Gene expression profiling of mesenteric lymph nodes from sheep with natural scrapie.
Prion diseases are characterized by the accumulation of the pathogenic PrPSc protein, mainly in the brain and the lymphoreticular system. Although prions multiply/accumulate in the lymph nodes without any detectable pathology, transcriptional changes in this tissue may reflect biological processes that contribute to the molecular pathogenesis of prion diseases. Little is known about the molecular processes that occur in the lymphoreticular system in early and late stages of prion disease. We performed a microarray-based study to identify genes that are differentially expressed at different disease stages in the mesenteric lymph node of sheep naturally infected with scrapie. Oligo DNA microarrays were used to identify gene-expression profiles in the early/middle (preclinical) and late (clinical) stages of the disease. In the clinical stage of the disease, we detected 105 genes that were differentially expressed (≥2-fold change in expression). Of these, 43 were upregulated and 62 downregulated as compared with age-matched negative controls. Fewer genes (50) were differentially expressed in the preclinical stage of the disease. Gene Ontology enrichment analysis revealed that the differentially expressed genes were largely associated with the following terms: glycoprotein, extracellular region, disulfide bond, cell cycle and extracellular matrix. Moreover, some of the annotated genes could be grouped into 3 specific signaling pathways: focal adhesion, PPAR signaling and ECM-receptor interaction. We discuss the relationship between the observed gene expression profiles and PrPSc deposition and the potential involvement in the pathogenesis of scrapie of 7 specific differentially expressed genes whose expression levels were confirmed by real time-PCR. The present findings identify new genes that may be involved in the pathogenesis of natural scrapie infection in the lymphoreticular system, and confirm previous reports describing scrapie-induced alterations in the expression of genes involved in protein misfolding, angiogenesis and the oxidative stress response. Further studies will be necessary to determine the role of these genes in prion replication, dissemination and in the response of the organism to this disease.


Platelets and endothelial cells.
The pathogenesis of atherosclerosis, the leading cause of morbidity and mortality in industrial countries, is multifactorial. Atherogenesis, the development of atherosclerotic lesions, is initiated by a mechanical or functional injury of the endothelium. The function of the endothelium is influenced by multiple factors as a consequence of cell-cell interactions. Cell-cell communication between endothelial cells with platelets has only recently begun to receive systematic study. In recent years it has been established that platelet-endothelial interactions are involved at all stages of atherosclerotic disease. This article reviews the interactions between endothelial cells and platelets in the context of their role to initiate and accelerate atherothrombosis, as well as in acute thrombotic occlusion (e.g., at the site of atherosclerotic plaque rupture or subsequent to coronary angioplasty). From a mechanistic standpoint, platelets and endothelial cells communicate on multiple levels. Cross-talk may occur over a distance (paracrine signaling), via transient interactions (so-called give-and-go mechanism), or through receptor-mediated cell-cell adhesion. Platelets may release or transfer substances that influence endothelial cell function, and vice versa. Among many others, adhesion molecules, such as P-selectin (CD62P), are of special interest because of their role in modulating interactions between blood cells and the endothelium, and also because of the possible use of the soluble form as a plasma predictor of adverse cardiovascular events. In addition to dietary, cholesterol and lipid lowering, and other pharmaceutical approaches, antiplatelet therapy plays an important part in the treatment of atherosclerosis and its multifactorial clinical manifestations. Understanding the specific interactions between platelets and the endothelium may lead to the development of novel therapeutic strategies.


Multiple mutualist effects on genomewide expression in the tripartite association between Medicago truncatula, nitrogen-fixing bacteria and mycorrhizal fungi.
While all species interact with multiple mutualists, the fitness consequences and molecular mechanisms underlying these interactions remain largely unknown. We combined factorial ecological experiments with genomewide expression analyses to examine the phenotypic and transcriptomic responses of model legume Medicago truncatula to rhizobia and mycorrhizal fungi. We found synergistic effects of these mutualists on plant performance and examined unique features of plant gene expression responses to multiple mutualists. There were genomewide signatures of mutualists and multiple mutualists on expression, with partners often affecting unique sets of genes. Mycorrhizal fungi had stronger effects on plant expression than rhizobia, with 70% of differentially expressed genes affected by fungi. Fungal and bacterial mutualists had joint effects on 10% of differentially expressed genes, including unexpected, nonadditive effects on some genes with important functions such as nutrient metabolism. For a subset of genes, interacting with multiple mutualists even led to reversals in the direction of expression (shifts from up to downregulation) compared to interacting with single mutualists. Rhizobia also affected the expression of several mycorrhizal genes, including those involved in nutrient transfer to host plants, indicating that partner species can also impact each other's molecular phenotypes. Collectively, these data illustrate the diverse molecular mechanisms and transcriptional responses associated with the synergistic benefits of multiple mutualists.


Oesophageal pressure-flow metrics in relation to bolus volume, bolus consistency, and bolus perception.
The utility of combined oesophageal pressure-impedance recording has been enhanced by automation of data analysis. To understand how oesophageal function as measured by automated impedance manometry (AIM) pressure-flow analysis varies with bolus characteristics and subjective perception of bolus passage. Oesophageal pressure-impedance recordings of 5 and 10 ml liquid or viscous swallows and 2 and 4 cm solid swallows from 20 healthy control subjects (five male; 25-73 years) were analysed. Metrics indicative of bolus pressurization (intrabolus pressure and intrabolus pressure slope) were derived. Bolus flow resistance, the relationship between bolus pressurization and flow timing, was assessed using a pressure-flow index. Bolus retention was assessed using the ratio of nadir impedance to peak pressure impedance (impedance ratio). Subjective perception of bolus passage was assessed swallow by swallow. Viscosity increased the bolus flow resistance and reduced bolus clearance. Responses to boluses of larger volume and more viscous consistency revealed a positive correlation between bolus pressurization and oesophageal peak pressure. Flow resistance was higher in subjects who perceived bolus hold up of solids. Bolus volume and bolus type alter oesophageal function and impact AIM analysis metrics descriptive of oesophageal function. Perception of bolus transit was associated with heightened bolus pressurization relative to bolus flow.


The Adaptive Behavioral Components (ABC) Model for Planning Longitudinal Behavioral Technology-Based Health Interventions: A Theoretical Framework.
A growing number of interventions incorporate digital and social technologies (eg, social media, mobile phone apps, and wearable devices) into their design for behavior change. However, because of a number of factors, including changing trends in the use of technology over time, results on the efficacy of these interventions have been mixed. An updated framework is needed to help researchers better plan behavioral technology interventions by anticipating the needed resources and potential changes in trends that may affect interventions over time. Focusing on the domain of health interventions as a use case, we present the Adaptive Behavioral Components (ABC) model for technology-based behavioral interventions. ABC is composed of five components: basic behavior change; intervention, or problem-focused characteristics; population, social, and behavioral characteristics; individual-level and personality characteristics; and technology characteristics. ABC was designed with the goals of (1) guiding high-level development for digital technology-based interventions; (2) helping interventionists consider, plan for, and adapt to potential barriers that may arise during longitudinal interventions; and (3) providing a framework to potentially help increase the consistency of findings among digital technology intervention studies. We describe the planning of an HIV prevention intervention as a case study for how to implement ABC into intervention design. Using the ABC model to plan future interventions might help to improve the design of and adherence to longitudinal behavior change intervention protocols; allow these interventions to adapt, anticipate, and prepare for changes that may arise over time; and help to potentially improve intervention behavior change outcomes. Additional research is needed on the influence of each of ABC's components to help improve intervention design and implementation.


Morphology and mechanical behaviour of pea-based starch-protein composites obtained by extrusion.
Starch-legume protein composites were obtained by extrusion of pea flour and pea starch-protein blend at various specific mechanical energies (100-2000 kJ/kg) and a temperature low enough to avoid expansion. The morphology of these composites displayed protein aggregates dispersed in a starch matrix, revealed by microscopy. Image analysis was used to determine the median width of protein aggregates (D50), their total perimeter and surface, from which a protein/starch interface index (Ii) was derived. The mechanical properties of composites were determined by a three-point bending test. The pea flour composites had a higher interface index Ii (1.8-3.1) with lower median particle width D50 (8-18 μm) and a more brittle behaviour than the blend composites that had a lower Ii (1-1.1) and higher D50 (22-31 μm). For both materials, rupture stress and strain were negatively correlated with Ii. This result suggested that there was a poor interfacial adhesion between the pea starch and proteins.


