The role of isoforms in the evolution of cryptic coloration in Peromyscus mice.
A central goal of evolutionary biology is to understand the molecular mechanisms underlying phenotypic adaptation. While the contribution of protein-coding and cis-regulatory mutations to adaptive traits has been well documented, additional sources of variation - such as the production of alternative RNA transcripts from a single gene, or isoforms - have been understudied. Here, we focus on the pigmentation gene Agouti, known to express multiple alternative transcripts, to investigate the role of isoform usage in the evolution of cryptic colour phenotypes in deer mice (genus Peromyscus). We first characterize the Agouti isoforms expressed in the Peromyscus skin and find two novel isoforms not previously identified in Mus. Next, we show that a locally adapted light-coloured population of P. maniculatus living on the Nebraska Sand Hills shows an upregulation of a single Agouti isoform, termed 1C, compared with their ancestral dark-coloured conspecifics. Using in vitro assays, we show that this preference for isoform 1C may be driven by isoform-specific differences in translation. In addition, using an admixed population of wild-caught mice, we find that variation in overall Agouti expression maps to a region near exon 1C, which also has patterns of nucleotide variation consistent with strong positive selection. Finally, we show that the independent evolution of cryptic light pigmentation in a different species, P. polionotus, has been driven by a preference for the same Agouti isoform. Together, these findings present an example of the role of alternative transcript processing in adaptation and demonstrate molecular convergence at the level of isoform regulation.


Chest radiographic image quality: comparison of asymmetric screen-film, digital storage phosphor, and digital selenium drum systems--preliminary study.
Conventional screen-film radiography does not display all regions of the thorax satisfactorily. Three chest radiographic techniques display both the lung and the mediastinum with good contrast. These techniques are asymmetric screen-film (ASF), digital storage phosphor (DSP), and digital selenium drum (DSD) imaging. ASF systems use two asymmetric screen-film combinations to produce a wide-latitude image of the thorax with good contrast in the lungs. In DSP systems, image data are acquired digitally with a wide dynamic range by using the optical output of a photostimulable phosphor plate; in DSD systems, the wide-range digital image data are acquired by using the electronic charge generated on a drum coated with a thin layer of amorphous selenium. The appearance of a DSP or DSD radiograph is then determined by user-selected image processing operations: tone scaling, spatial frequency processing, and dynamic range compensation. Digital chest radiographs processed with strong regional equalization provide both excellent contrast in the lungs and effective display of the mediastinum and chest wall. At visual comparison, the high lung contrast and good mediastinal, retrocardiac, and subdiaphragmatic detail provided by the DSD method distinguish it from the other two methods.


Cardiovascular regulation by skeletal muscle reflexes in health and disease.
Heart rate and blood pressure are elevated at the onset and throughout the duration of dynamic or static exercise. These neurally mediated cardiovascular adjustments to physical activity are regulated, in part, by a peripheral reflex originating in contracting skeletal muscle termed the exercise pressor reflex. Mechanically sensitive and metabolically sensitive receptors activating the exercise pressor reflex are located on the unencapsulated nerve terminals of group III and group IV afferent sensory neurons, respectively. Mechanoreceptors are stimulated by the physical distortion of their receptive fields during muscle contraction and can be sensitized by the production of metabolites generated by working skeletal myocytes. The chemical by-products of muscle contraction also stimulate metaboreceptors. Once activated, group III and IV sensory impulses are transmitted to cardiovascular control centers within the brain stem where they are integrated and processed. Activation of the reflex results in an increase in efferent sympathetic nerve activity and a withdrawal of parasympathetic nerve activity. These actions result in the precise alterations in cardiovascular hemodynamics requisite to meet the metabolic demands of working skeletal muscle. Coordinated activity by this reflex is altered after the development of cardiovascular disease, generating exaggerated increases in sympathetic nerve activity, blood pressure, heart rate, and vascular resistance. The basic components and operational characteristics of the reflex, the techniques used in human and animals to study the reflex, and the emerging evidence describing the dysfunction of the reflex with the advent of cardiovascular disease are highlighted in this review.


Fucoxanthin Alleviates Oxidative Stress through Akt/Sirt1/FoxO3α Signaling to Inhibit HG-Induced Renal Fibrosis in GMCs.
As one of the main marine carotenoids, fucoxanthin has strong antioxidant activity. FoxO3α, a member of the forkhead box O family of transcription factors, plays an important role in DN by regulating oxidative stress. The activity of FoxO3α is related to its phosphorylation and acetylation status, regulated by Akt and Sirt1, a lysine deacetylase. Our study aimed to investigate whether fucoxanthin could alleviate oxidative stress and fibrosis via FoxO3α in DN and whether Akt and Sirt1 were involved. We found that in GMCs cultured in HG, fucoxanthin treatment significantly reduced the expression of FN and collagen IV, as well as reactive oxygen species generation, suggesting that fucoxanthin is beneficial to alleviate both fibrosis and oxidative stress in DN. In addition, we found that fucoxanthin decreased the phosphorylation and acetylation level of FoxO3α, reversed the protein level of FoxO3α inhibited by HG, and then promoted the nuclear transport of FoxO3α. Besides, fucoxanthin promoted the expression of manganese superoxide dismutase, a downstream target of FoxO3α. Furthermore, we found that fucoxanthin reversed the activation of Akt and inhibition of Sirt1. However, the enhancement of fucoxanthin in FoxO3α expression and nuclear transport was significantly decreased by pretreatment with Akt activator SC79 or Sirt1 inhibitor EX527. In summary, our study explored fucoxanthin alleviated oxidative stress and fibrosis induced by HG through Akt/Sirt1/FoxO3α signaling in GMCs, suggesting fucoxanthin is a potential therapeutic strategy for DN.


Interleukin-10 receptor mutations in children with neonatal-onset Crohn's disease and intractable ulcerating enterocolitis.
Neonatal-onset inflammatory bowel disease (IBD) accounts for only 0.25% of pediatric IBD cases. The molecular pathogenesis of IBD remains unclear. Recently, rare Mendelian mutations have been identified in children with very early-onset Crohn's disease and ulcerative colitis. In this study, we report compound heterozygous mutations in the interleukin-10 receptor A (IL-10RA) gene in children with severe neonatal-onset IBD. Patient 1 had chronic diarrhea within the first month of life and had perianal fistulae. She was diagnosed with 'intractable ulcerating enterocolitis in infancy' and underwent subtotal colectomy at the age of 24 months because of poor response to immunosuppressant therapy. Compound heterozygous mutations, c.[301C>T];[350G>A](p.[R101W];[R117H]), were discovered in IL-10RA for this patient. Patient 2 presented symptoms within the first month of life and was diagnosed with Crohn's disease. Severe colitis and perianal and enteroenteric fistulae occurred repeatedly, and he underwent surgical management involving colectomy, colostomy, and ileostomy. We identified mutations in IL-10RA, c.[272A>G];[784C>T] (p.[Y91C];[R262C]). Patient 3 had chronic diarrhea and a rectovaginal fistula at 3 days of life and was diagnosed with Crohn's disease. She underwent fistulectomy and ileostomy, but experienced frequent relapses. Mutations, c.[272A>G];[301C>T] (p.[Y91C];[R101W]), were found in IL-10RA. This report confirms the genetic defect of IL-10RA in neonatal-onset IBD including 'intractable ulcerating enterocolitis in infancy'.


The effects of palm kernel cake based diet on spermatogenesis in Malin x Santa-Ines rams.
Testes from nine male Malin x Santa-Ines rams with an average body weight of 43.1+/-3.53 kg, were used to study the effects of palm kernel cake (PKC) based diet on spermatogenic cells and to assess copper (Cu) levels in liver, testis and plasma in sheep. Animals were divided into three groups and randomly assigned three dietary treatments using restricted randomization of body weight in completely randomized design. The dietary treatments were 60% palm kernel cake plus 40% oil palm frond (PKC), 60% palm kernel cake plus 40% oil palm frond supplemented with 23 mg/kg dry matter of molybdenum as ammonium molybdate [(NH(4))(6)Mo(7)O(24).4H(2)O] and 600 mg/kg dry matter of sulphate as sodium sulphate [Na(2)SO(4)] (PKC-MS) and 60% concentrate of corn-soybean mix+40% oil palm frond (Control), the concentrate was mixed in a ratio of 79% corn, 20% soybean meal and 1% standard mineral mix. The results obtained showed that the number of spermatogonia, spermatocytes, spermatids and Leydig cells were not significantly different among the three treatment groups. However, spermatozoa, Sertoli cells and degenerated cells showed significant changes, which, may be probably due to the Cu content in PKC. Liver and testis Cu levels in the rams under PKC diet was found to be significantly higher (P<0.05) than rams in Control and PKC-MS diets. Plasma Cu levels showed a significant increase (P<0.05) at the end of the experiment as compared to at the beginning of the experiment for PKC and Control. In conclusion, spermatogenesis is normal in rams fed the diet without PKC and PKC supplemented with Mo and S. However spermatogenesis was altered in the PKC based diet probably due to the toxic effects of Cu and the significant changes in organs and plasma. Thus, Mo and S play a major role in reducing the accumulation of Cu in organs.


