 was lost to follow-up after achieving SVR4. The most commonly reported adverse events were nausea (27%) and headache (23%). When compared with the exposure in adults treated in phase 2 and 3 sofosbuvir studies, the area under the curve and maximum concentration for sofosbuvir and GS-331007 in adolescents were within predefined pharmacokinetic equivalence boundaries of 50%-200%. Sofosbuvir and ribavirin was safe and highly effective in adolescents with chronic hepatitis C virus genotype 2 or 3 infection. (Hepatology 2017;66:1102-1110).


A 28-day, randomized, double-blind, placebo-controlled, parallel group study of nebulized revefenacin in patients with chronic obstructive pulmonary disease.
Revefenacin is a once-daily long-acting muscarinic antagonist (LAMA) in clinical development for the treatment of patients with chronic obstructive pulmonary disease (COPD). In a dose-ranging study, nebulized once-daily revefenacin had a long duration of action in patients after 7 days' administration of doses up to 700 μg. In this multiple-dose study, the bronchodilation efficacy and adverse events profile were characterized in patients administered nebulized revefenacin once daily for 28 days. A total of 355 COPD patients (mean age 62 years, mean forced expiratory volume in 1 s [FEV1] 44% of predicted) were randomized in a double-blind, placebo-controlled parallel group study. Inhaled corticosteroids as well as short-acting bronchodilators were permitted. Once-daily treatments (44, 88, 175 or 350 μg revefenacin or matching placebo) were administered by a standard jet nebulizer, for 28 days. The primary endpoint was change from baseline in D28 trough FEV1, and secondary endpoints included weighted mean FEV1 over 0 to 24 h and rescue medication (albuterol) use. Safety evaluations included adverse events, laboratory assessments, electrocardiograms and 24-h Holter profiles. Revefenacin (88, 175 and 350 μg) significantly improved D28 trough FEV1 over placebo (187.4, 166.6 and 170.6 mL, respectively, all p < 0.001); 44 μg produced a sub-therapeutic response. At doses ≥88 μg, more than 80% of patients achieved at least a 100-mL increase from baseline FEV1 in the first 4 h post dose compared with 33% of placebo patients. For doses ≥88 μg, D28 24 h weighted mean differences from placebo for FEV1 were numerically similar to respective trough FEV1 values, indicating bronchodilation was sustained for 24 h post dose. Doses ≥88 μg reduced the average number of albuterol puffs/day by more than one puff/day. The 350 μg dose did not demonstrate additional efficacy over that observed with 175 μg revefenacin. Revefenacin was generally well tolerated, with minimal reports of systemic anti-cholinergic effects. These data suggest that 88 and 175 μg revefenacin are appropriate doses for use in longer-term safety and efficacy trials. Revefenacin offers the potential for the first once-daily LAMA for nebulization in patients with COPD who require or prefer a nebulized drug delivery option. ClinicalTrials.gov NCT02040792 . Registered January 16, 2014.


Atypical cases of filariasis from a non-endemic area.
Filariasis can present in many different ways and pose significant dilemma to the clinician. We report four atypical cases of filariasis which presented as abdominal mass, cervical lymph node enlargement, fever in pregnancy and nosocomial febrile illness respectively. All the four cases were treated successfully with oral antifilarial agents. It is essential to be aware of such atypical presentations of filariasis so that prompt therapy can be initiated.


NAD(+)/NADH and skeletal muscle mitochondrial adaptations to exercise.
The pyridine nucleotides, NAD(+) and NADH, are coenzymes that provide oxidoreductive power for the generation of ATP by mitochondria. In skeletal muscle, exercise perturbs the levels of NAD(+), NADH, and consequently, the NAD(+)/NADH ratio, and initial research in this area focused on the contribution of redox control to ATP production. More recently, numerous signaling pathways that are sensitive to perturbations in NAD(+)(H) have come to the fore, as has an appreciation for the potential importance of compartmentation of NAD(+)(H) metabolism and its subsequent effects on various signaling pathways. These pathways, which include the sirtuin (SIRT) proteins SIRT1 and SIRT3, the poly(ADP-ribose) polymerase (PARP) proteins PARP1 and PARP2, and COOH-terminal binding protein (CtBP), are of particular interest because they potentially link changes in cellular redox state to both immediate, metabolic-related changes and transcriptional adaptations to exercise. In this review, we discuss what is known, and not known, about the contribution of NAD(+)(H) metabolism and these aforementioned proteins to mitochondrial adaptations to acute and chronic endurance exercise.