Negative regulation of the nuclear factor kappa B-inducing kinase by a cis-acting domain.
Nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK) participates in the activation of NF-kappaB, a family of eukaryotic transcription factors that mediate cell growth and transformation. NIK activates the IkappaB kinase both in vivo and in vitro, although how the activity of NIK is regulated has remained unclear. Here we show that the N-terminal region of NIK contains a negative-regulatory domain (NRD), which is composed of a basic motif and a proline-rich repeat motif. Deletion of these motifs leads to a marked enhancement of NIK function. We further demonstrate that the N-terminal NRD interacts with the C-terminal region of NIK, thereby inhibiting the binding of NIK to its substrate IkappaB kinase. Consistently, when expressed alone, the NRD potently inhibits NIK-mediated NF-kappaB signaling. These results provide a new insight into the mechanism of NIK regulation.


Repeated interaction model for diffusion-induced Ramsey narrowing.
In a recent paper [Y. Xiao et al., Phys. Rev. Lett. 96, 043601 (2006)] we characterized diffusion-induced Ramsey narrowing as a general phenomenon, in which diffusion of coherence in-and-out of an interaction region such as a laser beam induces spectral narrowing of the associated resonance lineshape. Here we provide a detailed presentation of the repeated interaction model of diffusion-induced Ramsey narrowing, with particular focus on its application to Electromagnetically Induced Transparency (EIT) of atomic vapor in a buffer gas cell. We compare this model both to experimental data and numerical calculations.


Dual quinone tagging for MALDI-TOF mass spectrometric quantitation of cysteine-containing peptide.
A dual quinone tagging strategy is designed for quantitation of cysteine-containing peptide (CCP) with MALDI-TOF mass spectrometry. The quinone compounds can rapidly and specifically bind to the thiol group of cysteine residues by a Michael addition reaction, which is used to identify both CCP and the number of cysteine residues in CCP through the direct observation of untagged and tagged products. After reduced with DL-dithiothreitol, the intramolecular disulfide bond can also be identified. Using benzoquinone (BQ) and methyl-p-benzoquinone (MBQ) as dual tags and a peptide with an amino acid sequence of SSDQFRPDDCT (C-pep1) as a model target, respectively, the quantitation strategy is performed through the intensity ratio of MBQ-tagged C-pep1 to BQ-tagged C-pep1 as the internal standard. The logarithm value of the intensity ratio is proportional to C-pep1 concentration in a range from 5.0 to 5000 nM. The limit of detection is as low as 2.0 nM. The proposed methodology provides a novel tool for rapid characterization, identification, and quantitation of biomolecules containing thiol reactive sites and has a promising application in the large-scale detection and analysis of cysteine-containing biomolecules.


Quantification of transmission in one-to-one experiments.
We study the statistical inference from data on transmission obtained from one-to-one experiments, and compare two algorithms by which the reproduction ratio can be quantified. The first algorithm, the transient state (TS) algorithm, takes the time course of the epidemic into account. The second algorithm, the final size (FS) algorithm, does not take time into account but is based on the assumption that the epidemic process has ended before the experiment is stopped. The FS algorithm is a limiting case of the TS algorithm for the situation where time tends to infinity. So far quantification of transmission has relied almost exclusively on the FS algorithm, even if the TS algorithm would have been more appropriate. Its practical use, however, is limited to experiments with only a few animals. Here, we quantify the error made when the FS algorithm is applied to data of one-to-one experiments not having reached the final size. We conclude that given the chosen tests, the FS algorithm underestimates the reproduction ratio R0, is liberal when testing H0: R0 > or = 1 against H1: R0 < 1, is conservative when testing H0: R0 < or = 1 against H1: R0 > 1 and calculates the same probability as the TS algorithm when testing H0: R(0-control) = R(0-treatment) against H1: R(0-control) > R(0-treatment) We show how the power of the test depends on the duration of the experiments and on the number of replicates. The methods are illustrated by an application to porcine reproductive and respiratory syndrome virus (PRRSV).


Computerized administration and scoring of the dichotic nonsense syllable test.
A user-friendly computer program was designed to administer the dichotic nonsense syllable test (DNST). Computer automation makes it easier for listeners to perform the response task appropriately and for audiologists to administer and accurately score the test. The program presents test stimuli in a user-controlled, self-paced sequence; provides an easy-to-use client interface to collect listener responses; verifies that appropriate responses are recorded before proceeding with the test; scores responses automatically; and stores the information in an electronic format that is easy to analyze and archive. The automated DNST protocol is described. Verification tests confirm the accuracy of stimulus sequencing, response recording, and test scoring. Computer automation makes administering the DNST easier, ensures the accuracy of test scoring, and simplifies the analysis and archiving of results. These refinements may make the DNST more useful to clinicians. Further research is required to establish normative data for this version of the DNST.


The impact of resistance training on strength and correlates of physical activity in youth.
Resistance training (RT) may have a positive impact on specific correlates of physical activity (PA) in inactive and/or obese youth, with strength as a possible underlying mechanism. The aim of this study was to investigate this. Twelve participants (aged 8.9 ± 1.0 years) were assigned to an experimental group (EG) or control group (CG). Pre and post intervention assessments for strength, physical self-perceptions (PSPs), weight status, fundamental movement skills (FMS), and PA levels were completed. The EG participated in a twice-weekly 10-week RT programme. There were significant group x time interactions for FMS (CAMSA total P = 0.016, CAMSA skill score P = 0.036) and stretch stature (P = 0.002) (EG larges changes than the CG). Large effect sizes for the differences in change scores between the EG and CG were evident for CAMSA total score (Hedges' g = 0.830, P = 0.138), CAMSA skill score (Hedges' g = 0.895, P = 0.112) and relative strength (Hedges' g = 0.825, P = 0.140). This study demonstrated that a 10-week RT intervention has a positive effect on strength and FMS, and may also benefit weight status and PSPs. This study supports the development of RT interventions to develop these correlates, and increase PA levels.


Tuning of human MAIT cell activation by commensal bacteria species and MR1-dependent T-cell presentation.
Human mucosal-associated invariant T (MAIT) cell receptors (TCRs) recognize bacterial riboflavin pathway metabolites through the MHC class 1-related molecule MR1. However, it is unclear whether MAIT cells discriminate between many species of the human microbiota. To address this, we developed an in vitro functional assay through human T cells engineered for MAIT-TCRs (eMAIT-TCRs) stimulated by MR1-expressing antigen-presenting cells (APCs). We then screened 47 microbiota-associated bacterial species from different phyla for their eMAIT-TCR stimulatory capacities. Only bacterial species that encoded the riboflavin pathway were stimulatory for MAIT-TCRs. Most species that were high stimulators belonged to Bacteroidetes and Proteobacteria phyla, whereas low/non-stimulator species were primarily Actinobacteria or Firmicutes. Activation of MAIT cells by high- vs low-stimulating bacteria also correlated with the level of riboflavin they secreted or after bacterial infection of macrophages. Remarkably, we found that human T-cell subsets can also present riboflavin metabolites to MAIT cells in a MR1-restricted fashion. This T-T cell-mediated signaling also induced IFNγ, TNF and granzyme B from MAIT cells, albeit at lower level than professional APC. These findings suggest that MAIT cells can discriminate and categorize complex human microbiota through computation of TCR signals depending on antigen load and presenting cells, and fine-tune their functional responses.


Molecular cloning, tissue distribution and ontogenetic expression of sodium proton exchanger isoform 2 ( NHE-2) mRNA in the small intestine of pigs.
Molecular cloning, tissue distribution and ontogenetic regulation of sodium/proton exchanger isoform 2 (NHE-2) mRNA expression were evaluated in the pig small intestine during postnatal development. The 2872-bp porcine full cDNA sequence of the NHE-2 (EF672046) cloned in this study showed 80% and 70% homology with known human and mouse gene sequence, respectively. Hydrophobic prediction suggests 13 putative membrane-spanning domains within porcine NHE-2. The porcine NHE-2 mRNA was detected in the brain, liver, kidney, heart, lung, small intestine and muscle. The small intestine had the highest NHE-2 mRNA abundance and the brain, lung and liver had the lowest NHE-2 mRNA abundance (P < 0.05). Along the longitudinal axis, the duodenum had the highest NHE-2 mRNA abundance and the ileum and colon had the lowest NHE-2 mRNA abundance (P < 0.05). The NHE-2 mRNA level was increased from day 1 to day 26 in the duodenum (P < 0.05) and dropped dramatically on day 30 (P < 0.05). There is no difference between day 1 and day 7 (P > 0.05). After day 30, the NHE-2 mRNA level remained the same except on day 90 (P > 0.05). The mRNA expression of NHE-2 was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages and growing stages of life, which may contribute to changes in NHE activity.