Impact of antimalarial treatment and chemoprevention on the drug sensitivity of malaria parasites isolated from ugandan children.
Changing treatment practices may be selecting for changes in the drug sensitivity of malaria parasites. We characterized ex vivo drug sensitivity and parasite polymorphisms associated with sensitivity in 459 Plasmodium falciparum samples obtained from subjects enrolled in two clinical trials in Tororo, Uganda, from 2010 to 2013. Sensitivities to chloroquine and monodesethylamodiaquine varied widely; sensitivities to quinine, dihydroartemisinin, lumefantrine, and piperaquine were generally good. Associations between ex vivo drug sensitivity and parasite polymorphisms included decreased chloroquine and monodesethylamodiaquine sensitivity and increased lumefantrine and piperaquine sensitivity with pfcrt 76T, as well as increased lumefantrine sensitivity with pfmdr1 86Y, Y184, and 1246Y. Over time, ex vivo sensitivity decreased for lumefantrine and piperaquine and increased for chloroquine, the prevalences of pfcrt K76 and pfmdr1 N86 and D1246 increased, and the prevalences of pfdhfr and pfdhps polymorphisms associated with antifolate resistance were unchanged. In recurrent infections, recent prior treatment with artemether-lumefantrine was associated with decreased ex vivo lumefantrine sensitivity and increased prevalence of pfcrt K76 and pfmdr1 N86, 184F, and D1246. In children assigned chemoprevention with monthly dihydroartemisinin-piperaquine with documented circulating piperaquine, breakthrough infections had increased the prevalence of pfmdr1 86Y and 1246Y compared to untreated controls. The noted impacts of therapy and chemoprevention on parasite polymorphisms remained significant in multivariate analysis correcting for calendar time. Overall, changes in parasite sensitivity were consistent with altered selective pressures due to changing treatment practices in Uganda. These changes may threaten the antimalarial treatment and preventive efficacies of artemether-lumefantrine and dihydroartemisinin-piperaquine, respectively.


Comparative efficacy and acceptability of first-generation and second-generation antidepressants in the acute treatment of major depression: protocol for a network meta-analysis.
Many antidepressants are indicated for the treatment of major depression. Two network meta-analyses have provided the most comprehensive assessments to date, accounting for both direct and indirect comparisons; however, these reported conflicting interpretation of results. Here, we present a protocol for a systematic review and network meta-analysis aimed at updating the evidence base and comparing all second-generation as well as selected first-generation antidepressants in terms of efficacy and acceptability in the acute treatment of major depression. We will include all randomised controlled trials reported as double-blind and comparing one active drug with another or with placebo in the acute phase treatment of major depression in adults. We are interested in comparing the following active agents: agomelatine, amitriptyline, bupropion, citalopram, clomipramine, desvenlafaxine, duloxetine, escitalopram, fluoxetine, fluvoxamine, levomilnacipran, milnacipran, mirtazapine, nefazodone, paroxetine, reboxetine, sertraline, trazodone, venlafaxine, vilazodone and vortioxetine. The main outcomes will be the proportion of patients who responded to or dropped out of the allocated treatment. Published and unpublished studies will be sought through relevant database searches, trial registries and websites; all reference selection and data extraction will be conducted by at least two independent reviewers. We will conduct a random effects network meta-analysis to synthesise all evidence for each outcome and obtain a comprehensive ranking of all treatments. To rank the various treatments for each outcome, we will use the surface under the cumulative ranking curve and the mean ranks. We will employ local as well as global methods to evaluate consistency. We will fit our model in a Bayesian framework using OpenBUGS, and produce results and various checks in Stata and R. We will also assess the quality of evidence contributing to network estimates of the main outcomes with the GRADE framework. This review does not require ethical approval. CRD42012002291.


Phylogeny of all major groups of cetaceans based on DNA sequences from three mitochondrial genes.
Traditionally, living cetaceans (order Cetacea) are classified into two highly distinct suborders: the echolocating toothed whales, Odontoceti, and the filter-feeding baleen whales, Mysticeti. A molecular phylogeny based on 1,352 base pairs of two mitochondrial ribosomal gene segments and the mitochondrial cytochrome b gene for all major groups of cetaceans contradicts this long-accepted taxonomic subdivision. One group of toothed whales, the sperm whales, is more closely related to the morphologically highly divergent baleen whales than to other odontocetes. This finding suggests that the suborder Odontoceti constitutes an unnatural grouping and challenges the conventional scenario of a long, independent evolutionary history of odontocetes and mysticetes. The superfamily Delphinoidea (dolphins, porpoises, and white whales) appears to be monophyletic; the Amazon River dolphin, Inia geoffrensis, is its sister species. This river dolphin is genetically more divergent from the morphologically similar marine dolphins than the sperm whales are from the morphologically dissimilar baleen whales. The phylogenetic relationships among the three families of Delphinoidea remain uncertain, and we suggest that the two cladogenetic events that generated these three clades occurred within a very short period of time. Among the baleen whales, the bowhead is basal, and the gray whale is the sister species to the rorquals (family Balaenopteridae). The phylogenetic position of beaked whales (Ziphioidea) remains weakly supported by molecular data. Based on molecular clock assumptions, the mitochondrial-DNA data suggest a more recent origin of baleen whales (approximately 25 mya) than has been previously assumed (> 40 mya). This revised phylogeny has important implications for the rate and mode of evolution of morphological and physiological innovations in cetaceans.


SSBP1 mutations cause mtDNA depletion underlying a complex optic atrophy disorder.
Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect the amount of SSBP1 protein and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids, and 7S-DNA amounts as well as mtDNA replication, affecting replisome machinery. The variable mtDNA depletion in cells was reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits, and complex amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex vivo in biopsies of affected tissues, such as kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by WT mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as a cause of human pathology.


Can Parental Engagement in Social Media Enhance Outcomes of an Online Healthy Lifestyle Program for Preschool-Aged Children?
This study explored parental engagement, child BMI and secondary outcomes from the social media component of an online healthy lifestyle program for parents of preschool-aged children. Intervention group participants received access to an online program and Facebook group. Data were collected at baseline and 3- and 6-months follow-up. Facebook usage data on comments and posts were used to determine total active engagement. There was a high level of Facebook group membership and most parents actively engaged at least once. Although there were varying levels of engagement between modules and cohorts, it was modest overall. User acceptability of the Facebook group was lower than expected. Children of parents in the intervention who engaged more in the Facebook group (by posting and commenting) demonstrated greater sleep duration over time (estimate 1.79, 95% CI 0.42 to 3.17, p = .01) Children of parents who engaged more in the Facebook group also participated in less moderate- to vigorous-intensity physical activity (estimate -0.14, 95% CI -0.26 to -0.01, p = .03). This study is one of the first parent-focussed healthy lifestyle interventions to include a social media component. Further research is recommended with larger sample sizes and longer duration to further explore the potential of social media in childhood obesity interventions.


Temporal changes in the prevalence and associates of diabetes-related lower extremity amputations in patients with type 2 diabetes: the Fremantle Diabetes Study.
To determine temporal changes in the prevalence and associates of lower extremity amputation (LEA) complicating type 2 diabetes. Baseline data from the longitudinal observational Fremantle Diabetes Study (FDS) relating to LEA and its risk factors collected from 1296 patients recruited to FDS Phase 1 (FDS1) from 1993 to 1996 and from 1509 patients recruited to FDS Phase 2 (FDS2) from 2008 to 2011 were analysed. Multiple logistic regression was used to determine associates of prevalent LEA in individual and pooled phases. Generalised linear modelling was used to examine whether diabetes related LEA prevalence and its associates had changed between Phases. There were 15 diabetes-related LEAs at baseline in FDS1 (1.2 %) and 15 in FDS2 (1.0 %; P = 0.22 after age, sex and race/ethnicity adjustment). In multivariable analysis, independent associates of a baseline LEA in FDS1 were a history of vascular bypass surgery or revascularisation, urinary albumin:creatinine ratio, peripheral sensory neuropathy and cerebrovascular disease (P ≤ 0.035). In FDS2, prevalent LEA was independently associated with a history of vascular bypass surgery or revascularisation, past hospitalisation for/current foot ulcer and fasting serum glucose (P ≤ 0.001). In pooled analyses, a history of vascular bypass or revascularisation, past hospitalisation for/current foot ulcer at baseline, urinary albumin:creatinine ratio (P < 0.001), as well as FDS Phase as a binary variable [odds ratio (95 % confidence interval): 0.28 (0.09-0.84) for FDS2 vs FDS1, P = 0.023] were associated with a lower risk of LEA at study entry. The risk of prevalent LEA in two cohorts of patients with type 2 diabetes from the same Australian community fell by 72 % over a 15-year period after adjustment for important between-group differences in diabetes-related and other variables. This improvement reflects primary care foot health-related initiatives introduced between Phases, and should have important individual and societal benefits against a background of a progressively increasing diabetes burden.


Late epilepsy following open surgery for aneurysmal subarachnoid haemorrhage.
To determine the risk for late epilepsy (>2 weeks postoperatively) following aneurysmal subarachnoid haemorrhage (SAH) treated by early aneurysm clipping. Subgroup analysis of the East Anglian regional audit of SAH (1994-2000; n = 872) with 12 month follow up. Prophylactic anticonvulsants were not routinely prescribed unless there was a perioperative seizure. 472 patients with aneurysmal SAH undergoing surgical clipping of the aneurysm were studied. Patients presenting in WFNS grade V, with space occupying haematomas requiring emergency surgery, or with posterior circulation aneurysms, rebleeds, and surgery after 21 days were excluded. Late epilepsy occurred in 23 patients (4.9%). There was a correlation between the incidence of late epilepsy and both the presenting WFNS grade (p<0.05) (grade 1, 1.4%; grade 2, 3.8%; grade 3, 9.6%; grade 4, 12.5%) and the Glasgow outcome score at discharge (p<0.01) (good recovery, 2.2%; moderate disability, 5.0%; severe disability, 15.5%). There was no relation between the incidence of late epilepsy and sex or the site of the aneurysm. The low incidence of late epilepsy following open surgery for aneurysmal SAH supports the withholding of prophylactic anticonvulsants. Patients with poor WFNS grade and poor recovery after surgery are at increased risk and should be closely monitored.