Erythropoietin treatment for methanol optic neuropathy.
To present the effect of erythropoietin for the treatment of methanol optic neuropathy. Two patients with methanol optic neuropathy were treated with 10,000 IU of intravenous erythropoietin twice a day for 3 days, 500 mg of methylprednisolone twice a day for 5 days (followed by 2 weeks of oral prednisolone [1 mg/kg per day]), and daily doses of vitamin B12, vitamin B6, and folic acid for 1 month. At presentation, the patients had no perception of light in both eyes, associated with mildly swollen optic discs. Both responded dramatically to the treatment regimen. In the first patient, visual acuity improved to 20/20 in both eyes within 3 days, whereas in the second patient, visual acuity returned to counting fingers at 6 feet, right eye, and 20/30, left eye, within 3 weeks. Intravenous erythropoietin may be an effective adjuvant when combined with current treatment for patients with methanol optic neuropathy.


Allergic Sensitization, Rhinitis and Tobacco Smoke Exposure in US Adults.
Tobacco exposure has been linked with sinonasal pathology and may be associated with allergic sensitization. This study evaluates the association between exposure to active smoking or secondhand smoke (SHS) and the prevalence of rhinitis and allergic sensitization in the US adult population. Cross-sectional study in 4,339 adults aged 20-85 in the National Health and Nutrition Examination Survey, 2005-2006. Never smoking was defined as reported lifetime smoking less than 100 cigarettes and serum cotinine levels <10ng/ml, while active smoking was defined as self-reported smoking or serum cotinine concentrations > 10 ng/mL. Self-reported rhinitis was based on symptoms during the past 12 months, and allergen sensitization was defined as a positive response to any of the 19 specific IgE antigens tested. Almost half of the population (43%) had detectable levels of IgE specific to at least one inhaled allergen and 32% reported a history of rhinitis. After multivariate adjustment, there was a statistically significant association between the highest serum cotinine tertile and rhinitis in active smokers (OR 1.42; 95%CI 1.00-2.00). The association between active smoking and rhinitis was stronger in individuals without allergic sensitization (OR 2.47; 95%CI 1.44-4.23). There was a statistically significant association between increasing cotinine tertiles and decreased odds of inhaled allergen sensitization (p-trend <.01). Tobacco smoke exposure was associated with increased prevalence of rhinitis symptoms, but not with allergic sensitization. The results indicate that the relationship between tobacco smoke exposure and sinonasal pathology in adults may be independent of allergic sensitization.


Anomalous behavior of nearly-entire visible band manipulated with degenerated image dipole array.
Recently, the control of anomalous light bending via flat gradient-phase metasurfaces has enabled many unprecedented applications. However, either low manipulation efficiency or challenging difficulties in fabrication hinders their practical applications, in particular in the visible range. Therefore, a concept of degenerated image dipole array is reported to realize anomalous light bending with high efficiency. A continuous phase delay varying rather than a discrete one, along with an in-plane wave vector is utilized to achieve anomalous light bending, by controlling and manipulating the mutual coupling between dipole array and the dipole array of its image. The anomalous light bending covers almost the entire visible range with broad incident angles, accompanied with preserved well-defined planar wavefront. In addition, this design is feasible to be fabricated with recent nanofabrication techniques due to its planarized surface configuration. The concept of imperfect image dipole array degenerated from ideal metamaterial absorbers surprisingly empowers significant enhancement in light manipulation efficiency for visible light in a distinct fashion.


Thioesterases: a new perspective based on their primary and tertiary structures.
Thioesterases (TEs) are classified into EC 220.127.116.11 through EC 184.108.40.206 based on their activities on different substrates, with many remaining unclassified (EC 3.1.2.-). Analysis of primary and tertiary structures of known TEs casts a new light on this enzyme group. We used strong primary sequence conservation based on experimentally proved proteins as the main criterion, followed by verification with tertiary structure superpositions, mechanisms, and catalytic residue positions, to accurately define TE families. At present, TEs fall into 23 families almost completely unrelated to each other by primary structure. It is assumed that all members of the same family have essentially the same tertiary structure; however, TEs in different families can have markedly different folds and mechanisms. Conversely, the latter sometimes have very similar tertiary structures and catalytic mechanisms despite being only slightly or not at all related by primary structure, indicating that they have common distant ancestors and can be grouped into clans. At present, four clans encompass 12 TE families. The new constantly updated ThYme (Thioester-active enzYmes) database contains TE primary and tertiary structures, classified into families and clans that are different from those currently found in the literature or in other databases. We review all types of TEs, including those cleaving CoA, ACP, glutathione, and other protein molecules, and we discuss their structures, functions, and mechanisms.