Genome-wide analysis of the interplay between chromatin-associated RNA and 3D genome organization in human cells.
The interphase genome is dynamically organized in the nucleus and decorated with chromatin-associated RNA (caRNA). It remains unclear whether the genome architecture modulates the spatial distribution of caRNA and vice versa. Here, we generate a resource of genome-wide RNA-DNA and DNA-DNA contact maps in human cells. These maps reveal the chromosomal domains demarcated by locally transcribed RNA, hereafter termed RNA-defined chromosomal domains. Further, the spreading of caRNA is constrained by the boundaries of topologically associating domains (TADs), demonstrating the role of the 3D genome structure in modulating the spatial distribution of RNA. Conversely, stopping transcription or acute depletion of RNA induces thousands of chromatin loops genome-wide. Activation or suppression of the transcription of specific genes suppresses or creates chromatin loops straddling these genes. Deletion of a specific caRNA-producing genomic sequence promotes chromatin loops that straddle the interchromosomal target sequences of this caRNA. These data suggest a feedback loop where the 3D genome modulates the spatial distribution of RNA, which in turn affects the dynamic 3D genome organization.


[Equilibrium disorders and their diagnosis in childhood].
Vertigo in children is rarer than in adults and the examiner in cases showing these symptoms must rely on the parents' or relatives' observations and details. Besides the equilibrium disorders caused by hereditary malabsorption or lesions in the peripheral or central vestibular structures, e.g. ototoxic drugs, tumours in the brain, meningitis, encephalitis, otitis, labyrinthine fistulas or head trauma, we only known of typical diseases, associated with vertigo, that develop during childhood. These are: so-called benign paroxysmal vertigo of childhood, benign paroxysmal torticollis, basilar migraine, spasmus nutans, visual-cliff phenomenon, and kinetosis. Careful examinations are necessary to differentiate these illnesses from vestibular epilepsy, brain tumours, and hereditary episodic vertigo. Neuro-otologic examination in children, especially small children, is a kind of "stepchild" in ENT departments. The reasons are the time-consuming examination necessary in the case of children and by problems connected with a plethora of troublesome individual tests. Additional difficulties arise in cases of sensory, mental, and other impairments. - The paper gives an overview of vestibular disturbances during childhood and diagnostic procedures for determination by means of Frenzel glasses, electronystagmography, cranio-corpography, and posturography.


Mitogenic response of adult rat olfactory ensheathing glia to four growth factors.
Olfactory ensheathing glia (EG) from adult rat proliferate slowly in vitro without added mitogens. The potential future use of EG in transplantation within the central nervous system to improve neural repair is dependent on identifying mitogens that will effectively expand EG without altering their phenotype. The mitogenic effects of heregulin (HRG), fibroblast growth factor 2 (FGF-2), platelet-derived growth factor BB (PDGF-BB), insulin-like growth factor 1 (IGF-1), and forskolin (FSK) on cultured adult-derived rat EG were monitored by tritiated-thymidine labeling and p75 immunostaining. In serum-containing medium, HRG, FGF-2, PDGF-BB, IGF-1, and FSK were capable of stimulating EG proliferation, and the stimulation by these growth factors was potentiated by FSK. The combinations of HRG + FGF-2, HRG + PDGF-BB, HRG + IGF-1, FGF-2 + PDGF-BB, and FGF-2 + IGF-1 all promoted EG proliferation in an additive manner. In serum-free medium, HRG and FGF-2 were mitogenic, but PDGF-BB, IGF-1 and FSK were not; however, FSK potentiated the stimulation by HRG and FGF-2, and the combination of HRG + FGF-2 promoted EG proliferation in an additive manner. This new information will be useful for the design of protocols to achieve sufficient numbers of adult-derived EG for clinical purposes. This study also further establishes similarities between EG and Schwann cells.


Acute interstitial pneumonia in mink kits: experimental reproduction of the disease.
Organ homogenates from kits that died of interstitial pneumonia were inoculated into adult Aleutian disease virus (ADV)-negative mink and shown to contain infectious ADV. Acute interstitial pneumonia was experimentally reproduced with the organ homogenate but only by inoculation of newborn kits born from ADV-negative dams. Older kits and kits from ADV-positive dams did not develop interstitial pneumonia, but later developed the classic form of Aleutian disease. Electron microscopic examination was done on purified suspensions of defined ADV isolates and on purified organ homogenates from kits with spontaneous or experimental interstitial pneumonia. In kits from both groups a virus, morphologically resembling the defined ADV isolates, was demonstrated. Findings of intranuclear inclusion bodies and intranuclear ADV antigen in alveolar type-II cells in affected lungs and the lack of immunologically mediated lesions suggest that lung lesions result from primary viral injury to alveolar type-II cells. Experiments also showed that infection of dams with ADV before pregnancy decreased the number of kits per mated dam and infection with ADV in mid-pregnancy caused fetal death, fetal resorption, or abortion.


Cloning and characterization of Daphnia mitochondrial DNA.
The mitochondrial genome of Daphnia pulex (Crustacea, Cladocera) was cloned as a single fragment into the plasmid vector pUC12. The genome size, estimated from restriction endonuclease fragment lengths, is 15,400 +/- 200 base pairs. The GC content, estimated from thermal denaturation studies, is 42%. The positions of 39 cleavage sites were mapped for 14 restriction enzymes. The distribution of these sites within the genome is random (P = 0.44). Heterologous hybridizations with Drosophila sylvestris mitochondrial DNA (mtDNA) probes indicate that gene orders within Daphnia and Drosophila mtDNAs are similar.


Reduced Structural Connectivity Between Left Auditory Thalamus and the Motion-Sensitive Planum Temporale in Developmental Dyslexia.
Developmental dyslexia is characterized by the inability to acquire typical reading and writing skills. Dyslexia has been frequently linked to cerebral cortex alterations; however, recent evidence also points toward sensory thalamus dysfunctions: dyslexics showed reduced responses in the left auditory thalamus (medial geniculate body, MGB) during speech processing in contrast to neurotypical readers. In addition, in the visual modality, dyslexics have reduced structural connectivity between the left visual thalamus (lateral geniculate nucleus, LGN) and V5/MT, a cerebral cortex region involved in visual movement processing. Higher LGN-V5/MT connectivity in dyslexics was associated with the faster rapid naming of letters and numbers (RANln), a measure that is highly correlated with reading proficiency. Here, we tested two hypotheses that were directly derived from these previous findings. First, we tested the hypothesis that dyslexics have reduced structural connectivity between the left MGB and the auditory-motion-sensitive part of the left planum temporale (mPT). Second, we hypothesized that the amount of left mPT-MGB connectivity correlates with dyslexics RANln scores. Using diffusion tensor imaging-based probabilistic tracking, we show that male adults with developmental dyslexia have reduced structural connectivity between the left MGB and the left mPT, confirming the first hypothesis. Stronger left mPT-MGB connectivity was not associated with faster RANln scores in dyslexics, but was in neurotypical readers. Our findings provide the first evidence that reduced cortico-thalamic connectivity in the auditory modality is a feature of developmental dyslexia and it may also affect reading-related cognitive abilities in neurotypical readers.SIGNIFICANCE STATEMENT Developmental dyslexia is one of the most widespread learning disabilities. Although previous neuroimaging research mainly focused on pathomechanisms of dyslexia at the cerebral cortex level, several lines of evidence suggest an atypical functioning of subcortical sensory structures. By means of diffusion tensor imaging, we here show that dyslexic male adults have reduced white matter connectivity in a cortico-thalamic auditory pathway between the left auditory motion-sensitive planum temporale and the left medial geniculate body. Connectivity strength of this pathway was associated with measures of reading fluency in neurotypical readers. This is novel evidence on the neurocognitive correlates of reading proficiency, highlighting the importance of cortico-subcortical interactions between regions involved in the processing of spectrotemporally complex sound.