Attachment of motile bacterial cells to prealigned holed microarrays.
Construction of biomotors is an exciting area of scientific research that holds great promise for the development of new technologies with broad potential applications in areas such as the energy industry and medicine. Herein, we demonstrate the fabrication of prealigned microarrays of motile Escherichia coli bacterial cells on SiOx substrates. To prepare these arrays, holed surfaces with a gold layer on the bottom of the holes were utilized. The attachment of bacteria to the holes was achieved via nonspecific interactions using poly-l-lysine hydrobromide (PLL). Our data suggest that a single motile bacterial cell can be selectively attached to an individual hole on a surface and bacterial cell binding can be controlled by altering the pH, with the greatest occupancy occurring at pH 7.8. Cells attached to hole arrays remained motile for at least 4 h. These data indicate that holed surface structures provide a promising footprint for the attachment of motile bacterial cells to form high-density site-specific functional bacterial microarrays.


Pharmacological exposure to ribavirin: a key player in the complex network of factors implicated in virological response and anaemia in hepatitis C treatment.
Ribavirin remains today a pivotal drug in the treatment of hepatitis C; in standard double therapy, as well as in triple combination with direct antiviral agents, ribavirin reduces relapse and can double the sustained virological response obtained with peginterferon alone or in association with direct antiviral agents. In the complex network of interacting factors determining sustained virological response independently of known predictive factors related to host and virus, two modern tools are emerging: polymorphisms in the IL28B gene and very early exposure to ribavirin. The use of a pharmacokinetic-pharmacodynamic model of early ribavirin exposure to adjust the dose individually would help promote a safer ribavirin use and improve sustained virological response. The variability of the influence of ribavirin exposure on anaemia is probably genetically determined; however, the low prevalence of the implicated protective alleles of the inosine triphosphate pyrophosphatase gene could explain their lack of influence on sustained virological response.


Phenotypic and functional characterisation of CCR7+ and CCR7- CD4+ memory T cells homing to the joints in juvenile idiopathic arthritis.
The aim of the study was to characterise CCR7+ and CCR7- memory T cells infiltrating the inflamed joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the functional and anatomical heterogeneity of these cell subsets in relation to the expression of the inflammatory chemokine receptors CXCR3 and CCR5. Memory T cells freshly isolated from the peripheral blood and synovial fluid (SF) of 25 patients with JIA were tested for the expression of CCR7, CCR5, CXCR3 and interferon-gamma by flow cytometry. The chemotactic activity of CD4 SF memory T cells from eight patients with JIA to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines was also evaluated. Paired serum and SF samples from 28 patients with JIA were tested for CCL21 concentrations. CCR7, CXCR3, CCR5 and CCL21 expression in synovial tissue from six patients with JIA was investigated by immunohistochemistry. Enrichment of CD4+, CCR7- memory T cells was demonstrated in SF in comparison with paired blood from patients with JIA. SF CD4+CCR7- memory T cells were enriched for CCR5+ and interferon-gamma+ cells, whereas CD4+CCR7+ memory T cells showed higher coexpression of CXCR3. Expression of CCL21 was detected in both SF and synovial membranes. SF CD4+ memory T cells displayed significant migration to both inflammatory and homeostatic chemokines. CCR7+ T cells were detected in the synovial tissue in either diffuse perivascular lymphocytic infiltrates or organised lymphoid aggregates. In synovial tissue, a large fraction of CCR7+ cells co-localised with CXCR3, especially inside lymphoid aggregates, whereas CCR5+ cells were enriched in the sublining of the superficial subintima. In conclusion, CCR7 may have a role in the synovial recruitment of memory T cells in JIA, irrespective of the pattern of lymphoid organisation. Moreover, discrete patterns of chemokine receptor expression are detected in the synovial tissue.


Dynamic and structural constraints in signal propagation by regulatory networks.
Regulatory networks are able to process complex signals and respond appropriately to the cellular context. Thus, an increasing effort by systems biology researchers is being focused on understanding which interactions are responsible for a given functional response. When translated into specific mathematical models, however, it has been repeatedly shown that this mapping between topology and function is not one-to-one, even for the simplest networks. Moreover, dynamical behavior may play an important role which is necessary to integrate in the general picture. We propose a unified theoretical/statistical approach to characterize the structure-function relationship in molecular networks when temporal features of both input signal and output response are important. The theory allows fast computation of network responses in terms of interaction strengths irrespective of molecular details, while statistical analysis identifies constraints between structural and dynamical features and network function. Investigating different feedback and feedforward loop architectures, we find that processing of temporal signals is strongly correlated to certain combinations of structural and dynamical characteristics, rather than to individual interactions. Our analysis offers new insight into the structure-function relationship in network motifs, quantifying how much the tuning of specific interactions affects network outcome, identifying key structural parameters for a given response and relating dynamics to network topology and function. This kind of analyses can be especially useful for synthetic biology approaches, where promoter libraries with a range of inputs and outputs can be engineered, and one has to choose the correct component needed to produce the desired network function.


Leisure-Time Screen-Based Sedentary Behavior and Leukocyte Telomere Length: Implications for a New Leisure-Time Screen-Based Sedentary Behavior Mechanism.
The field of sedentary behavior epidemiology is emerging. Short leukocyte telomere length (LTL) is a hallmark characteristic of aging, but LTL is also associated with morbidity and mortality independent of age. To my knowledge, only one study has examined the association between sedentary behavior and LTL. The purpose of this study was to examine the association between screen-based sedentary behavior and LTL. Data from the 1999-2002 National Health and Nutrition Examination Survey were used (N=6405; age, 20-84 years). Leisure-time screen-based sedentary behavior (television, video games, computer use) was assessed via questionnaire, and LTL was extracted from DNA in whole blood with the LTL assay performed using quantitative polymerase chain reaction. After adjustments (including age and physical activity), for every 1-hour increase in leisure-time screen-based sedentary behavior, participants had a 7% increased odds (odds ratio, 1.07; 95% CI, 1.00-1.13; P=.04) of having LTL in the lowest tertile (vs highest); leisure-time screen-based sedentary behavior was not associated with values in the middle (vs highest) tertile (odds ratio, 1.01; 95% CI, 0.95-1.07; P=.62). The results of this study revealed that greater leisure-time screen-based sedentary behavior is associated with shorter LTL.


Reverse inference of memory retrieval processes underlying metacognitive monitoring of learning using multivariate pattern analysis.
Monitoring of learning is only accurate at some time after learning. It is thought that immediate monitoring is based on working memory, whereas later monitoring requires re-activation of stored items, yielding accurate judgements. Such interpretations are difficult to test because they require reverse inference, which presupposes specificity of brain activity for the hidden cognitive processes. We investigated whether multivariate pattern classification can provide this specificity. We used a word recall task to create single trial examples of immediate and long term retrieval and trained a learning algorithm to discriminate them. Next, participants performed a similar task involving monitoring instead of recall. The recall-trained classifier recognized the retrieval patterns underlying immediate and long term monitoring and classified delayed monitoring examples as long-term retrieval. This result demonstrates the feasibility of decoding cognitive processes, instead of their content.


Trypanosoma cruzi chemical proteomics using immobilized benznidazole.
Benznidazole (Bzn) is a nitroimidazole drug currently used as first line treatment against Chagas disease, a neglected tropical disease caused by the flagellated protozoan Trypanosoma cruzi. Although the drug has been used since the late 1960s, its mechanism of action is not fully understood. In an attempt to study Bzn mode of action, a structurally modified derivative of the drug was synthesized and immobilized into a solid matrix. This allowed enrichment of T. cruzi proteins capable of binding immobilized Bzn, which were subsequently analysed by mass spectrometry. The proteins identified as specific non-covalent Bzn interactors were a homologue of the bacterial YjeF proteins, a Sec23A orthologue and the aldo-ketoreductase family member TcAKR. TcAKR is closely related to other enzymes previously associated with Bzn reductive activation such as NTRI and TcOYE. Thus, our untargeted search for Bzn binding partners allowed us to encounter proteins that could be related to drug reductive activation and/or resistance mechanisms.


Age-related cleavages of crystallins in human lens cortical fiber cells generate a plethora of endogenous peptides and high molecular weight complexes.
Low molecular weight peptides derived from the breakdown of crystallins have been reported in adult human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilization of crystallins. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of four human lenses representing young, middle and old-age human lenses. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C-terminus with significantly higher frequency compared to other residues, suggesting that trypsin-like proteolysis may be active in the lens cortical fiber cells. Selected reaction monitoring analysis of an endogenous αA-crystallin peptide (αA(57-65)) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA(57-65) formation escalated significantly. Using 2D gel electrophoresis/nanoLC-ESI-MS/MS, 12 protein complexes of 40-150 kDa consisting of multiple crystallin components were characterized from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross-linking, with these complexes potentially acting to stabilize degraded crystallins by sequestration into water soluble complexes.


Delivery of factor VIII gene into skeletal muscle cells using lentiviral vector.
This study was designed to investigate whether transduction of lentiviral vectors (LV) carrying human coagulation factor VIII (hFVIII) cDNA into skeletal muscle could increase circulating hFVIII concentrations. A LV containing bacterial LacZ gene as a control or human FVIII gene was intramuscularly administered into the thigh muscle of 5 weeks old Sparague-Dawley rats. The plasma human FVIII concentration and neutralizing anti-FVIII antibodies were measured for up to 12 weeks in these experimental animals. The plasma human FVIII levels in the rats injected with LV carrying FVIII cDNA peaked at post-injection 1st week (5.19 +/- 0.14 ng/mL vs. 0.21 +/- 0.05 ng/mL in control rats , p < 0.05). Elevated hFVIII concentrations were maintained for 4 weeks (2.52 +/- 0.83 ng/mL vs. 0.17 +/- 0.08 ng/mL in control rats, p < 0.05) after a single intramuscular injection. In the Bethesda assay, neutralizing antibodies for FVIII protein were detected only in FVIII-LV injected rats by the 10th week, but not in control rats. This study suggested that a single administration of an advanced generation LV carrying the human FVIII cDNA resulted in elevation of FVIII level in immune competent rats, and that this gene transfer approach to the skeletal muscle could be an effective tool in treatment of hemophilia A.


