Insights into plant metabolic networks from steady-state metabolic flux analysis.
Steady-state metabolic flux analysis (MFA) is an experimental approach that allows the measurement of multiple fluxes in the core network of primary carbon metabolism. It is based on isotopic labelling experiments, and although well established in the analysis of micro-organisms, and some mammalian systems, the extension of the method to plant cells has been challenging because of the extensive subcellular compartmentation of the metabolic network. Despite this difficulty there has been substantial progress in developing robust protocols for the analysis of heterotrophic plant metabolism by steady-state MFA, and flux maps have now been published that reflect the metabolic phenotypes of excised root tips, developing embryos and cotyledons, hairy root cultures, and cell suspensions under a variety of physiological conditions. There has been a steady improvement in the quality, extent and statistical reliability of these analyses, and new information is emerging on the performance of the plant metabolic network and the contributions of specific pathways. The principles of steady-state MFA are outlined here, the current status of the technique for characterizing primary metabolism in plants is described, and its complementary relationship to metabolomic analysis based on metabolite composition is discussed. It is argued that there is still considerable scope for further development of the technique, either by implementing refinements that have already been adopted in microbial investigations, or by developing techniques that are particularly relevant to the problems posed by plant tissues. If successful, these developments will lead to a more powerful phenotyping tool that will be faster to implement, and which will provide the basis for fully predictive mechanistic models of the network. This in turn will lead to an improved understanding of the regulation of plant metabolic networks, as well as a firm foundation for rational metabolic engineering.


Multilocus sequence typing of invasive group B Streptococcus in central area of Taiwan.
Group B Streptococcus (GBS) (Streptococcus agalactiae) is an important pathogen in neonates, pregnant women, and adults with underlying disease. Fifty clinical isolates were collected during the period 2001-2004 and analyzed by multilocus sequence typing and capsular serotyping. The six major sequence types (STs) identified by multilocus sequence typing were ST1, ST12, ST19, ST17, ST23, and ST10. Five major clonal complexes (CCs) and one single ST (ST61) from 11 different STs were found. CC1 (n=14) was the most common one, followed by CC12 (n=13), CC19 (n=9), CC17 (n=7), and CC23 (n=6). The most common serotypes were serotype III, followed by Ib, V, Ia, and IV. The most invasive strains in adults belonged to ST1 (CC1) and serotype V, and those in neonates belonged to ST17 (CC17) and serotype III. In addition, ST19 was distributed in adults and neonates. These results are similar to those of previous reports, but some geographic differences were found, suggesting that limited clonal lineages play important roles in GBS-associated diseases worldwide. Continued tracking of GBS in the population through clinical isolates is important for epidemiologic investigations and vaccine development.


Population genetic analysis of an Eastern U.S. wheat powdery mildew population reveals geographic subdivision and recent common ancestry with U.K. and Israeli populations.
The structure of the U.S. wheat powdery mildew population (Blumeria graminis f. sp. tritici) has not been previously investigated, and the global evolutionary history of B. graminis f. sp. tritici is largely unknown. After gathering 141 single-ascosporic B. graminis f. sp. tritici isolates from 10 eastern U.S. locations, 34 isolates from the United Kingdom, and 28 isolates from Israel, we analyzed pathogen population structure using presumptively neutral markers. DNA was extracted from conidia, primers for 12 "housekeeping" genes were designed, and amplicons were examined for polymorphism. Four genes were found to contain a total of 12 single-nucleotide polymorphisms in the U.S. population and were also analyzed in the U.K. and Israeli populations. In total, 25 haplotypes were inferred from the four concatenated genes, with 2 haplotypes comprising over 70% of the U.S. population. Using Hudson's tests and analysis of molecular variance, we found the wheat mildew isolates subdivided into four groups corresponding to distinct regions: the mid-Atlantic United States, the southern United States, the United Kingdom, and Israel. Genotypic diversity was greatest in samples from the United Kingdom, Israel, Virginia, and Kingston, NC. Using rarefaction, a procedure that compensates for differing sample sizes when estimating population richness and diversity, we found that cooler locations with greater conduciveness to regular powdery mildew epidemics had the greatest haplotype richness. Our results suggest that the eastern U.S. B. graminis f. sp. tritici population is young, descended recently from Old World populations with isolation and genetic drift, and is currently subdivided into northern and southern subpopulations.


Acceptability of an economic support component to reduce early pregnancy and school dropout in Zambia: a qualitative case study.
Background: Cash Transfer (CT) schemes have become popular poverty reducing measures in many low and middle-income countries. Research indicates that when provided to girls in resource poor settings, cash transfers can increase education and postpone marriage and pregnancy. However, a few studies indicate that they can also have negative effects which can affect their acceptability, such as generating intra-community tensions.Objective: Conceptually informed by Rogers' diffusion of innovation theory, this paper explores factors affecting the acceptability of economic support in a randomized controlled trial in rural Monze and Pemba Districts of Southern Province in Zambia.Methods: Qualitative data were collected through five focus group discussions and six in-depth, semi-structured interviews and analysed using thematic analysis. This study was done in the combined arm of a trial where girls received both economic support and participated in youth clubs offering sexuality and life-skills education.Results: In the study communities, acceptability was encouraging by the belief that economic support provided benefits beyond beneficiaries and that it improved access to education, and reduced teen pregnancies, marriages and school drop-out. However, provision of economic support only to selected girls and their parents and fear among some that the support was linked to satanic practices negatively affected acceptability. These fears were mitigated through community sensitisations.Conclusion: The study demonstrated that relative advantage, observability, simplicity and compatibility were key factors in influencing acceptability of the economic support. However, to enhance acceptability of cash transfer schemes aimed at addressing early marriage and pregnancy, it is important to explore socio-cultural factors that create suspicions and negative perceptions and to provide schemes that are perceived as relatively better than available similar schemes, understood, compatible and viable beyond the immediate beneficiary.


Surgical treatment of severe hidradenitis suppurativa of the axilla: thoracodorsal artery perforator (TDAP) flap versus split skin graft.
Hidradenitis suppurativa (HS) is a chronic, inflammatory disease affecting the apocrine glands of the axillary, groin and mammary regions with significant physical and psychosocial sequelae. Surgical excision of the affected tissue is the gold standard treatment. Severe axillary HS is associated with high rates of recurrence and requires extensive surgical resection with challenging reconstruction associated with risk of post-operative complications. The most effective method for reconstruction of the axilla after excision of HS is yet to be identified. We present a prospective observational study comparing thoraco-dorsal artery perforator (TDAP) flap and split-skin graft (SSG). Over 4 years, we enrolled 27 consecutive patients with Hurley's Stage III HS of the axilla who underwent surgical excision with reconstruction using either SSG (n=12) or TDAP flap reconstruction (n=15). We evaluated and compared intraoperative and post-operative data, quality of life (dermatology life quality index questionnaire) and pain/discomfort (visual analogue scale) before and after surgery. Patients who underwent TDAP flap reconstruction had significantly faster recovery, fewer complications and fewer overall number of procedures than those who underwent SSG reconstruction. All patients reported an improved quality of life (QOL) after their operation and the TDAP group showed significantly more improvement than the SSG group. All patients reported a reduction in pain/discomfort but there was no significant difference between groups. TDAP flap and SSG both improve QOL for patients with severe axillary HS. The TDAP flap showed greater benefits in terms of QOL, recovery, rate of complications and number of overall procedures.


Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis.
Staphylococcus aureus is one of the most important human pathogens and methicillin-resistant variants (MRSAs) are a major cause of hospital and community-acquired infection. We aimed to map the geographic distribution of the dominant clones that cause invasive infections in Europe. In each country, staphylococcal reference laboratories secured the participation of a sufficient number of hospital laboratories to achieve national geo-demographic representation. Participating laboratories collected successive methicillin-susceptible (MSSA) and MRSA isolates from patients with invasive S. aureus infection using an agreed protocol. All isolates were sent to the respective national reference laboratories and characterised by quality-controlled sequence typing of the variable region of the staphylococcal spa gene (spa typing), and data were uploaded to a central database. Relevant genetic and phenotypic information was assembled for interactive interrogation by a purpose-built Web-based mapping application. Between September 2006 and February 2007, 357 laboratories serving 450 hospitals in 26 countries collected 2,890 MSSA and MRSA isolates from patients with invasive S. aureus infection. A wide geographical distribution of spa types was found with some prevalent in all European countries. MSSA were more diverse than MRSA. Genetic diversity of MRSA differed considerably between countries with dominant MRSA spa types forming distinctive geographical clusters. We provide evidence that a network approach consisting of decentralised typing and visualisation of aggregated data using an interactive mapping tool can provide important information on the dynamics of MRSA populations such as early signalling of emerging strains, cross border spread, and importation by travel. In contrast to MSSA, MRSA spa types have a predominantly regional distribution in Europe. This finding is indicative of the selection and spread of a limited number of clones within health care networks, suggesting that control efforts aimed at interrupting the spread within and between health care institutions may not only be feasible but ultimately successful and should therefore be strongly encouraged.


Alcohol and endometrial cancer risk in the NIH-AARP diet and health study.
Previous investigations have provided conflicting results regarding whether alcohol consumption affects endometrial cancer risk, although in many of these studies the highest category of alcohol intake examined was limited. Further, most were unable to resolve how alcohol associations are affected by beverage type, the presence of other endometrial cancer risk factors, or tumor characteristics. To address these issues, we prospectively evaluated the association between alcohol intake and incident endometrial cancer (n = 1,491) in a cohort of 114,414 US women enrolled in the NIH-AARP Diet and Health Study. We calculated relative risks (RR) and 95% confidence intervals (CI) using Cox proportional hazards regression. After adjustment for age, body mass index (BMI), smoking and other potential confounders, the multivariable RRs (and 95% CIs) compared with nondrinkers were 0.97 (0.87-1.09) for >0-<12 g of alcohol/day, 1.06 (0.87-1.31) for 12-<24 g/day and 0.93 (0.71-1.20) for ≥ 24 g/day (p trend = 0.90). There was, however, some suggestion of higher risks associated with alcohol consumption among lean women (BMI, <25) and users of menopausal hormone therapy, with significant interactions with both parameters (respective interaction p-values of 0.002 and 0.005). The relationship was also enhanced, albeit nonsignificantly so, for low grade cancers. Our results do not support that alcohol is a strong contributor to endometrial cancer risk, but slight risk increases may prevail among some users or for selected tumor characteristics.