How strong are Malawi's family planning programs for adolescent and adult women? Results of a national assessment of implementation strength conducted by Malawi's National Evaluation Platform.
To assess the strength of implementation of family planning programs targeting youth (15-24) in Malawi with a specific focus on youth and the Youth-Friendly Health Services program. We conducted 9781 mobile phone interviews with facility in-Charge Nurses and health workers (health facility workers, health surveillance assistants [HSAs] and community-based distribution agent [CBDAs]) who provide family planning (FP) services across the 28 districts. Responses were entered in tablet using Open Data Kit. They were summarized and presented using R, Stata (College Station, TX, USA, StatsReport, JHU, Baltimore, MD, USA) and ArcView GIS (ESRI, Redlands, CA, USA). Availability of key products was a challenge across all health worker types as only 39% of health facilities, 29% of HSAs and 45% of CBDAs had all the FP methods they are supposed to provide on the day of the interview. About 50% of health workers were supervised within past 90 days preceding the study. Despite most facilities saying that they provide youth friendly health services, youth-specific FP guidelines or protocols were not available in 43% of facilities that provide these services and only 33% of facilities had special rooms and 58% have special days for youth. The commodity supply system needs to ensure that all facilities and workers have a consistent supply of all contraceptive methods. Government and program implementers should ensure availability of all FP guidelines and information, education, communication materials at all service delivery points and facilitate creation of special rooms or days for youth.


Dissipative metabolic patterns respond during neutrophil transmembrane signaling.
Self-organization is a common theme in biology. One mechanism of self-organization is the creation of chemical patterns by the diffusion of chemical reactants and their nonlinear interactions. We have recently observed sustained unidirectional traveling chemical redox [NAD(P)H - NAD(P)(+)] waves within living polarized neutrophils. The present study shows that an intracellular metabolic wave responds to formyl peptide receptor agonists, but not antagonists, by splitting into two waves traveling in opposite directions along a cell's long axis. Similar effects were noted with other neutrophil-activating substances. Moreover, when cells were exposed to an N-formyl-methionyl-leucyl-phenylalanine (FMLP) gradient whose source was perpendicular to the cell's long axis, cell metabolism was locally perturbed with reorientation of the pattern in a direction perpendicular to the initial cellular axis. Thus, extracellular activating signals and the signals' spatial cues are translated into distinct intracellular dissipative structures.


S. boulardii Fails to Hold Its Cell Wall Integrity against Nonpathogenic E. coli: Are Probiotic Yeasts Losing the Battle?
Probiotic yeast Saccharomyces boulardii exerts direct probiotic action on pathogenic E. coli by trapping them on surfaces and inactivating toxic lipopolysaccharides. Using optical dark-field microscopy, we show that nonpathogenic E. coli cells also readily bind probiotic S. boulardii. More importantly, the adhered nonpathogenic E. coli progressively damage S. boulardii cell walls and lyse them. Co-cultured methylene blue-supplemented agar-plate assay indicates that rough lipopolysaccharides might be playing a key role in S. boulardii cell wall damage. When experiments are repeated with lipopolysaccharide-depleted E. coli and also lipopolysaccharide-deficient E. coli, adhesion decreases substantially. The co-cultured assay further reveals that free lipopolysaccharides, released from E. coli, are also causing damage to S. boulardii walls like adhered E. coli. These new findings contradict the known S. boulardii-E. coli interaction mechanisms. We confirm that E. coli cells do not bind or damage human erythrocyte cell walls; therefore, they have not developed pathogenicity. The combined results demonstrate the first example of nonpathogenic E. coli being harmful to probiotic yeast S. boulardii. This finding is important because gut microbial flora contain large numbers of nonpathogenic E. coli. If they bind or damage probiotic S. boulardii cell walls, then the probiotic efficiency toward pathogenic E. coli will be compromised.


Developmental and endocrine regulation of kisspeptin expression in mouse Leydig cells.
Kisspeptin, encoded by the Kiss1 gene, binds to a specific G protein-coupled receptor (kisspeptin1 receptor) to regulate the central reproductive axis. Kisspeptin has also been reported to be expressed in peripheral tissues, including the testes. However, factors regulating testicular kisspeptin and its role in reproduction are unknown. Our objective herein was to begin to address kisspeptin function in the testis. In particular, we sought to determine the level of kisspeptin in the testis in comparison with the brain and other tissues, how these levels change from the prepubertal period through sexual maturation, and the factors involved in kisspeptin regulation in the testis. Immunohistochemical analysis of testis sections using a validated kisspeptin antibody localized kisspeptin to the Leydig cells. Kisspeptin was not detected in germ cells or Sertoli cells within the seminiferous tubules at any developmental time period studied, from prepuberty to sexual maturation. A developmental time course of testicular kisspeptin revealed that its mRNA and protein levels increased during development, reaching robust levels at postnatal day 28, correlating with pubertal onset. In vitro studies of primary mouse Leydig cells, as well as in vivo studies, indicated clearly that LH is involved in regulating levels of Leydig cell kisspeptin. Interestingly, gonadectomy resulted in elevated LH but reduced serum kisspeptin levels, suggesting that testicular kisspeptin may be secreted. These data document kisspeptin expression in mouse Leydig cells, its secretion into peripheral serum, and its regulation by changes in reproductive neuroendocrine function.


Distinct Adverse Clinical Outcomes of Small and Large Cribriform Patterns on Gleason 7 Prostate Cancer: A Preliminary Study.
We aimed to evaluate the effect of large and small cribriform morphology on survival following radical prostatectomy. We included 30 patients who underwent radical prostatectomy with curative intent between 2015 and 2022. Patients with the final pathology of Gleason 7 were included. Patients' radical prostatectomy specimens were reviewed by an experienced genitourinary pathologist. The diverse growth patterns of Gleason grade 4 were specified as poorly formed/fused glands, cribriform glands, and glomeruloid glands. The cribriform morphology was subdivided into small and large cribriform. Large cribriform growth morphology was defined by its size, which was double that of benign prostate glands. Small and large cribriform glands' percentages were indicated semiquantitatively. The cribriform morphology subtype present at 50% and higher was defined as the dominant pattern. The effect of histopathological patterns on biochemical recurrence and clinical progression was analyzed. Thirteen patients were small cribriform pattern dominant (group 1), whereas 14 of the patients were large cribriform pattern dominant (group 2). Pathological T, N stages, and surgical margin positivity were similar between groups. Biochemical recurrence and clinical progression rates were significantly higher in group 2. The large cribriform dominant patients had worse 2-year biochemical recurrence-free survival than small cribriform dominant patients (45.5% vs. 66.7%). In the univariate analysis, International Society of Urological Pathology grade, Gleason pattern 4 percentage, large cribriform pattern dominancy, and pT stage were predictors for biochemical recurrence-free survival. International Society of Urological Pathology grade was the only independent predictor for biochemical recurrence-free survival. Large cribriform pattern dominancy is associated with worse biochemical recurrence-free survival in Gleason 7 prostate cancer.


Causes of death among mental patients at Muhimbili Medical Centre, Dar es Salaam.
The clinical notes of the 146 patients who died during a five year period in the psychiatric ward at the Muhimbili Hospital were studied and analysed to identify gender, age, duration of hospital stay, psychiatric diagnosis and primary cause of mortality. There were 99 (67.8%) males and 47 (32.2%) females. The mean age, which could be established in only 105 (71.9%) instances, was 31.1 years. The average hospital stay was 21.6 days. The main psychiatric morbidity consisted of functional psychoses (52.7%), organic psychoses (37.6%), epilepsy (6.2%) and puerperal psychosis (2.1%). Mortality was primarily attributed to infectious diseases in at least half of the cases. Pyrexia of unknown origin was associated with death in 11.0% of cases and the cause of death could not be established in 17.1%. Clinical notes need to be improved and greater efforts made to diagnose and treat concomitant physical illnesses effectively.