Antigen processing via autophagy--not only for MHC class II presentation anymore?
T cells monitor intracellular and extracellular protein composition via proteolytic products that are displayed to them on major histocompatibility complex (MHC) molecules. For this purpose it has been documented that MHC class II molecules, which were originally thought to just display lysosomal products of endocytosed proteins to CD4(+) helper T cells, can also present intracellular substrates of autophagic pathways. This has triggered the interest of immunologists into the role of autophagy in antigen processing in general, and recently additional autophagic mechanisms for intracellular and extracellular antigen processing onto MHC class I molecules for presentation to CD8(+) cytolytic T cells have been revealed. Here, I will review the contribution of autophagy for MHC class I and class II antigen processing and presentation to T cells.


Human ghrelin ameliorates organ injury and improves survival after radiation injury combined with severe sepsis.
In the terrorist radiation exposure scenario, radiation victims are likely to suffer from additional injuries such as sepsis. Our previous studies have shown that ghrelin is protective in sepsis. However, it remains unknown whether ghrelin ameliorates sepsis-induced organ injury and mortality after radiation exposure. The purpose of this study is to determine whether human ghrelin attenuates organ injury and improves survival in a rat model of radiation combined injury (RCI) and, if so, the potential mechanism responsible for the benefit. To study this, adult male rats were exposed to 5-Gy whole body irradiation followed by cecal ligation and puncture (CLP, a model of sepsis) 48 h thereafter. Human ghrelin (30 nmol/rat) or vehicle (saline) was infused intravenously via an osmotic minipump immediately after radiation exposure. Blood and tissue samples were collected at 20 h after RCI (68 h after irradiation or 20 h after CLP) for various measurements. To determine the longterm effect of human ghrelin after RCI, the gangrenous cecum was removed at 5 h after CLP and 10-d survival was recorded. In addition, vagotomy or sham vagotomy was performed in sham and RCI animals immediately prior to ghrelin administration, and various measurements were performed at 20 h after RCI. Our results showed that serum levels of ghrelin and its gene expression in the stomach were decreased markedly at 20 h after RCI. Administration of human ghrelin attenuated tissue injury markedly, reduced proinflammatory cytokine levels, decreased tissue myeloperoxidase activity, and improved survival after RCI. Furthermore, elevated plasma levels of norepinephrine (NE) after RCI were reduced significantly by ghrelin. However, vagotomy prevented ghrelin's beneficial effects after RCI. In conclusion, human ghrelin is beneficial in a rat model of RCI. The protective effect of human ghrelin appears to be attributed to re-balancing the dysregulated sympathetic/parasympathetic nervous systems.


Genome-wide analysis of mRNAs regulated by Drosha and Argonaute proteins in Drosophila melanogaster.
RNA silencing pathways are conserved gene regulation mechanisms that elicit decay and/or translational repression of mRNAs complementary to short interfering RNAs and microRNAs (miRNAs). The fraction of the transcriptome regulated by these pathways is not known, but it is thought that each miRNA may have hundreds of targets. To identify transcripts regulated by silencing pathways at the genomic level, we examined mRNA expression profiles in Drosophila melanogaster cells depleted of four Argonaute paralogs (i.e., AGO1, AGO2, PIWI, or Aubergine) that play essential roles in RNA silencing. We also profiled cells depleted of the miRNA-processing enzyme Drosha. The results reveal that transcripts differentially expressed in Drosha-depleted cells have highly correlated expression in the AGO1 knockdown and are significantly enriched in predicted and validated miRNA targets. The levels of a subset of miRNA targets are also regulated by AGO2. Moreover, AGO1 and AGO2 silence the expression of a common set of mobile genetic elements. Together, these results indicate that the functional overlap between AGO1 and AGO2 in Drosophila is more important than previously thought.


Delivery of plasmid DNA to vascular tissue in vivo using catheter balloons coated with polyelectrolyte multilayers.
We report an approach for the localized delivery of plasmid DNA to vascular tissue from the surfaces of inflatable embolectomy catheter balloons. Using a layer-by-layer approach, ultrathin multilayered polyelectrolyte films were fabricated on embolectomy catheter balloons by alternately adsorbing layers of a hydrolytically degradable poly(β-amino ester) and plasmid DNA. Fluorescence microscopy revealed that the films coated the surfaces of the balloons uniformly. Coated balloons that were incubated in phosphate-buffered saline at 37 °C released ∼25 μg DNA/cm(2) over 24 h. Analysis of the DNA by gel electrophoresis showed that the DNA was released in open-circular ('nicked') and supercoiled conformations, and in vitro cell transfection assays confirmed that the released DNA was transcriptionally active. Arterial injury was induced in the left common, carotid arteries of Sprague-Dawley rats using uncoated balloons, followed by treatment with film-coated balloons for 20 min. X-gal, immunohistochemical, and immunofluorescence staining of sectioned arteries indicated high levels of β-galactosidase or enhanced green fluorescent protein (EGFP) expression in arteries treated with film-coated balloons. β-galactosidase and EGFP expression were observed throughout the medial layers of arterial tissue, and around approximately two-thirds of the circumference of the treated arteries. The layer-by-layer approach reported here provides a general platform for the balloon-mediated delivery of DNA to vascular tissue. Our results suggest the potential of this approach to deliver therapeutically relevant DNA to prevent complications such as intimal hyperplasia that arise after vascular interventions.


Micro-fusion inhibition tests: quantifying antibody neutralization of virus-mediated cell-cell fusion.
Although enveloped viruses canonically mediate particle entry through virus-cell fusion, certain viruses can spread by cell-cell fusion, brought about by receptor engagement and triggering of membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen in vivo, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell-cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell-cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell-cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP-Renilla luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell-cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.


Encoding instructions and stimulus presentation in local environmental context-dependent memory studies.
Murnane and Phelps (1993) recommend word pair presentations in local environmental context (EC) studies to prevent associations being formed between successively presented items and their ECs and a consequent reduction in the EC effect. Two experiments were conducted to assess the veracity of this assumption. In Experiment 1, participants memorised single words or word pairs, or categorised them as natural or man made. Their free recall protocols were examined to assess any associations established between successively presented items. Fewest associations were observed when the item-specific encoding task (i.e., natural or man made categorisation of word referents) was applied to single words. These findings were examined further in Experiment 2, where the influence of encoding instructions and stimulus presentation on local EC dependent recognition memory was examined. Consistent with recognition dual-process signal detection model predictions and findings (e.g., Macken, 2002; Parks & Yonelinas, 2008), recollection sensitivity, but not familiarity sensitivity, was found to be local EC dependent. However, local EC dependent recognition was observed only after item-specific encoding instructions, irrespective of stimulus presentation. These findings and the existing literature suggest that the use of single word presentations and item-specific encoding enhances local EC dependent recognition.


Spectral tuning in the eyes of deep-sea lanternfishes (Myctophidae): a novel sexually dimorphic intra-ocular filter.
Deep-sea fishes possess several adaptations to facilitate vision where light detection is pushed to its limit. Lanternfishes (Myctophidae), one of the world's most abundant groups of mesopelagic fishes, possess a novel and unique visual specialisation, a sexually dimorphic photostable yellow pigmentation, constituting the first record of a visual sexual dimorphism in any non-primate vertebrate. The topographic distribution of the yellow pigmentation across the retina is species specific, varying in location, shape and size. Spectrophotometric analyses reveal that this new retinal specialisation differs between species in terms of composition and acts as a filter, absorbing maximally between 356 and 443 nm. Microspectrophotometry and molecular analyses indicate that the species containing this pigmentation also possess at least 2 spectrally distinct rod visual pigments as a result of a duplication of the Rh1 opsin gene. After modelling the effect of the yellow pigmentation on photoreceptor spectral sensitivity, we suggest that this unique specialisation acts as a filter to enhance contrast, thereby improving the detection of bioluminescent emissions and possibly fluorescence in the extreme environment of the deep sea. The fact that this yellow pigmentation is species specific, sexually dimorphic and isolated within specific parts of the retina indicates an evolutionary pressure to visualise prey/predators/mates in a particular part of each species' visual field.


Quenchable high-pressure polymorph of zinc selenate.
A new quenchable high-pressure form of zinc selenate (ZnSeO(4)) was produced by subjecting the low-pressure modification to 40 kilobars at 400 degrees C for 30 minutes. The new form is orthorhombic, space group D(2h),(17)-Cmcm. The cell constants at 29 degrees C are: a, 5.511 angstroms; b, 8.110 angstroms; and c, 6.585 angstroms. The calculated density is 4.70 grams per cubic centimeter in comparison with 4.61 grams per cubic centimeter for the low-pressure modification. This implies a volume change of 2 percent at the transition.