A phase I/II trial of reduced intensity allogeneic hematopoietic cell transplant for hematologic malignancies using cladribine, thiotepa and rabbit antithymocyte globulin.
We conducted a phase I/II trial to assess the efficacy of cladribine, thiotepa and antithymocyte globulin as a reduced-intensity conditioning regimen for refractory or high-risk hematologic malignancy. The preparative regimen consisted of cladribine 5 mg/m(2)/day for 5 days, thiotepa 200 mg/m(2)/day for 3 days and ATG 3 mg/kg/day (day - 5) followed by allogeneic peripheral blood stem cell transplant. Twelve patients were transplanted from a human leukocyte antigen (HLA) matched family member. Two patients were treated at dose level I but both experienced grade IV dose limiting toxicities, and therefore the thiotepa dose was reduced to 133 mg/m(2)/day (dose level II). Only two of the next six patients experienced dose limiting toxicities. Median age was 46 years. At dose level II, the median time to neutrophil and platelet engraftment was 17 and 20 days, respectively. The incidence of acute and chronic graft-versus-host disease (GVHD) was 40% and 30%, respectively. Day + 100 non-relapse mortality was 0% and at 1 year was 20%. Median overall survival (OS) was 42 months and 2-year OS was 70%. Median progression-free survival (PFS) was 11 months and 2-year PFS was 40%. We conclude that the reduced-intensity conditioning regimen of cladribine, thiotepa and ATG achieved excellent donor chimerism with acceptable toxicity.


Interstitial pressure of subcutaneous nodules in melanoma and lymphoma patients: changes during treatment.
Interstitial pressure (IP) is a physiological variable that may have its greatest influence on the transport of high-molecular-weight therapeutic agents. IP in tumor nodules was measured in patients with metastatic melanoma or non-Hodgkin's lymphoma to determine the influence of this physiological variable on treatment outcome. The wick-in-needle technique was used to measure IP at time points before and after treatment with a variety of immunotherapy and chemotherapy regimens. Selected patients had IP measurements during chemotherapy or immunotherapy infusions. Ultrasound or computed tomography was used to evaluate the size of the studied lesions and their relationship to normal structures. The mean baseline IP in melanoma nodules (n = 22) and lymphoma nodules (n = 7) was 29.8 and 4.7 mm Hg, respectively (P = 0.013 for the difference between tumor types). In a subset of melanoma nodules for which IP had been measured before and after treatment, the IP increased significantly over time for nonresponding melanoma lesions from a baseline of 24.4 to 53.9 mm Hg after treatment (P = 0.005) and decreased in melanoma lesions that responded to treatment where the mean baseline and post-treatment IPs were 12.2 and 0 mm Hg, respectively (P = 0.001 for the difference in IP profiles between responding and nonresponding lesions). Six of seven lymphoma nodules responded completely to chemotherapy or radiation. The single nodule that did not respond had a baseline IP of 1 mm Hg that increased to 30 mm Hg after treatment. Tumor IP differs significantly between melanoma and non-Hodgkin's lymphoma. The changes in IP over time differ significantly between responding and nonresponding melanoma lesions. IP that increases during treatment appears to be associated with tumor progression in these tumor types.


Generation of anti-inflammatory adenosine by leukocytes is regulated by TGF-β.
Levels of anti-inflammatory extracellular adenosine are controlled by the sequential action of the ectonucleotidases CD39 and CD73, whose expression in CD4(+) T cells has been associated with natural regulatory T cells (nTregs). We here show that CD73 expression on activated murine CD4(+) T cells is induced by TGF-β independently of Foxp3 expression, operates at the transcriptional level and translates into gain of functional capacity to generate adenosine. In the presence of AMP, CD73 induced by TGF-β generates adenosine able to suppress proliferation of activated CD4(+) T cells in vitro. These effects are contextual and opposed by proinflammatory cytokines. CD73 is also upregulated by TGF-β in CD8(+) T cells, DCs and macrophages, so providing an amplification mechanism for adenosine generation in tissue microenvironments. Together, these findings expose a novel anti-inflammatory role for TGF-β.


Neutrophil serine proteases mediate inflammatory cell recruitment by glomerular endothelium and progression towards dysfunction.
Neutrophil recruitment into glomerular tissues and reduced capillary wall integrity has been implicated in the development of vasculitic glomerulonephritis (VGN). This study investigated the stages and mechanisms through which neutrophil serine proteases (SPs), proteinase 3 (PR3) or elastase contribute to endothelial dysfunction. Protease-induced damage to endothelium and adhesion molecule upregulation was measured by viability assays and ELISA. Neutrophil/platelet adhesion to human glomerular and umbilical vein endothelium was assessed using in vitro adhesion assays. PR3 and elastase (1 µg/mL, 2 h) significantly induced neutrophil adhesion to endothelial cells (EnC) whilst PR3 also enhanced platelet-EnC interactions. This neutrophil adhesion was associated with enhanced P-selectin expression and required CXCL8 receptor involvement, and could be inhibited by blocking the P-selectin ligand PSGL-1. SPs induced damage in a time- and dose-dependent fashion, decreasing cell monolayer integrity followed by cell membrane integrity, inducing caspase-3 activation and p21 cleavage. However, SPs caused significant EnC damage with increasing concentrations and prolonged exposures. Neutrophil SPs induce a pro-adhesive phenotype in glomerular endothelium primarily by inducing neutrophil and platelet adhesion that transits to dysfunction after high/prolonged exposures. Dysregulated release of these enzymes within glomeruli may contribute to injury during diseases such as VGN.


The Nature of Resistance of the Coagulation Factor XIII Structure to Hypochlorite-Induced Oxidation.
The damage to blood coagulation factor XIII (FXIII) at different stages of its enzymatic activation under the action of various physiological amounts of hypochlorite ion was studied. The results obtained by HPLC-MS/MS, SDS-PAGE, and colorimetry showed that, during the conversion of FXIII to FXIIIa, the vulnerability of FXIII to hypochlorite-induced oxidation increased. FXIII oxidized with 150 μM hypochlorite completely retained its enzymatic activity inherent to the intact protein, whereas FXIIIa treated with 50 μM hypochlorite showed sharply reduced enzymatic activity. It was shown that a number of methionine and cysteine residues on the catalytic subunit can perform antioxidant function; additionally, the regulatory subunits of FXIII-B contribute to the antioxidant protection of the catalytic center of the FXIII-A subunit, which, together with the tight packing of the tetrameric structure of the FXIII proenzyme, are the three factors that provide high protein resistance to the oxidizing agent.


Genome-wide analysis of PDX1 target genes in human pancreatic progenitors.
Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF) PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4). Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing β-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Also, comparative studies of PDX1 binding patterns in pancreatic progenitors and adult β-cells have not been conducted so far. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs) and T2DM, and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions include important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B, and MEIS1, which were activated during the differentiation process as revealed by the active chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation maintains PDX1 expression and initiates a pancreatic TF program. Remarkably, we identified several PDX1 target genes that have not been reported in the literature in human so far, including RFX3, required for ciliogenesis and endocrine differentiation in mouse, and the ligand of the Notch receptor DLL1, which is important for endocrine induction and tip-trunk patterning. The comparison of PDX1 profiles from PPs and adult human islets identified sets of stage-specific target genes, associated with early pancreas development and adult β-cell function, respectively. Furthermore, we found an enrichment of T2DM-associated SNPs in active chromatin regions from iPSC-derived PPs. Two of these SNPs fall into PDX1 occupied sites that are located in the intronic regions of TCF7L2 and HNF1B. Both of these genes are key transcriptional regulators of endocrine induction and mutations in cis-regulatory regions predispose to diabetes. Our data provide stage-specific target genes of PDX1 during in vitro differentiation of stem cells into pancreatic progenitors that could be useful to identify pathways and molecular targets that predispose for diabetes. In addition, we show that T2DM-associated SNPs are enriched in active chromatin regions at the pancreatic progenitor stage, suggesting that the susceptibility to T2DM might originate from imperfect execution of a β-cell developmental program.


The appraisal of facial beauty is rapid but not mandatory.
Facial attractiveness is an important source of social affective information. Here, we studied the time course and task dependence of evaluating attractive faces from a viewer's perspective. Event-related brain potentials (ERPs) were recorded while participants classified color portraits of unfamiliar persons according to gender and facial attractiveness. During attractiveness classification, enhanced ERP amplitudes for attractive and nonattractive faces relative to faces of intermediate attractiveness were found for an early component around 150 msec and for the late positive complex (LPC). Whereas LPC enhancement conforms to previous studies employing various types of affective stimuli, the finding of an early effect extends earlier research on rapid emotion processing to the dimension of facial attractiveness. Dipole source localization of this early ERP effect revealed a scalp distribution suggesting activation of posterior extrastriate areas. Importantly, attractiveness-related modulations of brain responses were only marginal during the gender decision task, arguing against the automaticity of attractiveness appraisal.