The Effect of Sustainability-Related Information on the Sensory Evaluation and Purchase Behavior towards Salami Products.
Consumer's interest in sustainable livestock farming methods has grown in response to concerns for the environment and animal welfare. The purpose of this study is to examine the different influences of sustainability product information on sensory characteristics and purchase behaviors. To accomplish this aim, the study used salami, which is an Italian-style sausage processed by fermentation and drying. Three different types of information were provided: salami made from the pork of an antibiotic-free pig (SMAFP), of an animal welfare pig (SMAWP), and of a grazing pig (SMGP). This study was conducted as an off-line experiment with Korean participants (n=140). As a result, there were sensory differences according to the sustainability information. For the SMAFP, it had a significant difference in, sourness (p<0.05). With the SMAWP, there was a difference in gumminess (p<0.10), and the SMGP had significant differences in sourness (p<0.01), sweetness (p<0.01), andmoisture (p<0.05). Moreover, the purchase intention and willingness to pay were significantly higher when the sustainability information was given. Especially, among the three types of salamis, participants were willing to pay the most for the SMAWP. This is one of the first consumer studies to investigate sensory evaluation and purchase behavior for various types of sustainable livestock production. These results contribute by helping sustainable meat producers and marketers become aware of the kind of sustainable information to which consumers are sensitive.


Perchlorate-reducing microorganisms isolated from contaminated sites.
An extensive microcosm survey of perchlorate-contaminated sites was undertaken to assess the ability of indigenous microorganisms to degrade perchlorate. Samples from 12 contaminated sites and from one pristine location were analysed. Perchlorate was degraded to below detection limit in all electron donor-amended microcosms. Perchlorate-reducing microorganisms (PRMs) were numerous at most of these sites. Sixteen distinct PRMs were isolated that were phylogenetically related to either Dechloromonas in the Beta Proteobacteria (9/16 isolates) or to Azospirillum in the Alpha Proteobacteria (7/16 isolates). The majority of previously isolated PRMs are in the Beta Proteobacteria related to Dechloromonas or Dechlorosoma. This study indicates that PRMs of the genus Azospirillum may be more prevalent at contaminated sites than the current record of isolates suggests. Cell yields, electron donor to perchlorate ratios and maximum specific growth rates were similar among the isolates and similar to the few previously published values. However, the Monod half-saturation constants for perchlorate for the two Azospirillum isolates characterized were lower than those measured for other genera, suggesting that they may be more effective at low concentrations of perchlorate. These results extend the current understanding of PRMs from diverse environments and provide added confidence that microbial perchlorate reduction is ubiquitous, even at highly contaminated sites, and can be harnessed effectively for bioremediation.


Lack of benefit of a primary care-based nurse-led education programme for people with osteoarthritis of the knee.
Osteoarthritis (OA) is the commonest cause of locomotor disability and forms a major element of the workload of the primary care team. There is evidence that patient education may improve quality of life, physical functioning, mental health and coping as well as reducing health service use. The aim of this study was to evaluate the effectiveness of a primary care-based patient education programme (PEP) using a randomised controlled trial. A cluster randomised controlled trial, involving 22 practices, was used to determine the efficacy of a nurse-led education programme. The programme consisted of a home visit and four 1-h teaching sessions. Patients were assessed at baseline and then 1, 3, 6 and 12 months post intervention using 36-item Short Form (SF-36), Western Ontario and McMaster Universities Arthritis Index (WOMAC), arthritis helplessness index and a patient knowledge questionnaire. Direct interviews were used at baseline and at the 12-month follow-up. There were no differences in depression, OA knowledge, pain or physical ability at either 1 month or 1 year between the two groups. Control practices (65 patients from 12 practices) recruited significantly fewer patients than intervention practices (105 patients from ten practices, p = 0.02). Control practices had more doctors (p = 0.02), more non-white patients (p = 0.007), fewer patients living alone (p = 0.005) and lower levels of disability (p = 0.008). We detected a lack of benefit of PEP for people with OA of the knee. This was thought to be due in part to the short intervention time employed and the heterogeneous nature of the disease and the population studied.


Long-term recognition memory for faces assessed by visual paired comparison in 3- and 6-month-old infants.
It has been argued that operant conditioning is the only type of long-term memory present in infants prior to 6 months of age. In this study, memory for faces was investigated in 3- and 6-month-old infants with a visual paired-comparison task. In Experiment 1, infants were habituated to a face presented in different poses; recognition was assessed after a 2-min or a 24-hr retention interval. The 6-month-old infants and the male but not the female 3-month-old infants exhibited novelty preferences. A 2nd experiment showed that 3-month-old female infants were delayed relative to male infants in their face-processing ability rather than in their memory capacity. The results of Experiment 3 demonstrated in 3-month-olds an electrophysiological correlate of delayed recognition memory. These findings are discussed in the context of the neural systems thought to be involved in visual recognition memory (but not in procedural memory), namely the limbic system.


The carotenoid-continuum: carotenoid-based plumage ranges from conspicuous to cryptic and back again.
Carotenoids are frequently used by birds to colour their plumage with green, yellow, orange or red hues, and carotenoid-based colours are considered honest signals of quality, although they may have other functions, such as crypsis. It is usually assumed that red through yellow colours have a signalling function while green is cryptic. Here we challenge this notion using the yellow and green colouration of blue tits (Cyanistes caeruleus), great tits (Parus major) and greenfinches (Carduelis chloris) as a model. The relationship between colouration (chroma, computed using visual sensitivities of conspecifics) and detectability (contrast against natural backgrounds as perceived by conspecifics and avian predators) followed a similar curvilinear pattern for yellow and green plumage with minimum detectability at intermediate levels of carotenoid deposition. Thus, for yellow and green plumage, colours at or close to the point of minimum detectability may aid in crypsis. This may be the case for blue and great tit green and yellow plumage, and greenfinch green plumage, all of which had comparably low levels of detectability, while greenfinch yellow plumage was more chromatic and detectable. As yellow and green blue tit colouration are strongly affected by carotenoid availability during moult, variation in pigment availability between habitats may affect the degree of background-matching or the costliness of producing cryptic plumage. Increasing carotenoid-deposition in the integument does not always lead to more conspicuous colours. In some cases, such as in blue or great tits, carotenoid deposition may be selected through enhanced background-matching, which in turn suggests that producing cryptic plumage may entail costs. We stress however, that our data do not rule out a signalling function of carotenoid-based plumage in tits. Rather, it shows that alternative functions are plausible and that assuming a signalling function based solely on the deposition of carotenoids in the integument may not be warranted.


Novel bleeding risk score for patients with atrial fibrillation on oral anticoagulants, including direct oral anticoagulants.
Balancing bleeding risk and stroke risk in patients with atrial fibrillation (AF) is a common challenge. Though several bleeding risk scores exist, most have not included patients on direct oral anticoagulants (DOACs). We aimed at developing a novel bleeding risk score for patients with AF on oral anticoagulants (OAC) including both vitamin K antagonists (VKA) and DOACs. We included patients with AF on OACs from a prospective multicenter cohort study in Switzerland (SWISS-AF). The outcome was time to first bleeding. Bleeding events were defined as major or clinically relevant non-major bleeding. We used backward elimination to identify bleeding risk variables. We derived the score using a point score system based on the β-coefficients from the multivariable model. We used the Brier score for model calibration (<0.25 indicating good calibration), and Harrel's c-statistics for model discrimination. We included 2147 patients with AF on OAC (72.5% male, mean age 73.4 ± 8.2 years), of whom 1209 (56.3%) took DOACs. After a follow-up of 4.4 years, a total of 255 (11.9%) bleeding events occurred. After backward elimination, age > 75 years, history of cancer, prior major hemorrhage, and arterial hypertension remained in the final prediction model. The Brier score was 0.23 (95% confidence interval [CI] 0.19-0.27), the c-statistic at 12 months was 0.71 (95% CI 0.63-0.80). In this prospective cohort study of AF patients and predominantly DOAC users, we successfully derived a bleeding risk prediction model with good calibration and discrimination.