Improved trabecular bone structure of 20-month-old male spontaneously hypertensive rats.
A few clinical studies have reported that elderly male participants with hypertensive disease frequently have higher bone mineral density (BMD) than the normotensive participants at several skeletal sites. The detailed mechanism is still unknown; therefore, a study of bone structure and density using the hypertensive animal models could be informative. We used micro-computed tomography to quantitatively evaluate the tibial and 3rd lumbar vertebral bones in the 20-month-old male spontaneous hypertensive rat (SHR). The BMD, volume fraction, and the microarchitecture changes of the SHR were compared to those of same-age normotensive controls (Wistar-Kyoto rat, WKY). We found that in the very old (20 month) male rats, the trabecular bone fraction and microstructure were higher than those in the same-age normotensive controls. The observation of the association of hypertension with BMD and bone strength in hypertensive rats warrants further investigations of bone mass and strength in elderly males with hypertension.


Managing hypoglycemia in diabetes may be more fear management than glucose management: a practical guide for diabetes care providers.
Diabetes management is complex and requires significant effort from the person with diabetes to achieve recommended self-management behaviours. Achieving guideline concordant self-management is made easier when the person with diabetes is committed to the behaviours. Ambivalence is the psychological state in which a person experiences inconsistent drives; both toward and away from the recommended behaviour. Ambivalence about achieving recommended control over blood glucose is expected in situations of hypoglycaemia, due to the associated dangers. In this paper we demonstrate that hypoglycaemia is a fear event and is likely to elicit strong drives to avoid future hypoglycaemia as a fear coping strategy. For many, this results in hyperglycaemia. If hyperglycaemia to avoid hypoglycaemia is a fear management strategy, then hypoglycaemia management should involve fear management. Few diabetes healthcare providers are trained, skilled and confident in fear management. The purpose of this paper is to review the evidence on the psychological consequences of hypoglycaemia and to outline fear management strategies that can be implemented by diabetes care providers. A step-by-step guide is provided to facilitate understanding of the process of the intervention.


Efficacy of Botulinum Toxin A Treatment for Pes Varus during Gait.
The efficacy of botulinum toxin A (BoNTA) injection on spasticity is usually measured using Modified Ashworth Scale (MAS), but this only evaluates muscle tone at rest and has poor reliability. There are no reports that quantitatively evaluate pes varus during walking after botulinum treatment. The purpose of this study was to evaluate the efficacy of BoNTA injection on pes varus during gait using 3-dimensional motion analysis. Twenty-four hemiplegic patients with spastic pes varus deformity during gait received BoNTA injection into lower limb muscles. MAS score, comfortable overground gait velocity, and pes varus angle during treadmill walking were evaluated before, 2, 6, and 12 weeks after the injection. Five healthy subjects were also recruited to develop the pes varus/valgus angle as a normal reference. The median MAS scores were significantly lower at 2 and 6 weeks after the injection. The maximum pes varus angle during the swing phase was significantly lower at 2, 6, and 12 weeks after the injection. It was significantly lower at 6 weeks after the injection during stance phase. The comfortable overground gait velocity was also improved after the injection. However, 2 patients experienced pain during gait and their pes varus angle increased during the follow-up period. BoNTA injection improved pes varus angle during gait. Evaluating motion in addition to spasticity at rest is recommended because improvements in limb function do not always parallel improvements in spasticity at rest.


RhoB controls endothelial cell morphogenesis in part via negative regulation of RhoA.
Recent studies have suggested a role for the small GTPase RhoB in the control of processes required for angiogenesis. However, the mechanisms whereby RhoB exerts control over these processes are not well understood. Given the role of vascular endothelial growth factor (VEGF) in pathological angiogenesis, we were interested in examining whether RhoB contributed to VEGF-induced angiogenic processes. To assess this, RhoB was specifically depleted in human umbilical vein endothelial cells (HUVEC), using siRNA-targeted strategies. The effects of RhoB depletion on VEGF-induced angiogenic activities were assessed using a variety of standard in vitro angiogenesis assays to assess endothelial cell viability, migration and capillary morphogenesis. Effects of RhoB depletion on signaling from other Rho family member proteins was also assessed using specific activity assays for RhoA and RhoC. We observed that although RhoB appeared dispensable for HUVEC viability, RhoB was required for endothelial cell migration, sprouting, and capillary morphogenesis. We also observed that siRNA-mediated depletion of RhoB in HUVEC resulted in increased RhoA activation in response to VEGF stimulation. This increased RhoA activation contributed to the cellular morphogenesis defects observed in RhoB-depleted cells, as inhibition of RhoA activity using C3 transferase, or inhibition of the activity of the downstream RhoA effectors Rho-dependent kinases I and II (ROCK I and II) led to a partial restoration of capillary morphogenesis in the absence of RhoB. Thus our data indicate that RhoB plays a significant role in VEGF-induced endothelial cell morphogenesis in part by negatively regulating the activity of RhoA and the RhoA/ROCK pathway.


One-bit-matching theorem for ICA, convex-concave programming on polyhedral set, and distribution approximation for combinatorics.
According to the proof by Liu, Chiu, and Xu (2004) on the so-called one-bit-matching conjecture (Xu, Cheung, and Amari, 1998a), all the sources can be separated as long as there is an one-to-one same-sign correspondence between the kurtosis signs of all source probability density functions (pdf's) and the kurtosis signs of all model pdf's, which is widely believed and implicitly supported by many empirical studies. However, this proof is made only in a weak sense that the conjecture is true when the global optimal solution of an independent component analysis criterion is reached. Thus, it cannot support the successes of many existing iterative algorithms that usually converge at one of the local optimal solutions. This article presents a new mathematical proof that is obtained in a strong sense that the conjecture is also true when any one of local optimal solutions is reached in helping to investigating convex-concave programming on a polyhedral set. Theorems are also provided not only on partial separation of sources when there is a partial matching between the kurtosis signs, but also on an interesting duality of maximization and minimization on source separation. Moreover, corollaries are obtained on an interesting duality, with supergaussian sources separated by maximization and subgaussian sources separated by minimization. Also, a corollary is obtained to confirm the symmetric orthogonalization implementation of the kurtosis extreme approach for separating multiple sources in parallel, which works empirically but lacks mathematical proof. Furthermore, a linkage has been set up to combinatorial optimization from a distribution approximation perspective and a Stiefel manifold perspective, with algorithms that guarantee convergence as well as satisfaction of constraints.


Primary human CD34+ hematopoietic stem and progenitor cells express functionally active receptors of neuromediators.
Recently, overlapping molecular phenotypes of hematopoietic and neuropoietic cells were described in mice. Here, we examined primary human CD34(+) hematopoietic stem and progenitor cells applying specialized cDNA arrays, real-time reverse-transcriptase-polymerase chain reaction (RT-PCR), and fluorescent-activated cell sorter (FACS) analysis focusing on genes involved in neurobiologic functions. We found expression of vesicle fusion and motility genes, ligand- and voltage-gated ion channels, receptor kinases and phosphatases, and, most interestingly, mRNA as well as protein expression of G protein-coupled receptors of neuromediators (corticotropin-releasing hormone 1 [CRH 1] and CRH 2 receptors, orexin/hypocretin 1 and 2 receptors, GABAB receptor, adenosine A(2)B receptor, opioid kappa 1 and mu 1 receptors, and 5-HT 1F receptor). As shown by 2-color immunofluorescence, the protein expression of these receptors was higher in the more immature CD38(dim) than in the CD38(bright) subset within the CD34(+) population, and completely absent in fully differentiated blood cells, suggesting that those receptors play a role in developmentally early CD34(+) stem and progenitor cells. The intracellular concentration of cyclic adenosine monophosphate (cAMP) in CD34(+) cells was diminished significantly upon stimulation of either CRH or orexin receptors, indicating that those are functionally active and coupled to inhibitory G proteins in human hematopoietic cells. In conclusion, these findings suggest a molecular interrelation of neuronal and hematopoietic signaling mechanisms in humans.


Novel TECTA mutations identified in stable sensorineural hearing loss and their clinical implications.
TECTA is a causative gene of autosomal dominant (DFNA8/A12) and autosomal recessive (DFNB 21) nonsyndromic sensorineural hearing loss (NSHL). Mutations in TECTA account for 4% of all autosomal dominant NSHL cases in some populations and are thus thought to be one of the major causes of autosomal dominant NSHL. A genotype-phenotype correlation for autosomal dominant mutations in the TECTA gene has been proposed. Two families (SB146 and SB149), which segregated moderate NSHL in an autosomal dominant fashion, were included in this study. We performed targeted resequencing of 134 known deafness genes (TRS-134) and bioinformatics analyses to find causative mutations for NSHL in these 2 families. Through TRS-134, we detected 2 novel mutations, i.e. c.3995G>T (p.C1332F) and c.5618C>T (p.T1873I), in the TECTA gene. These mutations cosegregated with NSHL in the studied families and were not detected in normal controls. The mutations c.3995G>T and c.5618C>T reside in the von Willebrand factor type D3-D4 (vWFD3-D4) interdomain of the zonadhesin (ZA) domain and the zona pellucida (ZP) domain, respectively. p.C1332F is the first mutation detected in the vWFD3-D4 interdomain of the ZA domain. The mutations p.C1332F and p.T1873I were associated with stable high-frequency and mid-frequency hearing loss, respectively. Notably, the cysteine residue mutated to phenylalanine in SB146 was not related to progression of sensorineural hearing loss, which argues against the previous hypothesis. Here we confirm a known genotype-phenotype correlation for the ZP domain and propose a hypothetical genotype-phenotype correlation which relates mutations in vWFD3-D4 to stable high-frequency NSHL in Koreans. This clinical feature makes subjects with the missense mutation in the vWFD3-D4 interdomain of TECTA potentially good candidates for middle ear implantation.