Overactivation of the nuclear factor (erythroid-derived 2)-like 2-antioxidant response element pathway in hepatocytes decreases hepatic ischemia/reperfusion injury in mice.
Hepatic ischemia/reperfusion injury (IRI) is a critical component of hepatic surgery. Oxidative stress has long been implicated as a key player in IRI. In this study, we examine the cell-specific role of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-antioxidant response element pathway in warm hepatic IRI. Nrf2 knockout (KO) and wild-type (WT) animals and novel transgenic mice expressing a constitutively active nuclear factor (erythroid-derived 2)-like 2 (caNrf2) mutant in hepatocytes (AlbCre+/caNrf2+) and their littermate controls underwent partial hepatic ischemia or sham surgery. The animals were killed 6 hours after reperfusion, and their serum and tissue were collected for analysis. As compared to WT animals after ischemia/reperfusion (IR), Nrf2 KO mice had increased hepatocellular injury with increased serum alanine aminotransferase and aspartate aminotransferase, Suzuki score, apoptosis, an increased inflammatory infiltrate, and enhanced inflammatory cytokine expression. On the other hand, AlbCre+/caNrf2+ that underwent IR had significantly reduced serum transaminases, less necrosis on histology, and a less pronounced inflammatory infiltrate and inflammatory cytokine expression as compared to the littermate controls. However, there were no differences in apoptosis. Taken together, Nrf2 plays a critical role in our murine model of warm hepatic IRI, with Nrf2 deficiency exacerbating hepatic IRI and hepatocyte-specific Nrf2 overactivation providing protection against warm hepatic IRI.


Determinants of willingness to donate living related and cadaveric organs: identifying opportunities for intervention.
Although low rates of cadaveric organ donation have been attributed to potential cadaveric donors' concerns regarding their religious beliefs and mistrust of the health care system, it is unclear whether similar concerns are important to potential living related donors. It is also not known which factors might be most responsible for low rates of cadaveric and living related donation among the general public. We conducted a cross-sectional study of households in Maryland, using a standardized telephone questionnaire, to assess factors associated with willingness to donate cadaveric and living related organs. We compared factors (demographic, cultural, attitudinal, and clinical) related to willingness to donate cadaveric organs with factors related to willingness to donate living related organs. In multivariate analyses, we assessed the independent relation of factors to willingness to donate cadaveric and living related organs, and we assessed the relative importance of these factors in explaining variation in the general public's willingness to donate. Of 385 participants (84% of randomized homes), 254 (66%) were extremely willing to donate to a sibling but only 179 (47%) had designated themselves a cadaveric donor on their drivers' licenses. In bivariate analysis, older age, comorbid conditions, mistrust in hospitals, and concerns about discrimination in hospitals were statistically significantly associated with less willingness to donate living related organs, although African-Americans, older age, lower education, lack of insurance, unemployment, comorbid conditions, and religion/spirituality were associated with less willingness to donate cadaveric organs. After adjusting for potential confounders, only mistrust in hospitals and concerns about discrimination remained strongly and independently associated with 50 to 60% less odds of willingness to donate living related organs [[relative odds [95% confidence intervals (CI)]: 0.4 (0.2-0.7) to 0.5 (0.3-1.0) and 0.4 (0.2-0.9), respectively]] although presence of dependents was associated with 70% higher odds of willingness to donate living related organs [relative odds (95% CI): 1.7 (1.0-3.0)]. In contrast, older age, employment status, religion/spirituality, and mistrust in hospitals were associated with 50 to 90% less odds of willingness to donate living related organs cadaveric organs [relative odds (95% CI): 0.3 (0.1-0.8), 0.4(0.2-0.8), 0.1 (0.1- 0.5) to 0.5 (0.2-0.9), and 0.3 (0.2-0.6), respectively]. Mistrust in hospitals and concerns about the surgical donation procedure contributed most to the variation in willingness to be a living related donor, although race contributed most to the variation in willingness to be a cadaveric donor. Many factors affect the general public's willingness to donate organs, but their relative contribution is different for living related versus cadaveric donation. Efforts to improve organ donation rates should be directed toward factors that are most important in explaining the existing variation in willingness to donate.


Otx2 is an intrinsic determinant of the embryonic stem cell state and is required for transition to a stable epiblast stem cell condition.
Mouse embryonic stem cells (ESCs) represent the naïve ground state of the preimplantation epiblast and epiblast stem cells (EpiSCs) represent the primed state of the postimplantation epiblast. Studies have revealed that the ESC state is maintained by a dynamic mechanism characterized by cell-to-cell spontaneous and reversible differences in sensitivity to self-renewal and susceptibility to differentiation. This metastable condition ensures indefinite self-renewal and, at the same time, predisposes ESCs for differentiation to EpiSCs. Despite considerable advances, the molecular mechanism controlling the ESC state and pluripotency transition from ESCs to EpiSCs have not been fully elucidated. Here we show that Otx2, a transcription factor essential for brain development, plays a crucial role in ESCs and EpiSCs. Otx2 is required to maintain the ESC metastable state by antagonizing ground state pluripotency and promoting commitment to differentiation. Furthermore, Otx2 is required for ESC transition into EpiSCs and, subsequently, to stabilize the EpiSC state by suppressing, in pluripotent cells, the mesendoderm-to-neural fate switch in cooperation with BMP4 and Fgf2. However, according to its central role in neural development and differentiation, Otx2 is crucially required for the specification of ESC-derived neural precursors fated to generate telencephalic and mesencephalic neurons. We propose that Otx2 is a novel intrinsic determinant controlling the functional integrity of ESCs and EpiSCs.


Light-driven production of ATP catalysed by F0F1-ATP synthase in an artificial photosynthetic membrane.
Energy-transducing membranes of living organisms couple spontaneous to non-spontaneous processes through the intermediacy of protonmotive force (p.m.f.)--an imbalance in electrochemical potential of protons across the membrane. In most organisms, p.m.f. is generated by redox reactions that are either photochemically driven, such as those in photosynthetic reaction centres, or intrinsically spontaneous, such as those of oxidative phosphorylation in mitochondria. Transmembrane proteins (such as the cytochromes and complexes I, III and IV in the electron-transport chain in the inner mitochondrial membrane) couple the redox reactions to proton translocation, thereby conserving a fraction of the redox chemical potential as p.m.f. Many transducer proteins couple p.m.f. to the performance of biochemical work, such as biochemical synthesis and mechanical and transport processes. Recently, an artificial photosynthetic membrane was reported in which a photocyclic process was used to transport protons across a liposomal membrane, resulting in acidification of the liposome's internal volume. If significant p.m.f. is generated in this system, then incorporating an appropriate transducer into the liposomal bilayer should make it possible to drive a non-spontaneous chemical process. Here we report the incorporation of F0F1-ATP synthase into liposomes containing the components of the proton-pumping photocycle. Irradiation of this artificial membrane with visible light results in the uncoupler- and inhibitor-sensitive synthesis of adenosine triphosphate (ATP) against an ATP chemical potential of approximately 12 kcal mol(-1), with a quantum yield of more than 7%. This system mimics the process by which photosynthetic bacteria convert light energy into ATP chemical potential.


US family physicians' intrauterine and implantable contraception provision: results from a national survey.
Establish a current cross-sectional national picture of intrauterine device (IUD) and implant provision by US family physicians and ascertain individual, clinical site and scope of practice level associations with provision. Secondary analysis of data from 2329 family physicians recertifying with the American Board of Family Medicine in 2014. Overall, 19.7% of respondents regularly inserted IUDs, and 11.3% regularly inserted and/or removed implants. Family physicians provided these services in a wide range of clinical settings. In bivariate analysis, almost all of the individual, clinical site and scope of practice characteristics we examined were associated with provision of both methods. In multivariate analysis, the scope of practice characteristics showed the strongest association with both IUD and implant provision. For IUDs, this included providing prenatal care with [adjusted odds ratio (aOR) 3.26, 95% confidence interval (95% CI)=1.93-5.49] or without (aOR=3.38, 95% CI=1.88-6.06) delivery, performance of endometrial biopsies (aOR=16.51, 95% CI=11.97-22.79) and implant insertion and removal (aOR=8.78, 95% CI=5.79-13.33). For implants, it was providing prenatal care and delivery (aOR=1.77, 95% CI=1.15-2.74), office skin procedures (aOR=3.07, 95% CI=1.47-6.42), endometrial biopsies (aOR=3.67, 95% CI=2.41-5.59) and IUD insertion (aOR=8.58, 95% CI=5.70-12.91). While a minority of family physicians regularly provided IUDs and/or implants, those who provided did so in a broad range of outpatient settings. Individual and clinical site characteristics were not largely predictive of provision. This connotes potential for family physicians to increase IUD and implant access in a variety of settings. Provision of both methods was strongly associated with scope of practice variables including performance of certain office procedures as well as prenatal and/or obstetrical care. These data provide a baseline from which to analyze change in IUD and implant provision in family medicine, identify gaps in care and ascertain potential leverage points for interventions to increase long-acting reversible contraceptive provision by family physicians. Interventions may be more successful if they first focus on sites and/or family physicians who already provide prenatal care, obstetrical care, skin procedures and/or endometrial biopsies.


Real-time cardiac synchronization with fixed volume frame rate for reducing physiological instabilities in 3D FMRI.
Although 2D echo-planar imaging (EPI) remains the dominant method for functional MRI (FMRI), 3D readouts are receiving more interest as these sequences have favorable signal-to-noise ratio (SNR) and enable imaging at a high isotropic resolution. Spoiled gradient-echo (SPGR) and balanced steady-state free-precession (bSSFP) are rapid sequences that are typically acquired with highly segmented 3D readouts, and thus less sensitive to image distortion and signal dropout. They therefore provide a powerful alternative for FMRI in areas with strong susceptibility offsets, such as deep gray matter structures and the brainstem. Unfortunately, the multi-shot nature of the readout makes these sequences highly sensitive to physiological fluctuations, and large signal instabilities are observed in the inferior regions of the brain. In this work a characterization of the source of these instabilities is given and a new method is presented to reduce the instabilities observed in 3D SPGR and bSSFP. Rapidly acquired single-slice data, which critically sampled the respiratory and cardiac waveforms, showed that cardiac pulsation is the dominant source of the instabilities. Simulations further showed that synchronizing the readout to the cardiac cycle minimizes the instabilities considerably. A real-time synchronization method was therefore developed, which utilizes parallel-imaging techniques to allow cardiac synchronization without alteration of the volume acquisition rate. The implemented method significantly improves the temporal stability in areas that are affected by cardiac-related signal fluctuations. In bSSFP data the tSNR in the brainstem increased by 45%, at the cost of a small reduction in tSNR in the cortical areas. In SPGR the temporal stability is improved by approximately 20% in the subcortical structures and as well as cortical gray matter when synchronization was performed.