[Various biochemical indicators of the cerebrospinal fluid in acute carbophos poisoning].
The study was performed to evaluate the levels of some enzymes (AST, ALT, HGTP, LDG) and free amino acids (aspartic, glutaminic, glycine, isoleucine, leucine) in liquor of 37 subjects who got poisoned with a Malathion insecticide. Their condition was diagnosed as moderate and severe. The liquor was obtained on poisoning day 1, 3, 10, 14 and 21. The liquor levels of enhancement mediatory amino acids, aspartic and glutaminic, rise early in poisoning, while concentration of inhibition mediator glycine tends to decline. Progress of intoxication brings about a rise in LDG and GGTP activity attributed to a membranotoxic effect of the insecticide.


Studies of interaction of a low-molecular-weight heparinoid (Org 10172) with cloxacillin and ticarcillin in healthy male volunteers.
Pharmacokinetic and pharmacodynamic interactions between Org 10172 (intravenous bolus injection of 3,250 anti-Xa units), which is a low-molecular-weight heparinoid, cloxacillin (500 mg orally four times daily for 3 days), and ticarcillin (4,000 mg intravenously four times daily for 2 days) were evaluated in two separate studies with healthy male volunteers (n = 18). Both cloxacillin and ticarcillin caused a significant increase in elimination half-life of anti-Xa activity, i.e., from 31 +/- 10 to 54 +/- 23 h and from 27 +/- 6 to 42 +/- 13 h, respectively (P less than 0.05). Ticarcillin decreased clearance (11%) and increased apparent volume of distribution (35%) (P less than 0.05), while for cloxacillin, these differences did not reach statistical significance. These changes in disposition of Org 10172 by the penicillins were not accompanied by important pharmacodynamic changes as evaluated by coagulation tests, platelet aggregation, and bleeding time. Cloxacillin appeared to influence blood coagulation (prolongation of the activated partial thromboplastin time and shortening of thrombin time; P less than 0.05) and facilitated thrombin-induced platelet aggregation, which coincided with a shorter bleeding time during the combined treatment in comparison with the time during treatment with Org 10172 alone (P less than 0.05). In conclusion, the disposition of Org 10172 was slightly changed by cloxacillin and ticarcillin, and, unexpectedly, cloxacillin appeared to have mild procoagulant effects.


Parent-of-origin effects on handedness and schizophrenia susceptibility on chromosome 2p12-q11.
Schizophrenia and non-right-handedness are moderately associated, and both traits are often accompanied by abnormalities of asymmetrical brain morphology or function. We have found linkage previously of chromosome 2p12-q11 to a quantitative measure of handedness, and we have also found linkage of schizophrenia/schizoaffective disorder to this same chromosomal region in a separate study. Now, we have found that in one of our samples (191 reading-disabled sibling pairs), the relative hand skill of siblings was correlated more strongly with paternal than maternal relative hand skill. This led us to re-analyse 2p12-q11 under parent-of-origin linkage models. We found linkage of relative hand skill in the RD siblings to 2p12-q11 with P=0.0000037 for paternal identity-by-descent sharing, whereas the maternally inherited locus was not linked to the trait (P>0.2). Similarly, in affected-sib-pair analysis of our schizophrenia dataset (241 sibling pairs), we found linkage to schizophrenia for paternal sharing with LOD=4.72, P=0.0000016, within 3 cM of the peak linkage to relative hand skill. Maternal linkage across the region was weak or non-significant. These similar paternal-specific linkages suggest that the causative genetic effects on 2p12-q11 are related. The linkages may be due to a single maternally imprinted influence on lateralized brain development that contains common functional polymorphisms.