An Experimental Model of Vasovagal Syncope Induces Cerebral Hypoperfusion and Fainting-Like Behavior in Awake Rats.
Vasovagal syncope, a contributing factor to elderly falls, is the transient loss of consciousness caused by decreased cerebral perfusion. Vasovagal syncope is characterized by hypotension, bradycardia, and reduced cerebral blood flow, resulting in fatigue, altered coordination, and fainting. The purpose of this study is to develop an animal model which is similar to human vasovagal syncope and establish an awake animal model of vasovagal syncope. Male Sprague-Dawley rats were subjected to sinusoidal galvanic vestibular stimulation (sGVS). Blood pressure, heart rate, and cerebral blood flow were monitored before, during, and post-stimulation. sGVS resulted in hypotension, bradycardia, and decreased cerebral blood flow. One cohort of animals was subjected to sGVS while freely moving. sGVS in awake animals produced vasovagal syncope-like symptoms, including fatigue and uncoordinated movements; two animals experienced spontaneous falling. Another cohort of animals was preconditioned with isoflurane for several days before being subjected to sGVS. Isoflurane preconditioning before sGVS did not prevent sGVS-induced hypotension or bradycardia, yet isoflurane preconditioning attenuated sGVS-induced cerebral blood flow reduction. The sGVS rat model mimics elements of human vasovagal syncope pathophysiology (hypotension, bradycardia, and decreased cerebral perfusion), including behavioral symptoms such as fatigue and altered balance. This study indicates that the sGVS rat model is similar to human vasovagal syncope and that therapies directed at preventing cerebral hypoperfusion may decrease syncopal episodes and reduce injuries from syncopal falls.


Early Lance-Adams syndrome after cardiac arrest: Prevalence, time to return to awareness, and outcome in a large cohort.
Early myoclonus after cardiac arrest (CA) is traditionally viewed as a poor prognostic sign (status myoclonus). However, some patients may present early Lance-Adams syndrome (LAS): under appropriate treatment, they can reach a satisfactory functional outcome. Our aim was to describe their profile, focusing on pharmacologic management in the ICU, time to return of awareness, and long-term prognosis. Adults with early LAS (defined as generalized myoclonus within 96h, with epileptiform EEG within 48h after CA) were retrospectively identified in our CA registry between 2006 and 2016. Functional outcome was assessed through cerebral performance categories (CPC) at 3 months, CPC 1-2 defined good outcome. Among 458 consecutive patients, 7 (1.5%) developed early LAS (4 women, median age 59 years). Within 72h after CA, in normothemia and off sedation, all showed preserved brainstem reflexes and localized pain. All patients were initially treated with valproate, levetiracetam and clonazepam; additional agents, including propofol and midazolam, were prescribed in the majority. First signs of awareness occurred after 3-23 days (median 11.8); 3/7 reached a good outcome at 3 months. Early after CA, myoclonus together with a reactive, epileptiform EEG, preserved evoked potentials and brainstem reflexes suggests LAS. This condition was managed with a combination of highly dosed, large spectrum antiepileptic agents including propofol and midazolam. Even if awakening was at times delayed, good outcome occurred in a substantial proportion of patients.


Transplantation of Human Amniotic Mesenchymal Stem Cells Promotes Functional Recovery in a Rat Model of Traumatic Spinal Cord Injury.
Human amniotic membrane mesenchymal stem cells (hAMSCs) are considered ideal candidate stem cells for cell-based therapy. In this study, we assessed whether hAMSCs transplantation promotes neurological functional recovery in rats after traumatic spinal cord injury (SCI). In addition, the potential mechanisms underlying the possible benefits of this therapy were investigated. Female Sprague-Dawley rats were subjected to SCI using a weight drop device and then hAMSCs, or phosphate-buffered saline (PBS) were immediately injected into the contused dorsal spinal cord at 2 mm rostral and 2 mm caudal to the injury site. Our results indicated that transplanted hAMSCs migrated in the host spinal cord without differentiating into neuronal or glial cells. Compared with the control group, hAMSCs transplantation significantly decreased the numbers of ED1+ macrophages/microglia and caspase-3+ cells. In addition, hAMSCs transplantation significantly increased the levels of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in the injured spinal cord, and promoted both angiogenesis and axonal regeneration. These effects were associated with significantly improved neurobehavioral recovery in the hAMSCs transplantation group. These results show that transplantation of hAMSCs provides neuroprotective effects in rats after SCI, and could be candidate stem cells for the treatment of SCI.


Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA.
Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis.


Social facilitation of long-lasting memory is mediated by CO2 in Drosophila.
How social interactions influence cognition is a fundamental question, yet rarely addressed at the neurobiological level. It is well established that the presence of conspecifics affects learning and memory performance, but the neural basis of this process has only recently begun to be investigated. In the fruit fly Drosophila melanogaster, the presence of other flies improves retrieval of a long-lasting olfactory memory. Here, we demonstrate that this is a composite memory composed of two distinct elements. One is an individual memory that depends on outputs from the α'β' Kenyon cells (KCs) of the mushroom bodies (MBs), the memory center in the insect brain. The other is a group memory requiring output from the αβ KCs, a distinct sub-part of the MBs. We show that social facilitation of memory increases with group size and is triggered by CO2 released by group members. Among the different known neurons carrying CO2 information in the brain, we establish that the bilateral ventral projection neuron (biVPN), which projects onto the MBs, is necessary for social facilitation. Moreover, we demonstrate that CO2-evoked memory engages a serotoninergic pathway involving the dorsal-paired medial (DPM) neurons, revealing a new role for this pair of serotonergic neurons. Overall, we identified both the sensorial cue and the neural circuit (biVPN>αβ>DPM>αβ) governing social facilitation of memory in flies. This study provides demonstration that being in a group recruits the expression of a cryptic memory and that variations in CO2 concentration can affect cognitive processes in insects.


Effects of prenatal exposure to air pollutants (polycyclic aromatic hydrocarbons) on the development of brain white matter, cognition, and behavior in later childhood.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and neurotoxic environmental contaminants. Prenatal PAH exposure is associated with subsequent cognitive and behavioral disturbances in childhood. To identify the effects of prenatal PAH exposure on brain structure and to assess the cognitive and behavioral correlates of those abnormalities in school-age children. Cross-sectional imaging study in a representative community-based cohort followed up prospectively from the fetal period to ages 7 to 9 years. The setting was urban community residences and an academic imaging center. Participants included a sample of 40 minority urban youth born to Latina (Dominican) or African American women. They were recruited between February 2, 1998, and March 17, 2006. Morphological measures that index local volumes of the surface of the brain and of the white matter surface after cortical gray matter was removed. We detected a dose-response relationship between increased prenatal PAH exposure (measured in the third trimester but thought to index exposure for all of gestation) and reductions of the white matter surface in later childhood that were confined almost exclusively to the left hemisphere of the brain and that involved almost its entire surface. Reduced left hemisphere white matter was associated with slower information processing speed during intelligence testing and with more severe externalizing behavioral problems, including attention-deficit/hyperactivity disorder symptoms and conduct disorder problems. The magnitude of left hemisphere white matter disturbances mediated the significant association of PAH exposure with slower processing speed. In addition, measures of postnatal PAH exposure correlated with white matter surface measures in dorsal prefrontal regions bilaterally when controlling for prenatal PAH. Our findings suggest that prenatal exposure to PAH air pollutants contributes to slower processing speed, attention-deficit/hyperactivity disorder symptoms, and externalizing problems in urban youth by disrupting the development of left hemisphere white matter, whereas postnatal PAH exposure contributes to additional disturbances in the development of white matter in dorsal prefrontal regions.


Development of dendritic cells from GM-CSF-/- mice in vitro : GM-CSF enhances production and survival of cells.
The production of dendritic cells (DC) from haemopoietic progenitors maintained in long term stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independently of added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility that cultures depend on endogenous GM-CSF produced in low levels was tested by attempting to generate LTC from spleen and BM of GM-CSF-/- mice. Multiple cultures from GM-CSF-/- and wild type mice were established and compared for cell production. GM-CSF-/- LTC developed more slowly, but by 16 weeks produced cells resembling DC in numbers comparable to wild type cultures. LTC maintained distinct populations of small and large cells, the latter resembling DC. Cells collected from GM-CSF-/- LTC were capable antigen presenting cells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressed the CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF to GM-CSF-/- LTC increased the proportion of large, mature DC present in culture. Stromal cells from GM-CSF-/- LTC could support the differentiation of DC from early progenitors maintained in LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells in increasing the survival of mature DC.


Mental Health Conditions and Medical and Surgical Hospital Utilization.
Mental health conditions are prevalent among children hospitalized for medical conditions and surgical procedures, but little is known about their influence on hospital resource use. The objectives of this study were to examine how hospitalization characteristics vary by presence of a comorbid mental health condition and estimate the association of a comorbid mental health condition with hospital length of stay (LOS) and costs. Using the 2012 Kids' Inpatient Database, we conducted a retrospective, nationally representative, cross-sectional study of 670 161 hospitalizations for 10 common medical and 10 common surgical conditions among 3- to 20-year-old patients. Associations between mental health conditions and hospital LOS were examined using adjusted generalized linear models. Costs of additional hospital days associated with mental health conditions were estimated using hospital cost-to-charge ratios. A comorbid mental health condition was present in 13.2% of hospitalizations. A comorbid mental health condition was associated with a LOS increase of 8.8% (from 2.5 to 2.7 days, P < .001) for medical hospitalizations and a 16.9% increase (from 3.6 to 4.2 days, P < .001) for surgical hospitalizations. For hospitalizations in this sample, comorbid mental health conditions were associated with an additional 31 729 (95% confidence interval: 29 085 to 33 492) hospital days and $90 million (95% confidence interval: $81 to $101 million) in hospital costs. Medical and surgical hospitalizations with comorbid mental health conditions were associated with longer hospital stay and higher hospital costs. Knowledge about the influence of mental health conditions on pediatric hospital utilization can inform clinical innovation and case-mix adjustment.