Art markets in crisis: how personal bonds and market subcultures mediate the effects of COVID-19.
We examine how the contemporary art market has changed as a result of the disruptions caused by the novel coronavirus. Based on interviews with artists, collectors, a dealer, and an auction house executive, we argue that the decline of face-to-face interaction, previously essential to art market transactions, has placed strain on each corner of the community. In the absence of physical co-presence with the artworks and art world actors, participants struggle to evaluate and appreciate artworks, make new social ties, develop trust, and experience a shared sense of pleasure and collective effervescence. These challenges especially impact the primary gallery market, where participants emphasize a communal commitment to art above instrumental speculation, which is more accepted in the secondary auction market. We find a transition to distant online communication, but the likelihood of this continuing after the lockdowns end and the virus dissipates varies according to the subcultures of these market segments.


Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II.
Angiotensin (Ang) II stimulates hypertrophic growth of vascular smooth muscle cells (VSMC). Accompanying this growth is the induction of the expression of growth-related protooncogenes (c-fos, c-jun, and c-myc), as well as the synthesis of the autocrine growth factors, such as PDGF-A and TGF-beta 1. In this study, we demonstrate further that Ang II also induces the synthesis of basic fibroblast growth factor (bFGF), a potent mitogen for VSMC. To examine how these factors interact to modulate the growth response of VSMC to Ang II, we used antisense oligomers to determine the relative contribution of these three factors. Treatment of confluent, quiescent smooth muscle cells with specific antisense oligomers complementary to bFGF, PDGF-A, and TGF-beta 1 efficiently inhibited the syntheses of these factors. Our results demonstrate that in these VSMC, TGF-beta 1 affects a key antiproliferative action, modulating the mitogenic properties of bFGF. Autocrine PDGF exerts only a minimal effect on DNA synthesis. An imbalance in these signals activated by Ang II may result in abnormal VSMC growth leading to the development of vascular disease.


Modulation of 7 T fMRI Signal in the Cerebellar Cortex and Nuclei During Acquisition, Extinction, and Reacquisition of Conditioned Eyeblink Responses.
Classical delay eyeblink conditioning is likely the most commonly used paradigm to study cerebellar learning. As yet, few studies have focused on extinction and savings of conditioned eyeblink responses (CRs). Saving effects, which are reflected in a reacquisition after extinction that is faster than the initial acquisition, suggest that learned associations are at least partly preserved during extinction. In this study, we tested the hypothesis that acquisition-related plasticity is nihilated during extinction in the cerebellar cortex, but retained in the cerebellar nuclei, allowing for faster reacquisition. Changes of 7 T functional magnetic resonance imaging (fMRI) signals were investigated in the cerebellar cortex and nuclei of young and healthy human subjects. Main effects of acquisition, extinction, and reacquisition against rest were calculated in conditioned stimulus-only trials. First-level β values were determined for a spherical region of interest (ROI) around the acquisition peak voxel in lobule VI, and dentate and interposed nuclei ipsilateral to the unconditioned stimulus. In the cerebellar cortex and nuclei, fMRI signals were significantly lower in extinction compared to acquisition and reacquisition, but not significantly different between acquisition and reacquisition. These findings are consistent with the theory of bidirectional learning in both the cerebellar cortex and nuclei. It cannot explain, however, why conditioned responses reappear almost immediately in reacquisition following extinction. Although the present data do not exclude that part of the initial memory remains in the cerebellum in extinction, future studies should also explore changes in extracerebellar regions as a potential substrate of saving effects. Hum Brain Mapp 38:3957-3974, 2017. © 2017 Wiley Periodicals, Inc.


AMP-activated protein kinase (AMPK)-induced preconditioning in primary cortical neurons involves activation of MCL-1.
Neuronal preconditioning is a phenomenon where a previous exposure to a sub-lethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP-activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischaemic and mitochondrial uncoupling-induced preconditioning in neurons, however, the molecular basis of AMPK-mediated preconditioning has been less well characterized. We investigated the effect of AMPK preconditioning using 5-aminoimidazole-4-carboxamide riboside (AICAR) in a model of NMDA-mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA-induced excitotoxicity. Analysing potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) in AICAR-preconditioned neurons. Interestingly, over-expression of MCL-1 protected neurons against NMDA-induced excitotoxicity while MCL-1 gene silencing abolished the effect of AICAR preconditioning. Monitored intracellular Ca²⁺ levels during NMDA excitation revealed that MCL-1 over-expressing neurons exhibited improved bioenergetics and markedly reduced Ca²⁺ elevations, suggesting a potential mechanism through which MCL-1 confers neuroprotection. This study identifies MCL-1 as a key effector of AMPK-induced preconditioning in neurons.


Scleractinian coral exoskeletons: surface microarchitecture and attachment scar patterns.
Scanning electron microscopic studies have revealed the configurations of the growth surfaces of scleractinian coral exoskeletons. Skeletal surfaces exhibit profuse growths of minute elongate aragonite crystals which, on basal and mural surfaces, are punctuated by scars. It is suggested that these scars are sites of attachment for the specialized processes that connect the living tissues of polyps to the nonliving skeleton. Patterns formed by the attachment scars are taxonomically significant.


Genetic mechanisms for loss of encapsulation in polysialyltransferase-gene-positive meningococci isolated from healthy carriers.
Encapsulated Neisseria meningitidis expressing serogroups A, B, C, W-135, or Y remain a major cause of morbidity and mortality globally. This bacterium is, however, a common commensal inhabitant of the human nasopharynx that causes disease infrequently. Isolates obtained from healthy carriers are frequently unencapsulated and therefore essentially avirulent. The lack of capsule can be due to inactivation of capsule synthesis genes by a variety of genetic mechanisms, or the absence of capsule synthesis genes. Analysis of inactivation mechanisms was undertaken in a diverse but representative set of 166 acapsulate meningococci isolated from carriage that possessed capsule synthesis genes. Slipped strand mispairing in the siaA and siaD genes of the capsule synthesis locus was observed in 39 isolates. Insertion sequence (IS) elements (IS1016-like, IS1106 and IS1301) were responsible for the loss of encapsulation in 46 isolates. Irreversible gene silencing events (insertions, deletions, base exchanges) were found in 47 isolates. Two non-synonymous mutations were identified in close vicinity of the putative active site of the UDP-N-acetylglucosamine 2-epimerase encoded by the siaA gene. The mechanisms for loss of encapsulation were not associated with particular meningococcal genotypes. There was no evidence for successive gene silencing events in the capsule genes, suggesting that the irreversible inactivation events observed were the result of short-term, within-host evolution. These observations are consistent with the postulate that particular meningococcal clonal complexes are associated with possession of a capsule and that this association is important for transmission success.


Hereditary nonpolyposis colorectal carcinoma (HNPCC) and HNPCC-like families: Problems in diagnosis, surveillance, and management.
To the authors' knowledge, hereditary nonpolyposis colorectal carcinoma (HNPCC) is the most commonly occurring hereditary disorder that predisposes to colorectal carcinoma (CRC), accounting for approximately 2-7% of all CRC cases diagnosed in the U.S each year. Its diagnosis is wholly dependent on a meticulously obtained family history of cancer of all anatomic sites, with particular attention to the pattern of cancer distribution within the family. The objective of the current study was to illustrate various vexing problems that can deter the diagnosis of HNPCC and, ultimately, its management. This was an observational cohort study. Sixteen HNPCC and HNPCC-like families were selected from a large resource of highly extended HNPCC families. High-risk patients were selected from these HNPCC families. An ascertainment bias was imposed by the lack of a population-based data set. Personal interviews and questionnaires were used for data collection. There was an array of difficulties highlighted by limitations in compliance, lack of a clinical or molecular basis for an HNPCC diagnosis, ambiguous DNA findings, problems in genetic counseling, failure to meet Amsterdam or Bethesda criteria, small families, lack of medical and pathologic documentation, poor cooperation of family members and/or their physicians, cultural barriers, economic stress, frequent patient fear and anxiety, perception of insurance discrimination, and limited patient and/or physician knowledge regarding hereditary cancer. The diagnosis and management of HNPCC is predicated on physician knowledge of its phenotypic and genotypic heterogeneity, in concert with the multifaceted problems that impact on patient compliance.


Human Social Behavior and Demography Drive Patterns of Fine-Scale Dengue Transmission in Endemic Areas of Colombia.
Dengue is known to transmit between humans and A. aegypti mosquitoes living in neighboring houses. Although transmission is thought to be highly heterogeneous in both space and time, little is known about the patterns and drivers of transmission in groups of houses in endemic settings. We carried out surveys of PCR positivity in children residing in 2-block patches of highly endemic cities of Colombia. We found high levels of heterogeneity in PCR positivity, varying from less than 30% in 8 of the 10 patches to 56 and 96%, with the latter patch containing 22 children simultaneously PCR positive (PCR22) for DEN2. We then used an agent-based model to assess the likely eco-epidemiological context of this observation. Our model, simulating daily dengue dynamics over a 20 year period in a single two block patch, suggests that the observed heterogeneity most likely derived from variation in the density of susceptible people. Two aspects of human adaptive behavior were critical to determining this density: external social relationships favoring viral introduction (by susceptible residents or infectious visitors) and immigration of households from non-endemic areas. External social relationships generating frequent viral introduction constituted a particularly strong constraint on susceptible densities, thereby limiting the potential for explosive outbreaks and dampening the impact of heightened vectorial capacity. Dengue transmission can be highly explosive locally, even in neighborhoods with significant immunity in the human population. Variation among neighborhoods in the density of local social networks and rural-to-urban migration is likely to produce significant fine-scale heterogeneity in dengue dynamics, constraining or amplifying the impacts of changes in mosquito populations and cross immunity between serotypes.