Effect of direct loading of phytoestrogens into the calcium phosphate scaffold on osteoporotic bone tissue regeneration.
3D porous calcium deficient hydroxyapatite (CDHA) scaffolds with phytoestrogens were fabricated for osteoporotic bone tissue regeneration through a combination of 3D printing techniques and cement chemistry as a room temperature process. Quercetin, one of the major phytoestrogens isolated from onions and apples, was directly incorporated into CDHA for local administration in place of bisphosphonates (BPs) (which are recognized as standard treatment in osteoporosis), to avoid drug side effects. The CDHA scaffolds with quercetin (QC-CDHA) showed favorable mechanical properties (compressive strength < 21 MPa) as well as pore morphology. Quercetin was steadily released with the biodegradation of CDHA scaffolds in vitro without any initial burst. The QC-CDHA scaffolds greatly influenced both osteoblast and osteoclast cell activities. The QC-CDHA scaffolds significantly increased pre-osteoblast cell (MC3T3-E1) proliferation, differentiation, and mineralization, whereas osteoclast cell (RANK treated RAW 264.7) proliferation and differentiation were dramatically suppressed. The influence of quercetin on bone tissue regeneration was superior to alendronate, which is one of the most commonly administered BPs. All results indicated that quercetin in CDHA scaffolds plays an important role in both enhancing bone formation and suppressing bone resorption. Consequently, this technology promises great potential in osteoporotic bone tissue regeneration.


Assessment of genetically modified cotton GHB811 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-ES-2018-154).
Cotton GHB811 was developed to confer tolerance to glyphosate and HPPD inhibitor herbicides. The molecular characterisation data and bioinformatic analyses do not identify issues requiring food/feed safety assessment. None of the identified differences in the agronomic/phenotypic and compositional characteristics tested between cotton GHB811 and its conventional counterpart needs further assessment, except for % lint, lint length and dihydrosterculic acid, which do not raise nutritional and safety concerns. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the 2mEPSPS and HPPD W336 proteins as expressed in cotton GHB811 and finds no evidence that the genetic modification would change the overall allergenicity of cotton GHB811. In the context of this application, the consumption of food and feed from cotton GHB811 does not represent a nutritional concern in humans and animals. The GMO Panel concludes that cotton GHB811 is as safe as the conventional counterpart and non-GM cotton reference varieties tested, and no post-market monitoring of food/feed is considered necessary. In the case of accidental release of viable cotton GHB811 seeds into the environment, this would not raise environmental safety concerns. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of cotton GHB811. The GMO Panel concludes that cotton GHB811 is as safe as its conventional counterpart and the tested non-GM cotton reference varieties with respect to potential effects on human and animal health and the environment.


A Ready-to-Use Single- and Duplex-TaqMan-qPCR Assay to Detect and Quantify the Biocontrol Agents Trichoderma asperellum and Trichoderma gamsii.
Trichoderma asperellum strain icc012 and Trichoderma gamsii strain icc080, the microbial active ingredients of RemedierTM (ISAGRO, Novara, Italy), are biocontrol agents (BCAs) employable for crop protection against a wide range of fungal pathogens, including soil-borne pathogens and fungi involved in grapevine trunk disease. In this study, single and duplex real-time quantitative PCR (qPCR) methods to detect and quantify T. asperellum and T. gamsii were developed. Primers/probe sets were designed on the T. asperellum and T. gamsii rpb2 genes and tested for specificity on a panel of microorganisms commonly associated with grape wood and soil. No differences were observed comparing single- and duplex-qPCR assays on different BCAs, 1 pg of target DNA was detected approximately at Cq = 34. R2-values and the efficiency were always equal to 0.99 and >80%, respectively. The detection limit of the duplex-qPCR assay on artificially inoculated samples was 2 × 103 and 4 × 104 conidia g-1 of grape wood tissue and soil, respectively. The methods will be useful to better schedule BCA application in the field and in grapevine nurseries, as well as for investigating the dynamic of BCA populations.