Deficiency of UDP-GlcNAc:Dolichol Phosphate N-Acetylglucosamine-1 Phosphate Transferase (DPAGT1) causes a novel congenital disorder of Glycosylation Type Ij.
Defects in the assembly of dolichol-linked oligosaccharide or its transfer to proteins result in severe, multi-system human diseases called Type I congenital disorders of glycosylation. We have identified a novel CDG type, CDG-Ij, resulting from deficiency in UDP-GlcNAc: dolichol phosphate N-acetyl-glucosamine-1 phosphate transferase (GPT) activity encoded by DPAGT1. The patient presents with severe hypotonia, medically intractable seizures, mental retardation, microcephaly, and exotropia. Metabolic labeling of cultured dermal fibroblasts from the patient with [2-(3)H]-mannose revealed lowered incorporation of radiolabel into full-length dolichol-linked oligosaccharides and glycoproteins. In vitro enzymatic analysis of microsomal fractions from the cultured cells indicated that oligosaccharyltransferase activity is normal, but the GPT activity is reduced to approximately 10% of normal levels while parents have heterozygous levels. The patient's paternal DPAGT1 allele contains a point mutation (660A>G) that replaces a highly conserved tyrosine with a cysteine (Y170C). The paternal allele cDNA produces a full-length protein with almost no activity when over-expressed in CHO cells. The maternal allele makes only about 12% normal mature mRNA, while the remainder shows a complex exon skipping pattern that shifts the reading frame encoding a truncated non-functional GPT protein. Thus, we conclude that the DPAGT1 gene defects are responsible for the CDG symptoms in this patient. Hum Mutat 22:144-150, 2003.


Conditional Deletion of Bmal1 Accentuates Microvascular and Macrovascular Injury.
The brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1 constitutes a major transcriptional regulator of the circadian clock. Here, we explored the impact of conditional deletion of Bmal1 in endothelium and hematopoietic cells in murine models of microvascular and macrovascular injury. We used two models of Bmal1fx/fx;Tek-Cre mice, a retinal ischemia/reperfusion model and a neointimal hyperplasia model of the femoral artery. Eyes were enumerated for acellular capillaries and were stained for oxidative damage markers using nitrotyrosine immunohistochemistry. LSK (lineage-negative, stem cell antigen-1-positive, c-Kit-positive) cells were quantified and proliferation assessed. Hematopoiesis is influenced by innervation to the bone marrow, which we assessed using IHC analysis. The number of acellular capillaries increased threefold, and nitrotyrosine staining increased 1.5-fold, in the retinas of Bmal1fx/fx;Tek-Cre mice. The number of LSK cells from the Bmal1fx/fx;Tek-Cre mice decreased by 1.5-fold and was accompanied by a profound decrease in proliferative potential. Bmal1fx/fx;Tek-Cre mice also exhibited evidence of bone marrow denervation, demonstrating a loss of neurofilament-200 staining. Injured femoral arteries showed a 20% increase in neointimal hyperplasia compared with similarly injured wild-type controls. Our study highlights the importance of the circadian clock in maintaining vascular homeostasis and demonstrates that specific deletion of BMAL1 in endothelial and hematopoietic cells results in phenotypic features similar to those of diabetes.


Degradation of aromatic compounds through the β-ketoadipate pathway is required for pathogenicity of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici.
Plant roots react to pathogen attack by the activation of general and systemic resistance, including the lignification of cell walls and increased release of phenolic compounds in root exudate. Some fungi have the capacity to degrade lignin using ligninolytic extracellular peroxidases and laccases. Aromatic lignin breakdown products are further catabolized via the β-ketoadipate pathway. In this study, we investigated the role of 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE), an enzyme of the β-ketoadipate pathway, in the pathogenicity of Fusarium oxysporum f. sp. lycopersici towards its host, tomato. As expected, the cmle deletion mutant cannot catabolize phenolic compounds known to be degraded via the β-ketoadipate pathway. In addition, the mutant is impaired in root invasion and is nonpathogenic, even though it shows normal superficial root colonization. We hypothesize that the β-ketoadipate pathway in plant-pathogenic, soil-borne fungi is necessary to degrade phenolic compounds in root exudate and/or inside roots in order to establish disease.


High-Throughput Approaches for the Identification of Pseudomonas aeruginosa Antivirulents.
Antimicrobial resistance is a serious medical threat, particularly given the decreasing rate of discovery of new treatments. Although attempts to find new treatments continue, it has become clear that merely discovering new antimicrobials, even if they are new classes, will be insufficient. It is essential that new strategies be aggressively pursued. Toward that end, the search for treatments that can mitigate bacterial virulence and tilt the balance of host-pathogen interactions in favor of the host has become increasingly popular. In this review, we will discuss recent progress in this field, with a special focus on synthetic small molecule antivirulents that have been identified from high-throughput screens and on treatments that are effective against the opportunistic human pathogen Pseudomonas aeruginosa.


Decreased fecundity among male lead workers.
To investigate time to pregnancy (TTP) in male lead workers in order to determine the dose-response relation between blood lead and decreased fecundity. A total of 163 currently employed married male lead battery workers were classified into five categories of exposure based on questionnaire information and annual individual blood lead measurement. Information pertaining to the TTP was collected using personal interviews with 133 men and their spouses, with 280 valid pregnancies. The fecundability ratios (FRs) were calculated with the Cox discrete proportional hazard regression technique to evaluate the effects of lead exposure. After other factors associated with TTP were controlled for, there was a dose-response relation between blood lead level and TTP. The measured FRs were 0.90 (95% CI 0.61 to 1.34), 0.72 (0.46 to 1.11), 0.52 (0.35 to 0.77), and 0.40 (0.27 to 0.59) for concurrent blood lead levels of <20, 20-29, 30-39, and > or =40 microg/dl, respectively. Paired self comparison was also performed for 41 couples that had pregnancies prior to lead exposure and pregnancies with male occupational lead exposure. The TTP was prolonged for 0.15 cycles by a 1 microg/dl increase in blood lead. These results corroborate the hypothesis that a raised blood lead level affects fecundity. A blood lead level of less than 40 microg/dl may still significantly prolong TTP.


Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of patients with multiple myeloma.
In this article, we review neoplastic contamination in the peripheral blood (PB) of patients with multiple myeloma (MM) upon stem cell mobilization. We first evaluated PB samples from pretreated MM patients following administration of high-dose cyclophosphamide (Cy, 7 g/m2 or 4 g/m2) and granulocyte colony-stimulating factor (G-CSF) for the presence of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic immunoglobulin (cIg) were counted by immunofluorescence microscopy after incubation with appropriate antisera against light and heavy chain Ig. Flow cytometry studies were performed to determine the presence of malignant B lineage elements, using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Prior to PBSC mobilization, circulating plasma cells were detected in all MM patients at 0.1%-1.8% of the mononuclear cell (MNC) fraction (mean value 0.7 +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after administration of chemotherapy and G-CSF. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors, with the peak coinciding with the optimal period for the collection of PBSC. The absolute number of plasma cells showed a 10-50-fold increase over the baseline value. Apheresis products contained 0.7 +/- 0.2% SD myeloma cells (range 0.2%-2.7%), which demonstrated the capacity of plasma cells to proliferate, differentiate, and mature in response to c-kit ligand (SCF), IL-3, IL-6, and a combination of IL-3 and IL-6. Subsequently, in an attempt to reduce tumor cell contamination prior to autologous transplantation, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute bone marrow (BM) function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51%-94%), with a 75-fold enrichment compared with the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33%-95%) and 45% (range 7%-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log depletion of plasma cells and CD 19+ B lineage cells as determined by immunofluorescence studies, although DNA analysis of the CDR III region of the IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high-dose melphalan (140 mg/m2) or melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis. The median time to 0.5 x 10(9) neutrophils, 20 x 10(9) and 50 x 10(9) platelets/L of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. Our data demonstrate the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring normal hematopoiesis following a TBI-containing conditioning regimen.


Infantile variant of Bartter syndrome and sensorineural deafness: a new autosomal recessive disorder.
The infantile variant of Bartter syndrome (IBS) is usually associated with maternal polyhydramnios, premature birth, postnatal polyuria and hypokalemic hypochloremic metabolic alkalosis and a typical appearance. IBS is thought to be an autosomal recessive trait. Several congenital tubular defects are associated with sensorineural deafness (SND). However, an association between the IBS and SND has not been reported so far. Here we describe 5 children of an extended consanguineous Bedouin family with IBS and SND. In 3 of the cases, the typical electrolyte imbalance and facial appearance were detected neonatally. SND was detected as early as age 1 month, suggesting either coincidental homozygotization of 2 recessive genes or a pleiotropic effect of one autosomal recessive gene. This association suggests that evaluation of SND is warranted in every case of IBS.