Does mindfulness meditation improve anxiety and mood symptoms? A review of the controlled research.
To review the impact of mindfulness-based stress reduction (MBSR) on symptoms of anxiety and depression in a range of clinical populations. Our review included any study that was published in a peer-reviewed journal, used a control group, and reported outcomes related to changes in depression and anxiety. We extracted the following key variables from each of the 15 studies identified: anxiety or depression outcomes after the MBSR program, measurement of compliance with MBSR instructions, type of control group included, type of clinical population studied, and length of follow-up. We also summarized modifications to the MBSR program. Measures of depression and anxiety were included as outcome variables for a broad range of medical and emotional disorders. Evidence for a beneficial effect of MBSR on depression and anxiety was equivocal. When active control groups were used, MBSR did not show an effect on depression and anxiety. Adherence to the MBSR program was infrequently assessed. Where it was assessed, the relation between practising mindfulness and changes in depression and anxiety was equivocal. MBSR does not have a reliable effect on depression and anxiety.


Urokinase plasminogen activator receptor (uPA-R): one potential characteristic of metastatic phenotypes in minimal residual tumor disease.
Evidence of dynamic development of cytokeratin (CK) 18-positive disseminated tumor cells in bone marrow of curatively resected cancer patients has implicated a subclinical minimal residual disease as a biologically relevant component in solid cancer. However, differentiation between irrelevant shed cells and those cells potentially capable of causing later recurrence has not yet been made. In parallel, accumulating data show functional association of the urokinase plasminogen activator (uPA) system and the membranous uPA receptor (uPA-R) with the capacity of a tumor cell for invasion and metastasis. The present study was designed to find descriptive evidence in vivo concerning whether uPA-R could be one potential characteristic for metastatically relevant phenotypes of disseminated tumor cells. An immunocytochemical double staining for uPA-R and CK18 (immunogold/alkaline phosphatase anti-alkaline phosphatase) was performed on perioperative and follow-up bone marrow aspirations of 78 curatively resected gastric cancer patients, if positive tumor cell status had been shown previously with the single alkaline phosphatase anti-alkaline phosphatase method. Bone marrow cells (10(6)) were examined in each assay. Postoperative qualitative and quantitative development of uPA-R-expressing disseminated tumor cells was followed in relation to uPA-R-negative cells and correlated with later clinical relapse. Double staining could be performed perioperatively or in follow-up, or both, in 58 of 78 patients. Expression of uPA-R on perioperatively disseminated tumor cells significantly correlated with later quantitative increases of tumor cells (P = 0.0009). Overall median tumor cell numbers with uPA-R expression significantly increased during follow-up from a median value of 5.5 to 10.0 in 10(6) cells (P = 0.008), and the mean relative percentage of uPA-R-positive, compared with uPA-R-negative, disseminated tumor cells also increased, from 47.9% at surgery to 68.6% in follow-up (P < 0.001). This was mainly due to patients with later tumor relapse (increase from 63.9 to 80.7%, P = 0.001). Patients without relapse showed slight increases at lower percentage levels (5.7% at surgery, 7.4% in follow-up). Differences for relapsing patients were significant (surgery, P = 0.006; follow-up, P < 0.001). Our results suggest from an in vivo model that uPA-R may be one antigen that enables identification and follow-up observations of metastatically relevant phenotypes of disseminated tumor cells, differentiating their individual potential for causing relapse.


Stress-related genetic polymorphisms in association with peripartum depression symptoms and stress hormones: A longitudinal population-based study.
Individual differences in the response of the stress system to hormonal changes during pregnancy and the postpartum period render some women susceptible to developing depression. The present study sought to investigate peripartum depression and stress hormones in relation to stress-related genotypes. The Edinburgh Postnatal Depression Scale was used to assess peripartum depressive symptoms in a sample of 1629 women, followed from pregnancy week seventeen to six months postpartum. Genotypes of ninety-four haplotype-tag single nucleotide polymorphisms (SNPs) in sixteen genes of the hypothalamus-pituitary-adrenal axis pathway were analyzed and data on psychosocial and demographic factors was collected. In sub-studies, salivary cortisol awakening response in gestational week 35-39, salivary evening cortisol levels in gestational week 36 and postpartum week 6, and blood cortisol and cortisone levels in gestational week 35-39 were analyzed. SNP-set kernel association tests were performed at the gene-level, considering psychosocial and demographic factors, followed by post-hoc analyses of SNPs of significant genes. Statistically significant findings at the 0.05 p-level included SNPs in the hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1) gene in relation to self-rated depression scores in postpartum week six among all participants, and serpin family A member 6 (SERPINA6) gene at the same time-point among women with de novo onset of postpartum depression. SNPs in these genes also associated with stress hormone levels during pregnancy. The present study adds knowledge to the neurobiological basis of peripartum depression by systematically assessing SNPs in stress-regulatory genes and stress-hormone levels in a population-based sample of women.


Use of magnetic resonance urography.
Magnetic resonance urography (MRU) is a new technique that uses heavily weighted T2 coronal images with fat suppression pulse. Urine appears white on MRU, resembling an intravenous urogram (IVU). Contrast agents are not necessary. This study describes the use of MRU in the diagnosis and treatment of patients with hematuria. One hundred six patients with microscopic or gross hematuria and 6 normal volunteers underwent MRU between 1992 and 1995. A modified, heavily weighted T2 technique with intravenous administration of furosemide and ureteral compression was used. Thirty-two patients had other imaging techniques as well for comparison. MRU provided high-resolution images in almost all cases; 73 (69%) had a normal MRU. Significant findings in the 33 patients with abnormalities included renal cysts in 17 (51%), renal cell carcinoma in 6 (18%), transitional cell carcinoma in 5 (15%), ureteropelvic junction obstruction in 3 (9%), and stones causing obstruction in 6 (18%). Five patients with renal failure also had good visualization of the entire urinary tract. MRU was comparable to other imaging modalities except in identifying nonobstructing calculi. MRU provides an alternative to conventional imaging of the urinary tract, especially in those patients who have contraindications to ionizing radiation and contrast agents. Improvements in resolution, technique, and cost have to be addressed before it can be used regularly in urologic practice.


Surface melt-induced acceleration of Greenland ice-sheet flow.
Ice flow at a location in the equilibrium zone of the west-central Greenland Ice Sheet accelerates above the midwinter average rate during periods of summer melting. The near coincidence of the ice acceleration with the duration of surface melting, followed by deceleration after the melting ceases, indicates that glacial sliding is enhanced by rapid migration of surface meltwater to the ice-bedrock interface. Interannual variations in the ice acceleration are correlated with variations in the intensity of the surface melting, with larger increases accompanying higher amounts of summer melting. The indicated coupling between surface melting and ice-sheet flow provides a mechanism for rapid, large-scale, dynamic responses of ice sheets to climate warming.


In search of wellness: allied health professionals' understandings of wellness in childhood disability services.
Wellness approaches are not routine in childhood disability services, despite theoretical and empirical support and an increasing demand for them from health consumers and disability activists. We aimed to investigate how health professionals define or understand wellness and its practice in the context of childhood disability. A qualitative, interpretive approach was taken. Semi-structured interviews were conducted with 23 health professionals (allied health therapists and managers) providing early intervention and ongoing therapy within four Australian childhood health and disability services. Years of experience providing services to children with disabilities and their families ranged from 6 months to 30 years (M=9.41, SD=9.04). The data revealed a noteworthy impediment to incorporating wellness into practice - the difficulties in the allied health professionals reaching consensus in defining wellness. There appeared to be distinct differences between the four services, while there appeared to be no appreciable difference based on the individual professional's years of experience or allied health discipline. The effect of organisational culture should be considered in efforts to embed wellness in childhood health and disability services in order to address client well-being, empowerment, choice, independence and rights to meaningful and productive lives.


Percutaneous Intramedullary Headless Screw Fixation and Wide-Awake Anesthesia to Treat Metacarpal Fractures: Early Results in 25 Patients.
Metacarpal fractures constitute 7.8% of the upper extremity fractures. The common treatments remain nonsurgical procedure, but high-demanding patients or unstable fractures require fixation with Kirschner wire (K-wires), plate, and screws. However, these approaches may cause scarring and adhesion with poor functional results. From 2014 to 2015, the authors used an intramedullary headless screw to treat 25 patients (24 men, 1 woman) with metacarpal bones fractures (20 V, 3 IV, 1 III, and 1 II). The fractures patterns were 23 fractures of distal third of metacarpal bone (16 oblique, 5 comminute configurations, and 2 transverse), 1 fracture of the base of the II metacarpal bone. One case presented a multiple metacarpal and phalangeal facture associated. The authors used wide-awake anesthesia (bupivacaine-epinephrine 1:100,000) and intramedullary titanium headless screw fixation percutaneously inserted (CCS Medartis and HCS Synthes 3 mm of diameter). No open reduction was needed. Early active mobilization started with a buddy strapping soon after surgery (0-3 days). The authors followed all patients until satisfactory function was achieved (4-6 weeks) and recorded the time till return to work. All fracture healed with less than 5 degrees of rotational or axial deformities. All patients return to work within 2.38 weeks after surgery (0.5-6 weeks). No cases of complex regional pain syndrome (CRPS), tendon lesions, nerve injuries, infection, hardware protruding, or mobilization were reported. Intramedullary screw fixation with wide-awake anesthesia for transverse, oblique, and select comminuted fractures treatment metacarpal fractures represent a reliable option to early active mobilization recovery and a quick return to the work and ordinary activities.