Characterization of the microbial community colonizing the anal and vulvar pores of helminths from the hindgut of zebras.
Scanning and transmission electron microscopy were used to examine the adherence and in situ morphology of the microbial community colonizing the anal and vulvar pores of the subfamily Cyathostominae (Nematoda: Strongylidae) from the colon of Burchell's zebra (Equus burchelli antiquorum). Two different morphological types of asporogenous rod were prominent in the microbial community. One was a thin, septate, filamentous organism (0.4 to 0.5 micron by 2 to 3 microns) with blunt ends, which was more prominent at the site of attachment. The other was a larger (1.8 to 2.4 microns by 5 to 10 microns) multicellular rod with round ends in the form of a trichome. Spiral- and vibrio-shaped bacteria were also present in the thin sections. The septate filaments were shown to contain a cell spacer similar to those described in Methanospirillum hungatei. Attachment to the cuticle was by means of an amorphous electron-dense material with fibrillar appearance and not by specialized holdfast segments. Ten isolates were obtained from a habitat-simulating medium on which a homogenate from the posterior region was plated. Antibodies were raised to whole cells of five rod-shaped isolates in rabbits and fluorescein isothiocyanate labeled. Positive bright-yellow fluorescence was obtained with one of the clostridial isolates. The results are discussed with reference to other bacteria with similar morphology, the nature of this unique interrelationship between the microbial community and its parasitic host inside the equine hindgut, and the possibility of biological control of parasitic helminths.


Perspectives on paediatric sleep-disordered breathing in the UK: a qualitative study.
There is limited understanding of treatment pathways for paediatric sleep-disordered breathing. This study explored current UK pathways and what is important to well-being for parents and children. The study comprised in-depth qualitative interviews (n = 22) with parents of children (2-9 years) with symptoms of sleep-disordered breathing referred to a regional ENT clinic (n = 11), general practitioners who might refer these children to ENT (n = 5) and hospital doctors involved in treating these children (n = 6). Interviews were audio recorded, transcribed verbatim, anonymised and analysed thematically. General practitioners rarely identify seeing children with sleep-disordered breathing; conversely hospital doctors identify unsuspected issues. Parents are worried their child will stop breathing, but routes to referral and diagnosis are not straightforward. Modern technology can aid investigation and diagnosis. Patient weight is an issue for general practitioners and hospital doctors. Adenotonsillectomy is the treatment of choice, and information on paediatric sleep-disordered breathing is needed. Guidelines for the management of paediatric sleep-disordered breathing are needed.


Effects of docosahexaenoic acid on serum lipoproteins in patients with combined hyperlipidemia: a randomized, double-blind, placebo-controlled trial.
The objective of this study was to evaluate the effects of daily dietary supplementation with 1.25 g or 2.5 g of docosahexaenoic (DHA), in the absence of eicosapentaenoic acid (EPA), on serum lipids and lipoproteins in persons with combined hyperlipidemia (CHL) [serum low-density lipoprotein cholesterol (LDL-C) 130 to 220 mg/dL and triglycerides 150 to 400 mg/dL]. After a 6-week dietary stabilization period, subjects entered a 4-week single-blind placebo (vegetable oil) run-in phase. Those with adequate compliance during the the run-in were randomized into one of three parallel groups (placebo, 1.25, or 2.5 g/day DHA) for 6 weeks of treatment. Supplements were administered in a triglyceride form contained in gelatin capsules. Primary outcome measurements were plasma phospholipid DHA content, serum triglycerides, high-density lipoprotein cholesterol (HDL-C). LDL-C and non-HDL-C. The DHA content of plasma phospholipids increased dramatically (2 to 3 fold) in a dose-dependent manner. Significant (p < 0.05) changes were observed in serum triglycerides (17 to 21% reduction) and HDL-C (6% increase) which were of similar magnitude in both DHA groups. Non-HDL-C [+1.6 (NS) and +5.7% (p < 0.04)] and LDL-C [+9.3% (NS) and +13.6% (p < 0.001)] increased in the DHA treatment groups. All lipid effects reached an apparent steady state within the first 3 weeks of treatment. Dietary DHA, in the absence of EPA, can affect lipoprotein cholesterol and triglyceride levels in patients with combined hyperlipidemia. The desirable triglyceride and HDL-C changes were present at a dose which did not significantly increased non-HDL-C or LDL-C. These preliminary findings suggest that dietary supplementation with 1.25 g DHA/day, provided in a triglyceride form, may be an effective tool to aid in the management of hypertriglyceridemia.


Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age.
Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10-12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss.


Use of stable isotope-labelled cells to identify active grazers of picocyanobacteria in ocean surface waters.
Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open-ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA-SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA-SIP is a useful method to identity specific predators for picocyanobacteria in situ, and that the method could possibly be used to identify other bacterial predators important in the microbial food-web.