Temporalization of peak electric generation particulate matter emissions during high energy demand days.
Underprediction of peak ambient pollution by air quality models hinders development of effective strategies to protect health and welfare. The U.S. Environmental Protection Agency's community multiscale air quality (CMAQ) model routinely underpredicts peak ozone and fine particulate matter (PM2.5) concentrations. Temporal misallocation of electricity sector emissions contributes to this modeling deficiency. Hourly emissions are created for CMAQ by use of temporal profiles applied to annual emission totals unless a source is matched to a continuous emissions monitor (CEM) in the National Emissions Inventory (NEI). More than 53% of CEMs in the Pennsylvania-New Jersey-Maryland (PJM) electricity market and 45% nationally are unmatched in the 2008 NEI. For July 2006, a United States heat wave with high electricity demand, peak electric sector emissions, and elevated ambient PM2.5 mass, we match hourly emissions for 267 CEM/NEI pairs in PJM (approximately 49% and 12% of unmatched CEMs in PJM and nationwide) using state permits, electricity dispatch modeling and CEMs. Hourly emissions for individual facilities can differ up to 154% during the simulation when measurement data is used rather than default temporalization values. Maximum CMAQ PM2.5 mass, sulfate, and elemental carbon predictions increase up to 83%, 103%, and 310%, at the surface and 51%, 75%, and 38% aloft (800 mb), respectively.


Anxiety Severity Levels and Coping Strategies during the COVID-19 Pandemic among People Aged 15 Years and Above in Gonabad, Iran.
The coronavirus disease 2019 (COVID-19) outbreak is a global health problem. It is necessary to provide evidence on its unprecedented psychological effects to develop effective psychological interventions. The current study aims to determine the anxiety severity level, coping strategies, and influencing factors in response to the COVID-19 pandemic among people aged 15 years and above in Gonabad, Iran. We conducted a cross-sectional survey via online questionnaires between February and March 2020. We evaluated the anxiety severity levels and coping strategies using the Corona Disease Anxiety Scale (CDAS) and Coping Inventory for Stressful Situations-Short Form (CISS-SF), respectively. Multinomial and ordinal logistic regression models were used to identify the predictors of coping strategies and anxiety. Totally, 500 people completed the questionnaires (response rate: 73%). Of them, 53.4% (95% confidence interval [CI]: 48.9%- 57.8%) suffered moderate to severe levels of anxiety. More than half of the respondents (52.0%; 95% CI: 47.5%-56.4%) utilized emotional-based or avoidant coping strategies. People with no academic education (odds ratio [OR]: 2.16; 95% CI: 1.41- 3.31) and without physical exercise (OR: 2.04; 95% CI: 1.22-3.33) preferred emotional-based coping instead of problem-based coping strategy. Female gender (OR: 1.60, 95%, CI: 1.13-2.28), underlying medical conditions (OR: 2.52, 95% CI: 1.65-3.87), and emotional-based coping (OR: 4.06, 95% CI: 2.76-5.99) were associated with higher severity levels of anxiety. The severity of anxiety during the COVID-19 pandemic was significant among participants. Further attention is needed to enhance the mental health of the vulnerable population during the COVID-19 pandemic. Our findings also identified some factors related to the severity level of anxiety related to COVID-19 that could help formulate better psychological interventions.


Assessment of myocardial viability with 99mTc-sestamibi tomography before coronary bypass graft surgery: correlation with histopathology and postoperative improvement in cardiac function.
Assessment of myocardial viability by 99mTc-sestamibi remains controversial. Accordingly, we investigated the use of sestamibi as a marker of myocardial viability, defined by histopathology, and for predicting improvement of myocardial function after coronary artery bypass graft surgery (CABG). 99mTc-sestamibi perfusion tomography and radionuclide angiography were performed within 2 days before CABG in 21 patients with > or = 75% stenosis of the left anterior descending coronary artery and resting anterior wall dyssynergy. During CABG, transmural myocardial biopsies were obtained from the dyssynergic anterior wall and from normal myocardial segments to determine the extent of viable myocardium by histopathology. Improvement of regional left ventricular function was evaluated by radionuclide angiography at 6 to 8 weeks after CABG. There was a good correlation (r=.85, P<.001) between the quantified sestamibi activity and the extent of viable myocardium determined morphometrically. Among 21 biopsied dyssynergic myocardial segments, 11 improved their function after CABG and 10 failed to improve. Biopsied segments with improved postoperative function had significantly higher sestamibi activity (81+/-5% versus 49+/-16%, P<.0001) and significantly lower extent of interstitial fibrosis (7+/-4% versus 31+/-21%, P=.0002) than segments that failed to improve. A 55% threshold of 99mTc-sestamibi activity had positive and negative predictive values of 79% and 100%, respectively, for recovery of function after CABG in the biopsied segments. Myocardial 99mTc-sestamibi activity correlates well with the extent of viable myocardium and predicts improvement in regional function after CABG. This lends support to the use of sestamibi as a myocardial viability agent.


Gastrointestinal transit and prolonged ambulatory colonic motility in health and faecal incontinence.
Colonic motor function has not been studied in the ambulatory setting over a prolonged period in the unprepared state. Furthermore, the disturbance of this function in patients with faecal incontinence is unknown. To study colonic function over two to three days in the ambulatory, unprepared state in health and in patients with idiopathic faecal incontinence. Six healthy women and six women with faecal incontinence and a structurally intact anal sphincter ingested a dual radioisotope meal, and had a six sensor, solid state manometric probe colonoscopically inserted into the left colon. Scanning was performed until radioisotope left the gut and pressure was recorded for a median of 44 hours. Three of six patients showed abnormal gastric emptying. Patients showed no disturbance of colonic radioisotope transit. Controls had a median of 12, whereas patients had a median of 16, high amplitude propagated waves per 24 hours. In three patients urge incontinence was associated with high amplitude (up to 500 cm water) propagated waves which often reached the rectum. These high pressure waves were identical to those occurring in healthy subjects, the only difference being the lack of adequate sphincter response. Passive incontinence was not associated with colonic motor activity. Defaecation in all subjects was associated with identical propagated waves, and distal movement of 13% (median) of right colonic content and excretion of 32% from the left colon and rectum. The urge to defaecate was associated with either propagated waves (45%) or non-propagated contractions (55%). Rectal motor complexes were recorded in both groups of subjects, but similar rhythmic activity was also recorded in the sigmoid and descending colon. Normal colonic function consists of frequent high pressure propagated waves. Rhythmic activity occurs both proximal to and in the rectum. Defaecation is characterised by high pressure propagated waves associated with coordinated anal sphincter relaxation. Patients with faecal incontinence may have a widespread disturbance of gut function. Urge incontinence, an urge to defaecate, and defaecation can all be associated with identical high amplitude propagated pressure waves.


Carotid endarterectomy: characterization of recent increases in procedure rates.
Recent increases in the rate of carotid endarterectomies (CEAs) have been attributed to results of clinical trials demonstrating efficacy when CEA is performed in centers of excellence. Subsequent population-based data suggest that trial results may not be matched in the community. This study was undertaken to characterize trends in CEA procedure rates after the dissemination of trial data and to describe any change in patient outcomes with population-based data from a single state. Hospital administrative data on CEAs from 1992 to 1996 (n = 45,744) were obtained for the state of Florida. Annualized CEA rates per 100, 000 Florida residents were analyzed to determine trends in patient age, sex, admission type, size of hospital beds, ownership type and teaching status, and annual hospital and surgeon CEA volume. Outcomes were examined to track trends in complication rates. The annual number of CEA procedures increased 74% from 63.7 per 100,000 residents per year to 110.8 per 100,000 residents per year between 1992 and 1996. A single large increase occurred during the second half of 1994 when CEAs increased 73.5% from 16.6 per 100, 000 residents per quarter to 28.8 per 100,000 residents per quarter after a clinical alert on benefits to CEAs in asymptomatic patients. Over 5 years, there were significant trends toward more nonemergent admissions, and more procedures were performed in high-volume hospitals and by high-volume surgeons. Procedure rates in both women and very elderly patients increased more than 70%, which was in step with younger patients and men. The incidence of inpatient stroke and death declined over the 5-year period, whereas the rate of perioperative myocardial infarction remained constant. Experience from Florida indicates that CEA rates increased as results of the Asymptomatic Carotid Artery Study disseminated. Trial results have been broadly interpreted to include women and very elderly patients. More patients are being referred to busier hospitals and to high-volume surgeons, which should continue to result in better patient outcomes.