Lactoferrin expression in mammary epithelial cells is mediated by changes in cell shape and actin cytoskeleton.
Lactoferrin is a secreted iron binding protein which is expressed during normal functional development of mammary epithelium. Murine mammary epithelial cell lines competent for milk protein expression were used to identify microenvironmental factors that regulate lactoferrin expression. While lactoferrin was not expressed in adherent monolayer cultures under standard subconfluent conditions on plastic, lactoferrin mRNA and protein steadily accumulated when the cells aggregated to form spheroids on a reconstituted basement membrane gel. However, unlike other milk proteins such as beta-casein, lactoferrin expression was also induced at high cell density in the absence of exogenously added basement membrane or prolactin. These results led us to examine whether changes in cell growth, cell-cell interactions and/or cell shape were responsible for regulation of lactoferrin gene expression. Rounded, non-proliferating cells in suspension in serum-free medium expressed lactoferrin even as single cells. Conversely, lactoferrin expression could be inhibited in non-proliferative cells in serum-free medium by maintaining them in contact with an air-dried extracellular matrix which caused the cells to retain flat, spread morphologies. These findings indicated that cessation of cell growth was not sufficient, that cell-cell interactions were not required, and that cell culture conditions which minimize cell spreading may be important in maintaining lactoferrin expression. Additional data supporting this latter concept were generated by treating spread cells with cytochalasin D. The resulting disruption of microfilament assembly induced both cell rounding and lactoferrin expression. Shape-dependent regulation of lactoferrin mRNA was both transcriptional and post-transcriptional. Surprisingly, treatment of rounded cells with a transcription inhibitor, actinomycin D, produced a stabilization of lactoferrin mRNA, suggesting that transcription of an unstable factor is required for degradation of lactoferrin mRNA. Importantly, lactoferrin mRNA expression was regulated similarly in early passage normal human mammary epithelial cells. In vivo, the changing extracellular matrix components of the mammary gland during different stages of normal and abnormal growth and differentiation may provide different physical constraints on the configurations of cell surface molecules. These physical constraints may be communicated to the cell interior through mechanical changes in the cytoskeleton. Unlike beta-casein whose expression is upregulated by specific integrin-mediated signals, lactoferrin may be representative of a class of proteins synthesized in the mammary gland using basal transcriptional and translational machinery. The suppression of lactoferrin expression that is observed in monolayer culture and in malignant tissues may reflect inappropriate cell shapes and cytoskeletal structures that are manifested under these conditions.


The differential effects of prolonged exercise upon executive function and cerebral oxygenation.
The acute-exercise effects upon cognitive functions are varied and dependent upon exercise duration and intensity, and the type of cognitive tasks assessed. The hypofrontality hypothesis assumes that prolonged exercise, at physiologically challenging intensities, is detrimental to executive functions due to cerebral perturbations (indicated by reduced prefrontal activity). The present study aimed to test this hypothesis by measuring oxygenation in prefrontal and motor regions using near-infrared spectroscopy during two executive tasks (flanker task and 2-back task) performed while cycling for 60min at a very low intensity and an intensity above the ventilatory threshold. Findings revealed that, compared to very low intensity, physiologically challenging exercise (i) shortened reaction time in the flanker task, (ii) impaired performance in the 2-back task, and (iii) initially increased oxygenation in prefrontal, but not motor regions, which then became stable in both regions over time. Therefore, during prolonged exercise, not only is the intensity of exercise assessed important, but also the nature of the cognitive processes involved in the task. In contrast to the hypofrontality hypothesis, no inverse pattern of oxygenation between prefrontal and motor regions was observed, and prefrontal oxygenation was maintained over time. The present results go against the hypofrontality hypothesis.


Fiber-based polarization-sensitive optical coherence tomography of a minimalistic system configuration.
We present a very simple method of constructing a polarization-sensitive optical coherence tomography (PS-OCT) system. An ordinary fiber-based swept-source OCT system was reconfigured for PS-OCT by adding a long section of polarization-maintaining fiber in the sample arm. Two polarization modes of a large group-delay difference formed spatially distinguished polarization channels. The depth-encoded information on the polarization states was retrieved by an amplitude-based analysis. We found that our method provides an economic scheme of PS-OCT. It demonstrates that an ordinary OCT system can be easily reconfigured for PS-OCT imaging if it has sufficient margins in the imaging range.


Antillatoxin, a novel lipopeptide, enhances neurite outgrowth in immature cerebrocortical neurons through activation of voltage-gated sodium channels.
Antillatoxin (ATX) is a structurally novel lipopeptide that activates voltage-gated sodium channels (VGSC) leading to sodium influx in cerebellar granule neurons and cerebrocortical neurons 8 to 9 days in vitro (Li et al., 2001; Cao et al., 2008). However, the precise recognition site for ATX on the VGSC remains to be defined. Inasmuch as elevation of intracellular sodium ([Na(+)](i)) may increase N-methyl-d-aspartate receptor (NMDAR)-mediated Ca(2+) influx, Na(+) may function as a signaling molecule. We hypothesized that ATX may enhance neurite outgrowth in cerebrocortical neurons by elevating [Na(+)](i) and augmenting NMDAR function. ATX (30-100 nM) robustly stimulated neurite outgrowth, and this enhancement was sensitive to the VGSC antagonist, tetrodotoxin. To unambiguously demonstrate the enhancement of NMDA receptor function by ATX, we recorded single-channel currents from cell-attached patches. ATX was found to increase the open probability of NMDA receptors. Na(+)-dependent up-regulation of NMDAR function has been shown to be regulated by Src family kinase (SFK) (Yu and Salter, 1998). The Src kinase inhibitor PP2 abrogated ATX-enhanced neurite outgrowth, suggesting a SFK involvement in this response. ATX-enhanced neurite outgrowth was also inhibited by the NMDAR antagonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), and the calmodulin-dependent kinase kinase (CaMKK) inhibitor, 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), demonstrating the requirement for NMDAR activation with subsequent downstream engagement of the Ca(2+)-dependent CaMKK pathway. These results with the structurally and mechanistically novel natural product, ATX, confirm and generalize our earlier results with a neurotoxin site 5 ligand. These data suggest that VGSC activators may represent a novel pharmacological strategy to regulate neuronal plasticity through NMDAR-dependent mechanisms.


Loss-of-function mutations in the EGF-CFC gene CFC1 are associated with human left-right laterality defects.
All vertebrates display a characteristic asymmetry of internal organs with the cardiac apex, stomach and spleen towards the left, and the liver and gall bladder on the right. Left-right (L-R) axis abnormalities or laterality defects are common in humans (1 in 8,500 live births). Several genes (such as Nodal, Ebaf and Pitx2) have been implicated in L-R organ positioning in model organisms. In humans, relatively few genes have been associated with a small percentage of human situs defects. These include ZIC3 (ref. 5), LEFTB (formerly LEFTY2; ref. 6) and ACVR2B (encoding activin receptor IIB; ref. 7). The EGF-CFC genes, mouse Cfc1 (encoding the Cryptic protein; ref. 9) and zebrafish one-eyed pinhead (oep; refs 10, 11) are essential for the establishment of the L-R axis. EGF-CFC proteins act as co-factors for Nodal-related signals, which have also been implicated in L-R axis development. Here we identify loss-of-function mutations in human CFC1 (encoding the CRYPTIC protein) in patients with heterotaxic phenotypes (randomized organ positioning). The mutant proteins have aberrant cellular localization in transfected cells and are functionally defective in a zebrafish oep-mutant rescue assay. Our findings indicate that the essential role of EGF-CFC genes and Nodal signalling in left-right axis formation is conserved from fish to humans. Moreover, our results support a role for environmental and/or genetic modifiers in determining the ultimate phenotype in humans.


FSH-producing macroadenoma associated in a patient with Cushing's disease.
We encountered a Cushing's disease patient whose surgically removed pituitary macroadenoma was not an ACTH-producing, but rather a gonadotroph adenoma. Cure was obtained only after a tiny microadenoma, overlooked on preoperative studies, was removed by a 2nd operation from a compressed thin normal anterior pituitary gland. This 45-year-old woman with Cushing syndrome presented with diabetes mellitus and steroid psychosis. Endocrinological examinations suggested Cushing's disease and MRI disclosed an invasive macroadenoma (22 mm in diameter) with suprasellar extension. Despite total removal of the invasive macroadenoma by transsphenoidal surgery, her elevated serum cortisol- and ACTH levels failed to decrease. Histologic study of the surgical specimen disclosed that the tumor was a silent FSH-producing, rather than an ACTH-producing adenoma. Detailed re-evaluation of pre- and postoperative MRI suggested the presence of a 3-mm microadenoma on the left side of a thin compressed normal gland. Venous sampling of the cavernous sinus confirmed this suspicion. In a 2nd operation an ACTH-producing microadenoma was removed from inside the thin remaining compressed normal pituitary gland and endocrinological cure of Cushing's disease was achieved. Although double adenomas, being a non-ACTH producing macroadenoma associated with an ACTH producing tiny microadenoma, are extremely rare in patients with Cushing's disease, detailed preoperative MRI evaluation is necessary to avoid missing tiny adenomas hidden in a compressed normal pituitary gland which is the cause of Cushing's disease, especially when a macroadenoma is found in patient with Cushing's disease.


Houttuynia cordata Thunb extract induces apoptosis through mitochondrial-dependent pathway in HT-29 human colon adenocarcinoma cells.
The Houttuynia cordata Thunb (HCT) extract has been used as a traditional Chinese herb medicine and as well as an effective drug for treating allergic inflammation for thousands of years. In this study, we investigated the anti-cancer activity of HCT and its molecular mechanisms in the human colon adenocarcinoma cell line HT-29. HCT inhibited HT-29 cell viability in a dose- and time-dependent manner by MTT assay. Treatment with 450 microg/ml of HCT for 48 and 72 h led to DNA damage and apoptosis by DAPI staining and comet assay. HCT increased reactive oxygen species production and decreased the levels of mitochondria membrane potential (MMP) in HT-29 cells by flow cytometry analysis. HCT caused the release of cytochrome c, Apaf-1, pro-caspase-9 and AIF from mitochondria via a decrease of the MMP. The decrease of MMP was then associated with a decrease in the ratio of Bax/Bcl-2 and activation of caspase-9 and -3 by Western blotting and caspase activity assay. Caspase-9 and -3 inhibitors almost completely suppressed HCT-induced caspase-9 and -3 activities. Our results demonstrated that the HCT-induced apoptosis in human colon adenocarcinoma cell line HT-29 might be related to a mitochondrial-dependent pathway.