Fracture Strength of Endodontically Treated Teeth with Different Access Cavity Designs.
The purpose of this study was to compare in vitro the fracture strength of root-filled and restored teeth with traditional endodontic cavity (TEC), conservative endodontic cavity (CEC), or ultraconservative "ninja" endodontic cavity (NEC) access. Extracted human intact maxillary and mandibular premolars and molars were selected and assigned to control (intact teeth), TEC, CEC, or NEC groups (n = 10/group/type). Teeth in the TEC group were prepared following the principles of traditional endodontic cavities. Minimal CECs and NECs were plotted on cone-beam computed tomographic images. Then, teeth were endodontically treated and restored. The 160 specimens were then loaded to fracture in a mechanical material testing machine (LR30 K; Lloyd Instruments Ltd, Fareham, UK). The maximum load at fracture and fracture pattern (restorable or unrestorable) were recorded. Fracture loads were compared statistically, and the data were examined with analysis of variance and the Student-Newman-Keuls test for multiple comparisons. The mean load at fracture for TEC was significantly lower than the one for the CEC, NEC, and control groups for all types of teeth (P < .05), whereas no difference was observed among CEC, NEC, and intact teeth (P > .05). Unrestorable fractures were significantly more frequent in the TEC, CEC, and NEC groups than in the control group in each tooth type (P < .05). Teeth with TEC access showed lower fracture strength than the ones prepared with CEC or NEC. Ultraconservative "ninja" endodontic cavity access did not increase the fracture strength of teeth compared with the ones prepared with CEC. Intact teeth showed more restorable fractures than all the prepared ones.


Immune cell phenotype and functional defects in Netherton syndrome.
Netherton syndrome (NS) is a rare life-threatening syndrome caused by SPINK5 mutations leading to a skin barrier defect and a severe atopic diathesis. NS patients are prone to bacterial infections, but the understanding of the underlying immune deficiency is incomplete. We analyzed blood lymphocyte phenotypes and function in relation to clinical infections in 11 Finnish NS patients, aged 3 to 17 years, and healthy age-matched controls. The proportion of B cells (CD19+) and naïve B cells (CD27-, IgD+) were high while memory B cells (CD27+) and switched memory B cells (CD27+IgM-IgD-), crucial for the secondary response to pathogens, was below or in the lowest quartile of the reference values in 8/11 (73%) and 9/11 (82%) patients, respectively. The proportion of activated non-differentiated B cells (CD21low, CD38low) was below or in the lowest quartile of the reference values in 10/11 (91%) patients. Despite normal T cell counts, the proportion of naïve CD4+ T cells was reduced significantly and the proportion of CD8+ T central memory significantly elevated. An increased proportion of CD57+ CD8+ T cells indicated increased differentiation potential of the T cells. The proportion of cytotoxic NK cells was elevated in NS patients in phenotypic analysis based on CD56DIM, CD16+ and CD27- NK cells but in functional analysis, decreased expression of CD107a/b indicated impaired cytotoxicity. The T and NK cell phenotype seen in NS patients also significantly differed from that of age-matched atopic dermatitis (AD) patients, indicating a distinctive profile in NS. The frequency of skin infections correlated with the proportion of CD62L+ T cells, naïve CD4+ and CD27+ CD8+ T cells and with activated B cells. Clinically beneficial intravenous immunoglobulin therapy (IVIG) increased naïve T cells and terminal differentiated effector memory CD8+ cells and decreased the proportion of activated B cells and plasmablasts in three patients studied. This study shows novel quantitative and functional aberrations in several lymphocyte subpopulations, which correlate with the frequency of infections in patients with Netherton syndrome. IVIG therapy normalized some dysbalancies and was clinically beneficial.


Financial burden, distress, and toxicity in cardiovascular disease.
Cardiovascular disease (CVD) is a major source of financial burden and distress, which has 3 main domains: (1) psychological distress; (2) cost-related care non-adherence or medical care deferral, and (3) tradeoffs with basic non-medical needs. We propose 4 ways to reduce financial distress in CVD: (1) policymakers can expand insurance coverage and curtail underinsurance; (2) health systems can limit expenditure on low-benefit, high-cost treatments while developing services for high-risk individuals; (3) physicians can engage in shared-decision-making for high-cost interventions, and (4) community-based initiatives can support patients with system navigation and financial coping. Avenues for research include (1) analysis of how healthcare policies affect financial burden; (2) comparative effectiveness studies examining high and low-cost strategies for CVD management; and (3) studying interventions to reduce financial burden, financial coaching, and community health worker integration.