Quantitative analysis of resistance to natural killer attacks reveals stepwise killing kinetics.
Molecular mechanisms can protect cancer cells from immune attacks. At the level of bulk tissue, these survival mechanisms are often indistinguishable and simply appear as reduced cell death. However, by tracking individual cell survival and death times, we found broad variation in the kinetics of immune evasion. In response to attacks by natural killer cells, we observed that some cancer lines exhibited exponential survival time distributions. Slowly killed cancer lines had reduced exponential rate constants. In contrast, a line engineered to express the serpin protein PI-9, which is known to promote resistance to immune killing, exhibited a markedly nonexponential survival time distribution. By following the histories of individual cancer cells with multiplexed reporters, we obtained evidence that two or more immune attacks are likely required to kill serpin-expressing cells. Thus, resistance is a finite and measurable quantity, with a distinct kinetic signature. A quantitative model based on independently measured parameters is consistent with our conclusions.


Differential constraints on the working memory and reading abilities of individuals with learning difficulties and typically developing children.
This study examined the factors that constrain the working memory span performance and reading ability of individuals with generalized learning difficulties. In the study, 50 individuals with learning difficulties (LD) and 50 typically developing children (TD) matched for reading age completed two working memory span tasks. Participants also completed independent measures of the processing and storage operations involved in each working memory span task and Raven's Coloured Progressive Matrices. The results showed that despite an equivalent level of working memory span, the relative importance of the constraints on working memory differed between the groups. In addition, working memory span was not closely related to word recognition or sentence comprehension performance in the LD group. These results suggest that the working memory span performance of LD and TD individuals may reflect different working memory limitations and that individuals with generalized learning difficulties may approach cognitive tasks in a qualitatively different way from that of typically developing individuals.


Fatal Salmonella septicaemia with disseminated intravascular coagulation and renal failure.
Four elderly patients with salmonella septicaemia complicated by irreversible endotoxic shock and acute renal failure are described. The organism isolated from three of the patients with Salmonella typhimurium, which is not usually invasive; the fourth patient was infected with S. abony. The factors associated with the severe systemic infections are discussed. Clinical and haematological findings suggested disseminated intravascular coagulation, and this was subsequently confirmed histologically in each patient by the identification of haematoxylinophil bodies in the pulmonary and renal microcirculations.


Egg oiling to reduce hatch-year ring-billed gull numbers on Chicago's beaches during swim season and water quality test results.
A burgeoning ring-billed gull population along Chicago's Lake Michigan beaches contributes to degraded water quality through fecal contamination. Egg oiling was conducted at Chicago's gull colonies to reduce production and the influx of hatch-year (HY) gulls using Chicago's beaches, with a second, long-term objective of eventually reducing adult gull numbers through attrition. We also investigated swim season water quality trends through the course of this work. From 2007 to 2009, 52, 80, and 81%, of nests at the two primary nest colonies had their eggs rendered inviable by corn oil application. Counts of HY and after hatch-year (AHY) gulls were analyzed during treatment years for 10 beaches. Water quality data were available from the Chicago Park District during our three treatment years and the prior year (baseline) for 19 beaches. HY counts declined at all 10 surveyed beaches from the initial year (52% nests with oiled eggs) to subsequent years with ~80% of nests oiled. Overall, HY gulls numbers on beaches decreased 86% from 2007 to 2009. Decreases in beach usage by AHY gulls were not detected. Compared to pretreatment, the number of beaches with improved water quality test rates increased each year through the course of the study. The frequency of water quality tests showing bacterial exceedances compared to 2006 declined at 18 of 19 beaches by 2009. Egg oiling resulted in fewer HY gulls using Chicago's beaches and was likely a beneficial factor for reduced frequencies of swim advisories and swim bans.


Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase.
Increased superoxide production contributes to reduced vascular nitric oxide (NO) bioactivity and endothelial dysfunction in experimental models of diabetes. We characterized the sources and mechanisms underlying vascular superoxide production in human blood vessels from diabetic patients with coronary artery disease compared with nondiabetic patients. Vascular superoxide production was quantified in both saphenous veins and internal mammary arteries from 45 diabetic and 45 matched nondiabetic patients undergoing coronary artery bypass surgery. NAD(P)H-dependent oxidases were important sources of vascular superoxide in both diabetic and nondiabetic patients, but both the activity of this enzyme system and the levels of NAD(P)H oxidase protein subunits (p22phox, p67phox, and p47phox) were significantly increased in diabetic veins and arteries. In nondiabetic vessels, endothelial NO synthase produced NO that scavenged superoxide. However, in diabetic vessels, the endothelium was an additional net source of superoxide production because of dysfunctional endothelial NO synthase that was corrected by intracellular tetrahydrobiopterin supplementation. Furthermore, increased superoxide production in diabetes was abrogated by the protein kinase C inhibitor chelerythrine. These observations suggest important roles for NAD(P)H oxidases, endothelial NO synthase uncoupling, and protein kinase C signaling in mediating increased vascular superoxide production and endothelial dysfunction in human diabetes mellitus.


Informed choice and the nanny state: learning from the tobacco industry.
To examine the 'nanny state' arguments used by tobacco companies, explore the cognitive biases that impede smokers' ability to make fully informed choices, and analyse the implications for those working to limit the harmful effects of other risk products. A critical analysis of the practices engaged in by the tobacco industry, the logic on which they relied, and the extent to which their work has informed approaches used by other industries. The tobacco industry's deliberate strategy of challenging scientific evidence undermines smokers' ability to understand the harms smoking poses and questions arguments that smoking is an informed choice. Cognitive biases predispose smokers to discount risk information, particularly when this evidence is disputed and framed as uncertain. Only state intervention has held the tobacco industry to account and begun ameliorating the effects of their sustained duplicity. Evidence other industries are now adopting similar tactics, particularly use of 'nanny state' claims to oppose proportionate interventions, is concerning. Some marketing strategies have deliberately mis-informed consumers thus directly contributing to many public health problems. Far from removing free choice, government policies that restrain commercial communications and stimuli are prerequisites necessary to promote free choice.


Horizontal Synchronization of Neuronal Activity in the Barrel Cortex of the Neonatal Rat by Spindle-Burst Oscillations.
During development, activity in the somatosensory cortex is characterized by intermittent oscillatory bursts at gamma (early gamma-oscillations, EGOs) and alpha-beta (spindle-bursts, SBs) frequencies. Here, we explored the topography of EGOs and SBs in the neighbor barrels of the whisker-related barrel cortex of neonatal rats (P4-7) during responses evoked by simultaneous activation of multiple whiskers as it occurs during natural conditions. We found that brief simultaneous deflection of all whiskers evoked complex neuronal responses comprised of EGOs and SBs. In contrast to EGOs, that specifically synchronized neuronal activity in each individual barrel, SBs efficiently synchronized activity between neighboring barrels. After plucking a single whisker, synchronous stimulation of spared whiskers evoked EGO-lacking responses in the whisker-deprived barrel, even though the remaining neuronal activity was synchronized by SBs in neighboring barrels. Thus, EGOs specifically support topographic synchronization of neuronal activity within barrels, whereas SBs support horizontal synchronization between neighboring barrels during stimulation of multiple whiskers. We suggest that these two co-existing activity patterns coordinate activity-dependent formation of topographic maps and support the emergence of integrative functions in the primary somatosensory cortex during the critical period of somatosensory maps development.


Single-cell transcriptome analysis of CD8 + T-cell memory inflation.
Background: Persistent viruses such as murine cytomegalovirus (MCMV) and adenovirus-based vaccines induce strong, sustained CD8 + T-cell responses, described as memory "inflation". These retain functionality, home to peripheral organs and are associated with a distinct transcriptional program. Methods: To further define the nature of the transcriptional mechanisms underpinning memory inflation at different sites we used single-cell RNA sequencing of tetramer-sorted cells from MCMV-infected mice, analyzing transcriptional networks in virus-specific populations in the spleen and gut intra-epithelial lymphocytes (IEL). Results: We provide a transcriptional map of T-cell memory and define a module of gene expression, which distinguishes memory inflation in spleen from resident memory T-cells (T RM) in the gut. Conclusions: These data indicate that CD8 + T-cell memory in the gut epithelium induced by persistent viruses and vaccines has a distinct quality from both conventional memory and "inflationary" memory which may be relevant to protection against mucosal infections.