A Core Outcome Set for Evaluation of Interventions to Prevent Preterm Birth.
To develop a consensus on a set of key clinical outcomes for the evaluation of preventive interventions for preterm birth in asymptomatic pregnant women. A two-stage web-based Delphi survey and a face-to-face meeting of key stakeholders were used to develop a consensus on a set of critical and important outcomes. We approached five stakeholder groups (parents, midwives, obstetricians, neonatologists, and researchers) from middle- and high-income countries. Outcomes subjected to the Delphi survey were identified by systematic literature review and stakeholder input. Survey participants scored each outcome on a 9-point Likert scale anchored between 1 (limited importance) and 9 (critical importance). They had the opportunity to reflect on total and stakeholder subgroup feedback between survey stages. For consensus, defined a priori, outcomes required at least 70% of participants of each stakeholder group to score them as "critical" and less than 15% as "limited." A total of 228 participants from five stakeholder groups from three lower middle-income countries, seven upper middle-income countries, and 17 high-income countries were asked to score 31 outcomes. Of these participants, 195 completed the first survey and 174 the second. Consensus was reached on 13 core outcomes: four were related to pregnant women: maternal mortality, maternal infection or inflammation, prelabor rupture of membranes, and harm to mother from intervention. Nine were related to offspring: gestational age at birth, offspring mortality, birth weight, early neurodevelopmental morbidity, late neurodevelopmental morbidity, gastrointestinal morbidity, infection, respiratory morbidity, and harm to offspring from intervention. This core outcome set for studies that evaluate prevention of preterm birth developed with an international multidisciplinary perspective will ensure that data from trials that assess prevention of preterm birth can be compared and combined. COMET Initiative, http://www.comet-initiative.org/studies/details/603, REGISTRATION NUMBER: 603.


Progressive Recruitment of the Frontoparietal Multiple-demand System with Increased Task Complexity, Time Pressure, and Reward.
A distributed, frontoparietal "multiple-demand" (MD) network is involved in tasks of many different kinds. Integrated activity across this network may be needed to bind together the multiple features of a mental control program (Duncan, 2013). Previous data suggest that, especially with low cognitive load, there may be some differentiation between MD regions (e.g., anterior vs. posterior regions of lateral frontal cortex), but with increasing load, there is progressive recruitment of the entire network. Differentiation may reflect preferential access to different task features, whereas co-recruitment may reflect information exchange and integration. To examine these patterns, we used manipulations of complexity, time pressure, and reward while participants solved a spatial maze. Complexity was manipulated by combining two simple tasks. Time pressure was added by fading away the maze during route planning, and on some of these trials, there was the further possibility of a substantial reward. Simple tasks evoked activity only in posterior MD regions, including posterior lateral frontal cortex, pre-supplementary motor area/anterior cingulate, and intraparietal sulcus. With increasing complexity, time pressure, and reward, increases in activity were broadly distributed across the MD network, though with quantitative variations. Across the MD network, the results show a degree of functional differentiation, especially at low load, but strong co-recruitment with increased challenge or incentive.


The cellular basis for different fetal hemoglobin levels among sickle cell individuals with two, three, and four alpha-globin genes.
Fetal hemoglobin (HbF) levels vary widely among individuals with sickle cell anemia (SS). Previous studies have suggested that HbF levels in SS individuals with alpha-thalassemia (two or three functional alpha-globin genes) are lower than HbF levels in SS individuals with four normal alpha-globin genes. Using immunocytochemical techniques, we studied F cell production as measured by % F reticulocytes, the amount of HbF per F cell, and the preferential survival of F cells versus non-F cells in 51 subjects with four alpha genes, 32 subjects with three alpha genes, and 18 subjects with two alpha genes. Comparison between alpha-globin gene groups was performed for the total sample as well as for a subset of 82 individuals who had replicate samples and a further subset of 39 age-matched individuals. %HbF levels were 6.8, 4.9, and 4.5 percent for the total four-, three-, and two-alpha-globin-gene groups, respectively. The percentage of F reticulocytes, percentage HbF per F cell, and the enrichment ratio (% F cell/% F reticulocytes) did not change significantly with alpha-globin gene number. Moreover, no correlation existed between alpha-globin gene number and the absolute number of F cells in any group studied. However, there was a strong inverse correlation (r = -0.407, P = .0001) between non-F cell levels (1.7 +/- 2, 2.2 +/- 5, 3.0 +/- 1.0 X 10(12)/L) and decreasing alpha-globin gene number. These data suggest that falling HbF levels among SS individuals with lessened numbers of alpha-globin genes reflect prolonged survival of non-F cells and are not due to intrinsic differences in F cell production or in the amount of HbF per F cell. The improved survival of non-F cells in SS alpha-thalassemia is presumed to be due to the lower MCHC observed in such individuals.


Can exercise increase fitness and reduce weight in patients with schizophrenia and depression?
Psychiatric patients have a reduced life expectancy of 15-20 years compared with the general population. Most years of lost life are due to the excess mortality from somatic diseases. Sedentary lifestyle and medication is partly responsible for the high frequency of metabolic syndrome in this patient group and low levels of physical activity is associated with increased risk of cardiovascular disease, diabetes, and all-cause mortality. This study aimed to review trials allocating patients with either schizophrenia or depression to exercise interventions for effect on cardiovascular fitness, strength, and weight. We searched PubMed, Embase, and PsycINFO including randomized clinical trial allocating patients with either schizophrenia or depression to isolated exercise interventions. We identified five trials including patients with schizophrenia (n = 94) and found little evidence that exercise could increase cardiovascular fitness or decrease weight. Nine exercise trials for patients with depression (n = 892) were identified increasing cardiovascular fitness by 11-30% and strength by 33-37%. No evidence in favor of exercise for weight reduction was found. Based on the current evidence isolated exercise interventions are unlikely to improve cardiovascular fitness or induce weight loss in patients with schizophrenia. In patients with depression, exercise interventions are likely to induce clinically relevant short term effects, however, due to lack of reporting, little is known about the effect on weight reduction and cardiovascular fitness. Future exercise trials regarding patients with mental illness should preferably measure changes in cardiovascular strength, repetition maximum, and anthropometric outcomes. Ideally, participants should be assessed beyond the intervention to identify long lasting effects.


Characterization of lipoprotein profiles in patients with hypertriglyceridemic Fredrickson-Levy and Lees dyslipidemia phenotypes: the Very Large Database of Lipids Studies 6 and 7.
The association between triglycerides (TG) and cardiovascular diseases is complex. The classification of hypertriglyceridemic (HTG) phenotypes proposed by Fredrickson, Levy and Lees (FLL) helps inform treatment strategies. We aimed to describe levels of several lipoprotein variables from individuals with HTG FLL phenotypes from the Very Large Database of Lipids. We included fasting samples from 979,539 individuals from a contemporary large study population of US adults. Lipids were directly measured by density-gradient ultracentrifugation using the Vertical Auto Profile test while TG levels were measured in whole plasma using the Abbott ARCHITECT C-8000 system. Hyperchylomicronemic (Hyper-CM) and non-chylomicronemic (non-CM) phenotypes were defined using computationally derived models. Individuals with FLL type IIa phenotype were excluded. Distributions of lipid variables were compared using medians and Kruskal-Wallis test. A total of 11.9% (n = 116,925) of individuals met criteria for HTG FLL phenotypes. Those with hyper-CM phenotypes (n = 5, < 0.1% of population) had two-fold higher TG levels compared with non-chylomicronemic (non-CM) individuals (11.9% of population) (p < 0.001). Type IIb individuals had the highest non-HDL-C levels (median 242 mg/dl). Cholesterol in large VLDL1+2 particles was higher than in small VLDL3 particles in all phenotypes except FLL type III. Hyper-CM phenotypes had significantly lower HDL-C levels but greater HDL2/HDL3-C ratio compared to non-CM phenotypes. Cholesterol content of the lipoprotein (a) peak was significantly higher in the hyper-CM groups compared to non-CM phenotypes (p < 0.0001). This observational hypothesis-generating study provides insight into the complexity of lipid metabolism in HTG phenotypes, including less traditional lipid measures such as LDL density, HDL subclasses and Lp(a)-C.


De novo deletion within the telomeric region flanking the human alpha globin locus as a cause of alpha thalassaemia.
We have identified and characterized a Scottish individual with alpha thalassaemia, resulting from a de novo 48 kilobase (kb) deletion from the telomeric flanking region of the alpha globin cluster which occurred as a result of recombination between two misaligned repetitive elements that normally lie approximately 83 kb and 131 kb from the 16p telomere. The deletion removes two previously described putative regulatory elements (HS-40 and HS-33) but leaves two other elements (HS-10 and HS-8) intact. Analysis of this deletion, together with eight other published deletions of the telomeric region, showed that they all severely downregulated alpha globin expression. Together they defined a 20.4-kb region of the human alpha cluster, which contains all of the positive cis-acting elements required to regulate alpha globin expression. Comparative analysis of this region with the corresponding segment of the mouse alpha globin cluster demonstrated conserved non-coding sequences corresponding to the putative regulatory elements HS-40 and HS-33. Although the role of HS-40 as an enhancer of alpha globin expression is fully established, these observations suggest that the role of HS-33 and other sequences in this region should be more fully investigated in the context of the natural human and mouse alpha globin loci.


A randomized controlled trial testing the effectiveness of a paramedic-delivered care transitions intervention to reduce emergency department revisits.
Approximately 20% of community-dwelling older adults discharged from the emergency department (ED) return to an ED within 30 days, an occurrence partially resulting from poor care transitions. Prior published interventions to improve the ED-to-home transition have either lacked feasibility or effectiveness. The Care Transitions Intervention (CTI) has been validated to decrease rehospitalization among patients transitioning from the hospital to the home but has never been tested for patients transitioning from the ED to the home. Paramedics, traditionally involved only in emergency care, are well-positioned to deliver the CTI, but have never been previously evaluated in this role. This single-blinded randomized controlled trial tests whether the paramedic-delivered ED-to-home CTI reduces community-dwelling older adults' ED revisits in the 30 days after an index visit. We are prospectively recruiting patients aged≥ 60 years at 3 EDs in Rochester, NY and Madison, WI to enroll 2400 patient subjects. Subjects are randomized into control and treatment groups, with the latter receiving the adapted CTI. The intervention consists of the paramedic performing one home visit and up to three follow-up phone calls. During these interactions, the paramedic follows the CTI approach by coaching patients toward their goals, with a focus on their personal health record, medication management, red flags, and primary care follow-up. We follow patient participants for 30 days. All receive a survey during the index ED visit to capture baseline demographic and health information and two telephone-based surveys to assess process objectives and outcomes. We also perform a medical record review. The primary outcome is the odds of ED revisit within 30 days after discharge from the index ED visit. This is the first study to test whether the CTI, applied to the ED-to-home transition and delivered by community paramedics, can decrease the rate at which older adults revisit an ED. Outcomes from this research will help address a major emergency care challenge by supporting older adults in the transition from the ED to home, thereby improving health outcomes for this population and reducing potentially avoidable ED visits. ClinicalTrials.gov Registration: NCT02520661 . Trial registration date: August 13, 2015.


Cross-linked porous polyurethane materials featuring dodecaborate clusters as inorganic polyol equivalents.
We report the discovery that a perhydroxylated dodecaborate cluster ([B12(OH)12]2-) can act as an inorganic polyol, serving as a molecular cross-linker in the synthesis of polyurethane-based materials. We further demonstrate how the inherent robustness of the utilized boron cluster can effectively enhance the thermal stability of the produced polyurethane materials incorporating [B12(OH)12]2- building blocks compared to analogous polymers made from carbon-based polyols. Ultimately, this approach provides a potential route to tune the chemical and physical properties of soft materials through incorporation of polyhedral boron-rich clusters into the polymer network.


An antibody to RGMa promotes regeneration of cochlear synapses after noise exposure.
Auditory neuropathy is caused by the loss of afferent input to the brainstem via the components of the neural pathway comprising inner hair cells and the first order neurons of the spiral ganglion. Recent work has identified the synapse between cochlear primary afferent neurons and sensory hair cells as a particularly vulnerable component of this pathway. Loss of these synapses due to noise exposure or aging results in the pathology identified as hidden hearing loss, an initial stage of cochlear dysfunction that goes undetected in standard hearing tests. We show here that repulsive axonal guidance molecule a (RGMa) acts to prevent regrowth and synaptogenesis of peripheral auditory nerve fibers with inner hair cells. Treatment of noise-exposed animals with an anti-RGMa blocking antibody regenerated inner hair cell synapses and resulted in recovery of wave-I amplitude of the auditory brainstem response, indicating effective reversal of synaptopathy.


Recovery of impaired songs following unilateral but not bilateral lesions of nucleus uvaeformis of adult zebra finches.
Zebra finches utilize neural circuits in both cerebral hemispheres to produce their learned songs. Although direct reciprocal connections do not exist between song control nuclei across hemispheres, premotor activity in these nuclei during singing is precisely and continuously coordinated between the hemispheres. We hypothesized that this interhemispheric coordination is mediated by bilateral feedback projections from medullary and midbrain song control nuclei to the thalamic song control nucleus uvaeformis (Uva). Consistent with our hypothesis, bilateral lesions of Uva severely impaired singing. This impairment was long-lasting, as it persisted for at least 35 days after the lesions. Unilateral lesions of Uva on either side also resulted in an immediate singing impairment. However, song recovered substantially after less than 15 days, suggesting a possible compensation by the unlesioned side. Although the acoustic structure of individual syllables recovered fully after unilateral lesioning, subtle changes in the sequencing of syllables were observed after song recovery, suggesting that the lesion led to an alteration in the functioning of the remaining song control network. These results demonstrate that the adult songbird brain can adjust to damage to certain parts of the song control network and recover from their associated singing deficits. The well-identified and localized central neural pathways mediating birdsong production provide an advantageous model system to analyze systematically the sensorimotor contexts and the specific sites and mechanisms for behavioral recovery following partial damage to a behavior-producing neural circuit.


Small Molecule Inhibitor Screen Reveals Calcium Channel Signaling as a Mechanistic Mediator of Clostridium difficile TcdB-Induced Necrosis.
Clostridioides difficile is the leading cause of nosocomial diarrhea in the United States. The primary virulence factors are two homologous glucosyltransferase toxins, TcdA and TcdB, that inactivate host Rho-family GTPases. The glucosyltransferase activity has been linked to a "cytopathic" disruption of the actin cytoskeleton and contributes to the disruption of tight junctions and the production of pro-inflammatory cytokines. TcdB is also a potent cytotoxin that causes epithelium necrotic damage through an NADPH oxidase (NOX)-dependent mechanism. We conducted a small molecule screen to identify compounds that confer protection against TcdB-induced necrosis. We identified an enrichment of "hit compounds" with a dihydropyridine (DHP) core which led to the discovery of a key early stage calcium signal that serves as a mechanistic link between TcdB-induced NOX activation and reactive oxygen species (ROS) production. Disruption of TcdB-induced calcium signaling (with both DHP and non-DHP molecules) is sufficient to ablate ROS production and prevent subsequent necrosis in cells and in a mouse model of intoxication.


Fluoroquinolone Antibiotics and Tendon Injury in Adolescents.
To estimate the association between fluoroquinolone use and tendon injury in adolescents. We conducted an active-comparator, new-user cohort study using population-based claims data from 2000 to 2018. We included adolescents (aged 12-18 years) with an outpatient prescription fill for an oral fluoroquinolone or comparator broad-spectrum antibiotic. The primary outcome was Achilles, quadricep, patellar, or tibial tendon rupture identified by diagnosis and procedure codes. Tendinitis was a secondary outcome. We used weighting to adjust for measured confounding and a negative control outcome to assess residual confounding. The cohort included 4.4 million adolescents with 7.6 million fills for fluoroquinolone (275 767 fills) or comparator (7 365 684) antibiotics. In the 90 days after the index antibiotic prescription, there were 842 tendon ruptures and 16 750 tendinitis diagnoses (crude rates 0.47 and 9.34 per 1000 person-years, respectively). The weighted 90-day tendon rupture risks were 13.6 per 100 000 fluoroquinolone-treated adolescents and 11.6 per 100 000 comparator-treated adolescents (fluoroquinolone-associated excess risk: 1.9 per 100 000 adolescents; 95% confidence interval -2.6 to 6.4); the corresponding number needed to treat to harm was 52 632. For tendinitis, the weighted 90-day risks were 200.8 per 100 000 fluoroquinolone-treated adolescents and 178.1 per 100 000 comparator-treated adolescents (excess risk: 22.7 per 100 000; 95% confidence interval 4.1 to 41.3); the number needed to treat to harm was 4405. The excess risk of tendon rupture associated with fluoroquinolone treatment was extremely small, and these events were rare. The excess risk of tendinitis associated with fluoroquinolone treatment was also small. Other more common potential adverse drug effects may be more important to consider for treatment decision-making, particularly in adolescents without other risk factors for tendon injury.


Selection and evolution of enzymes from a partially randomized non-catalytic scaffold.
Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA, to select for functional proteins from an in vitro translated protein library of >10(12 )independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand, from both random-sequence libraries and libraries based on a known protein fold. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold.


Expression of oncostatin M in chronic obstructive sialadenitis of the submandibular gland.
We investigated the expression of oncostatin M messenger RNA (mRNA) and protein in normal submandibular glands and those with chronic obstructive sialadenitis and localized the expression of oncostatin M protein. Submandibular glands from 10 patients with chronic obstructive sialadenitis as a study group and 10 normal submandibular glands as a control group were examined. Oncostatin M mRNA extracted from submandibular gland was used for reverse transcription-polymerase chain reaction and analyzed semiquantitatively. The difference in expression level of oncostatin M protein between the 2 groups was analyzed through Western blot analysis, and oncostatin M protein was localized immunohistochemically. The expression levels of oncostatin M mRNA and protein were significantly increased in the study group. The protein was predominantly localized in ductal epithelia and infiltrating inflammatory cells and was more strongly expressed in the study group also. Oncostatin M is expressed in both chronic obstructive sialadenitis and normal submandibular gland, and is up-regulated in chronic obstructive sialadenitis. These results suggest that oncostatin M is involved in the pathologic process of chronic obstructive sialadenitis. However, the physiologic role in normal glands, as well as a possible role in the development of chronic obstructive sialadenitis, remains to be elucidated.


Alcohol and injury: a risk function analysis from the Emergency Room Collaborative Alcohol Analysis Project (ERCAAP).
While emergency room (ER) studies have documented a strong association of alcohol and injury, less is known about the level of risk at which various quantities of alcohol or particular patterns of drinking place the individual for injury. Comparative risk function analyses are carried out in ER samples in seven countries that cover sites in 14 studies included in the Emergency Room Collaborative Alcohol Analysis Project (ERCAAP). Risk of injury is analyzed for the mean number of drinks consumed per day and the number of occasions in which 5 or more drinks were consumed at one time (5+) during the last year. All countries showed similar increases in injury risk to an average volume of about 2 drinks per day, with a leveling off of risk at higher average daily volumes, with the exception of Italy. Risk of injury increased to 12 or more 5+ days for the USA, Canada and Mexico, but leveled off after only 3 5+ days for Argentina and Spain. Poland showed increased risk to 30 5+ days. Similar risk curves were found for both males and females, although females were at lower risk of injury in all countries expect Spain and Poland. In low detrimental drinking pattern societies, risk curves showed higher risk for any drinking and any frequency of 5+ but at higher levels of each, risk levels decreased nearly to levels found for abstainers. Risk functions were also consistent across gender and age groups in low detrimental drinking pattern societies, with higher risk for males and those <30. For those countries with high detrimental drinking patterns, injury risk increased with volume and 5+ drinking primarily among males. This ER-based risk function analysis suggests that risk of injury increases proportionally with increased alcohol consumption at lower consumption levels, but a threshold effect is achieved at relatively low levels of mean daily consumption and higher consumption times. Risk may be culturally specific, dependent, in part, on the manner in which alcohol is used in the culture.