Accumulation of mitochondrial DNA deletions within dopaminergic neurons triggers neuroprotective mechanisms.
Acquired alterations in mitochondrial DNA are believed to play a pathogenic role in Parkinson's disease. In particular, accumulation of mitochondrial DNA deletions has been observed in substantia nigra pars compacta dopaminergic neurons from patients with Parkinson's disease and aged individuals. Also, mutations in mitochondrial DNA polymerase gamma result in multiple mitochondrial DNA deletions that can be associated with levodopa-responsive parkinsonism and severe substantia nigra pars compacta dopaminergic neurodegeneration. However, whether mitochondrial DNA deletions play a causative role in the demise of dopaminergic neurons remains unknown. Here we assessed the potential pathogenic effects of mitochondrial DNA deletions on the dopaminergic nigrostriatal system by using mutant mice possessing a proofreading-deficient form of mitochondrial DNA polymerase gamma (POLGD257A), which results in a time-dependent accumulation of mitochondrial DNA deletions in several tissues, including the brain. In these animals, we assessed the occurrence of mitochondrial DNA deletions within individual substantia nigra pars compacta dopaminergic neurons, by laser capture microdissection and quantitative real-time polymerase chain reaction, and determined the potential deleterious effects of such mitochondrial DNA alterations on mitochondrial function and dopaminergic neuronal integrity, by cytochrome c oxidase histochemistry and quantitative morphology. Nigral dopaminergic neurons from POLGD257A mice accumulate mitochondrial DNA deletions to a similar extent (∼40-60%) as patients with Parkinson's disease and aged individuals. Despite such high levels of mitochondrial DNA deletions, the majority of substantia nigra pars compacta dopaminergic neurons from these animals did not exhibit mitochondrial dysfunction or degeneration. Only a few individual substantia nigra pars compacta neurons appeared as cytochrome c oxidase-negative, which exhibited higher levels of mitochondrial DNA deletions than cytochrome c oxidase-positive cells (60.38±3.92% versus 45.18±2.83%). Survival of dopaminergic neurons in POLGD257A mice was associated with increased mitochondrial DNA copy number, enhanced mitochondrial cristae network, improved mitochondrial respiration, decreased exacerbation of mitochondria-derived reactive oxygen species, greater striatal dopamine levels and resistance to parkinsonian mitochondrial neurotoxins. These results indicate that primary accumulation of mitochondrial DNA deletions within substantia nigra pars compacta dopaminergic neurons, at an extent similar to that observed in patients with Parkinson's disease, do not kill dopaminergic neurons but trigger neuroprotective compensatory mechanisms at a mitochondrial level that may account for the high pathogenic threshold of mitochondrial DNA deletions in these cells.


A genetic analysis of nitrosative stress.
Nitrosative stress is induced by pathophysiological levels of nitric oxide (NO) and S-nitrosothiols (e.g., S-nitrosoglutathione, GSNO) and arises, at least in significant part, from the nitrosylation of critical protein Cys thiols (S-nitrosylation) and metallocofactors. However, the mechanisms by which NO and GSNO mediate nitrosative stress are not well understood. Using yeast Saccharomyces cerevisiae strains lacking NO- and/or GSNO-consuming enzymes (flavohemoglobin and GSNO reductase, respectively), we measured the individual and combined effects of NO and GSNO on both cell growth and the formation of protein-bound NO species. Our results suggest an intracellular equilibrium between NO and GSNO, dependent in part on cell-catalyzed release of NO from GSNO (i.e., "SNO-lyase" activity). However, whereas NO induces multiple types of protein-based modifications, levels of which correlate with inhibition of cell growth, GSNO mainly affects protein S-nitrosylation, and the relationship between S-nitrosylation and nitrosative stress is more complex. These data support the idea of multiple classes of protein-SNO, likely reflected in divergent routes of synthesis and degradation. Indeed, a significant fraction of protein S-nitrosylation by NO occurs in the absence of O(2), which is commonly assumed to drive this reaction but instead is apparently dependent in substantial part upon protein-bound transition metals. Additionally, our findings suggest that nitrosative stress is mediated principally via the S-nitrosylation of a subset of protein targets, which include protein SNOs that are stable to cellular glutathione (and thus are not metabolized by GSNO reductase). Collectively, these results provide new evidence for the mechanisms through which NO and GSNO mediate nitrosative stress as well as the cellular pathways of protein S-nitrosylation and denitrosylation involving metalloproteins, SNO lyase(s) and GSNO reductase.


Partner characteristics predicting HIV-1 set point in sexually acquired HIV-1 among African seroconverters.
Plasma HIV-1 RNA set point is an important predictor of HIV-1 disease progression. We hypothesized that inoculum size and HIV-1 exposure prior to HIV-1 transmission may modulate set point. We evaluated predictors of set point among 141 African HIV-1 seroconverters and their HIV-1-infected study partners. We compared characteristics of seroconverters and their HIV-1-infected partners and HIV-1 set point. Data were from a clinical trial of genital HSV-2 suppression with acyclovir to reduce HIV-1 transmission in HIV-1 serodiscordant couples with HIV-1 transmission linkage assigned through virus sequencing. Our analysis includes data from all transmissions including those with transmission linkage to the HIV-1-infected "source partner" and those that were not linked to their HIV-1-infected study partner. In multivariable analysis, higher plasma HIV-1 in source partners was associated with higher seroconverter set point ( + 0.44 log10 copies/ml per log(10) source partner plasma HIV-1, p < 0.001). In addition, bacterial vaginosis (BV) among female source partners near the time of infection was associated with higher set point in their male seroconverters ( + 0.49 log(10), p = 0.04). Source partner characteristics associated with lower set point included male circumcision ( - 0.63 log(10), p = 0.03) and assignment to acyclovir ( - 0.44 log10, p = 0.02). The proportion of variation in set point explained by plasma HIV-1 RNA of the source partner, after controlling for other factors, was 0.06. Source partner plasma HIV-1 level is the most significant predictor of seroconverter set point, possibly reflecting characteristics of the transmitted virus. Acyclovir use, BV among women source partners, and circumcision among male source partners may alter the set point by affecting transmitted virus inoculum in the source partners' genital compartment.


Use of colchicine in cortical area 1 of the hippocampus impairs transmission of non-motivational information by the pyramidal cells.
Colchicine, a potent neurotoxin derived from plants, has been recently introduced as a degenerative toxin of small pyramidal cells in the cortical area 1 of the hippocampus (CA1). In this study, the effect of the alkaloid in CA1 on the behaviors in the conditioning task was measured. Injections of colchicine (1,5 µg/rat, intra-CA1) was performed in the male Wistar rats, while the animals were settled and cannulated in a stereotaxic apparatus. In the control group solely injection of saline (1 µl/rat, intra-CA1) was used. One week later, all the animals passed the saline conditioning task using a three-day schedule of an unbiased paradigm. They were administered saline (1 ml/kg, s.c.) twice a day throughout the conditioning phase. To evaluate the possible effects of cell injury by the toxin on the pyramidal cells, both the motivational signals while in the conditioning box and the non-motivational locomotive signs of the treated and control rats were measured. Based on the present study the alkaloid caused no change in the score of place conditioning, but affected both the sniffing and grooming behaviors in the group that received colchicine. However, the alkaloid did not show the significant effect on the rearing or compartment entering in the rats. According to the findings, the intra-CA1 injection of colchicine may impair the neuronal transmission of non-motivational information by the pyramidal cells in the dorsal hippocampus.


Cytokeratins Biosensing Using Tilted Fiber Gratings.
Optical fiber gratings have widely proven their applicability in biosensing, especially when they are coupled with antibodies for specific antigen recognition. While this is customarily done with fibers coated by a thin metal film to benefit from plasmonic enhancement, in this paper, we propose to study their intrinsic properties, developing a label-free sensor for the detection of biomarkers in real-time without metal coatings for surface plasmon resonances. We focus on the inner properties of our modal sensor by immobilizing receptors directly on the silica surface, and reporting the sensitivity of bare tilted fiber Bragg gratings (TFBGs) used at near infrared wavelengths. We test different strategies to build our sensing surface against cytokeratins and show that the most reliable functionalization method is the electrostatic adsorption of antibodies on the fiber, allowing a limit of detection reaching 14 pM by following the guided cladding modes near the cut-off area. These results present the biodetection performance that TFBGs bring through their modal properties for different functionalizations and data processing strategies.


Dynamics of Cdk1 substrate specificity during the cell cycle.
Cdk specificity is determined by the intrinsic selectivity of the active site and by substrate docking sites on the cyclin subunit. There is a long-standing debate about the relative importance of these factors in the timing of Cdk1 substrate phosphorylation. We analyzed major budding yeast cyclins (the G1/S-cyclin Cln2, S-cyclin Clb5, G2/M-cyclin Clb3, and M-cyclin Clb2) and found that the activity of Cdk1 toward the consensus motif increased gradually in the sequence Cln2-Clb5-Clb3-Clb2, in parallel with cell cycle progression. Further, we identified a docking element that compensates for the weak intrinsic specificity of Cln2 toward G1-specific targets. In addition, Cln2-Cdk1 showed distinct consensus site specificity, suggesting that cyclins do not merely activate Cdk1 but also modulate its active-site specificity. Finally, we identified several Cln2-, Clb3-, and Clb2-specific Cdk1 targets. We propose that robust timing and ordering of cell cycle events depend on gradual changes in the substrate specificity of Cdk1.


pyTFM: A tool for traction force and monolayer stress microscopy.
Cellular force generation and force transmission are of fundamental importance for numerous biological processes and can be studied with the methods of Traction Force Microscopy (TFM) and Monolayer Stress Microscopy. Traction Force Microscopy and Monolayer Stress Microscopy solve the inverse problem of reconstructing cell-matrix tractions and inter- and intra-cellular stresses from the measured cell force-induced deformations of an adhesive substrate with known elasticity. Although several laboratories have developed software for Traction Force Microscopy and Monolayer Stress Microscopy computations, there is currently no software package available that allows non-expert users to perform a full evaluation of such experiments. Here we present pyTFM, a tool to perform Traction Force Microscopy and Monolayer Stress Microscopy on cell patches and cell layers grown in a 2-dimensional environment. pyTFM was optimized for ease-of-use; it is open-source and well documented (hosted at https://pytfm.readthedocs.io/) including usage examples and explanations of the theoretical background. pyTFM can be used as a standalone Python package or as an add-on to the image annotation tool ClickPoints. In combination with the ClickPoints environment, pyTFM allows the user to set all necessary analysis parameters, select regions of interest, examine the input data and intermediary results, and calculate a wide range of parameters describing forces, stresses, and their distribution. In this work, we also thoroughly analyze the accuracy and performance of the Traction Force Microscopy and Monolayer Stress Microscopy algorithms of pyTFM using synthetic and experimental data from epithelial cell patches.