Embelin Alleviates Severe Airway Inflammation in OVA-LPS-Induced Rat Model of Allergic Asthma.
Asthma is a chronic lung disease, which causes wheezing, tightness in the chest, shortness of breath and coughing. In the wake of coronavirus disease 2019 (COVID-19), which affect the lungs, asthma patients are at high risk. Embelin, a natural benzoquinone obtained mainly from Embelia ribes Burm, has excellent biological properties, including protection against acute asthma. However, since asthma is a chronic and multi-factorial inflammatory disease, asthma conferred by a single allergen in an animal may not be clinically significant. Therefore, the purpose of the current study was to evaluate the effectiveness of embelin against ovalbumin (OVA)-lipopolysaccharide (LPS)-induced severe airway inflammation in experimental animals and to investigate the plausible mechanism of action. Rats (n=36) were divided into six groups. Group I served as a normal control. Groups II-VI were sensitised with severe allergens (OVA and LPS) on day 7, 14 and 21, followed by OVA and LPS challenge for 30 min three times/week for 3 weeks. Group II acted as an asthmatic disease control and received only vehicle. On the other hand, groups III-V received embelin (12.5, 25 and 50 mg/kg, P.O. respectively) while group VI received a standard dexamethasone (2.5 mg/kg, P.O.) for 15 days from day 27. Lung function parameters, including the respiratory rate, tidal volume and airflow rate were measured at the end of the experiment (day 42). The total and differential counts of leukocytes in the blood and bronchoalveolar fluid (BALF) were calculated. Th2-mediated serum pro-inflammatory cytokines such as interleukin (IL)-4, IL-5 and IL-13 levels were analyzed. At the end of the study protocol, the lung tissues were removed for a histopathology study. Additionally, a molecular docking simulation on embelin and standard dexamethasone was applied to support the in vivo findings. Significant inhibition of eosinophils, neutrophils, lymphocytes and monocytes in the blood and the BALF was seen in the groups, which received embelin (25 and 50 mg/kg) and dexamethasone (2.5 mg/kg). Moreover, the lung function parameters were normalised by embelin (25 and 50 mg/kg) treatment significantly. The lung histopathological changes confirmed the protective effect of embelin against severe airway inflammation. The docking findings indicated good binding efficacy of embelin to IL-13. Overall, our findings indicate that embelin can alleviate severe airway inflammation in OVA-LPS-induced model of allergic asthma occurring by suppression of Th2-mediated immune response. Due to its promising anti-asthmatic effect, it is recommended that embelin should be investigated in clinical trials against asthma. It should also be further explored against COVID-19 or COVID-like diseases due to its ameliorative effects on cytokines and immune cell infiltration.


Development of Abuse-Deterrent Formulations Using Sucrose Acetate Isobutyrate.
The objective of the present investigation was to understand the effect of sucrose acetate isobutyrate (SAIB) on abuse-deterrent properties (ADPs) of abuse-deterrent formulations (ADFs) based on Polyox™. SAIB would enhance ADPs of Polyox™-based formulations due to its glassy liquid and hydrophobic properties. Formulations were prepared by granulation followed by compression and heat curing at 90°C. The formulations were evaluated for surface morphology, hardness, manipulation in coffee grinder, particle size distribution, drug (pseudoephedrine hydrochloride) extraction in water, alcohol, 0.1 N HCl, 0.1 N NaOH at room temperature and elevated temperature using microwave and oven, syringeability and injectability, and dissolution. The heat curing of formulations significantly increased the hardness (> 490 N). Addition of SAIB imparted elasticity to formulations and decreased brittleness as indicated by lower values of work done and gradient compared to control formulations. After grinding, about 7.7-25.6% of the powder remained on the sieve (1 mm pore opening), D90 was 53.1-136.7 μm more, and Q (fraction < 500 μm) was 17.8-40.7% less in SAIB-based formulations compared to control formulations. Drug extraction between control and test intact formulations was similar. However, drug extraction was 23.9-42.5% (water), 20.6-26.1% (0.1 N HCl), and 37.4-50.6% (0.1 N NaOH) less in SAIB-based powder cured and uncured formulations compared to control formulations. Dissolution varied from 65.6 ± 4.2 to 97.6 ± 4.0% in 9 h from the formulations. In conclusion, addition of SAIB to Polyox™-based ADFs has synergistic effect on ADPs. This would further decrease potential of drug abuse/misuse by various routes.


Constructions of gene libraries of Rana nigromaculata and Mus musculus with cosmid pHC79.
Gene libraries of Rana nigromaculata and Mus musculus (C57J6) have been constructed by the cosmid system developed by Collins using BamHl and Pstl as cloning restriction enzymes. A simplified formula, which is a modification of Clarke and Carbon derived from approximation, was used for calculating the number of colonies or recombinants necessary for the construction of a gene library. Pro+ and leu+ complementary to the recipient cells (HB101) with the genetic constitution of pro- and leu- have been selected from the BamH1 gene libraries of both Rana nigromaculata and the mouse in the present work.


The role of Schwann cells, T cells and Mycobacterium leprae in the immunopathogenesis of nerve damage in leprosy.
Damage to peripheral nerves is the major complication of reversal (type I) reactions in leprosy. The underlying mechanism of nerve damage remains largely unresolved; however, an important role for type-1 T cells has been suggested. Mycobacterium leprae has a remarkable tropism for the Schwann cells that surround peripheral axons. Because reversal reactions in leprosy are often accompanied by severe and irreversible nerve destruction, and are associated with increased cellular immune reactivity against M. leprae, a likely immunopathogenic mechanism of damage to Schwann cells and peripheral nerves in leprosy is that infected Schwann cells process and present antigens of M. leprae to antigen-specific, inflammatory, type-1 T cells, and that these T cells subsequently damage and lyse infected Schwann cells. Previous animal studies with CD8+ T cells revealed evidence for the existence of such a mechanism. A similar role has been suggested for CD4+ T cells. These latter cells may be more important in causing nerve damage in vivo, given the predilection of M. leprae for Schwann cells, and the dominant role of CD4+, serine esterase+ Th1 cells in the lesions of leprosy. Antagonism of the molecular interactions among M. leprae, Schwann cells and inflammatory T cells may therefore provide a rational strategy for prevention of damage of Schwann cell and nerves in leprosy.


RELATIVE CONTRIBUTIONS OF PARENT-PERCEIVED CHILD CHARACTERISTICS TO VARIATION IN CHILD FEEDING BEHAVIOR.
Few studies have examined the relative impact of co-occurring child characteristics on problematic feeding behavior. The aim of the current study was to assess the relative contributions of parent-perceived child characteristics in multivariable models of child feeding behavior. One hundred sixty-one mothers reported on their child's feeding behavior and a number of key child characteristics. These characteristics were entered into controlled multivariable models of child feeding behavior, using child and parent frequency domains of the Behavioral Pediatrics Feeding Assessment Scale (BPFAS; W. Crist et al., 1994) as outcome measures. Child feeding problems were positively associated with food neophobia and external behavioral and social issues, but not with most domains of temperamental difficulty or sensory sensitivity. Feeding problem frequency was associated with externalizing symptoms whereas parental perceptions of problems and coping were associated with social-interaction problems in the child. Population feeding problems appear to be external and interactive problems rather than driven by innate or internalizing factors. The association with externalizing symptoms suggests that feeding problems at this level may fall within a wider profile of challenging behavior; however, the existence of problematic feeding behaviors may constitute a challenge for parents only when the child's social interactions also are seen to be deficient.


Lower limb muscle strength (LLMS): why sedentary life should never start? A review.
Aging coincides with a decline in LLMS. Preserving LLMS may be considered a very important determinant of functional independence in the elderly. To maintain LLMS the question arises whether habitual physical activities (HPA) can prevent a decline in LLMS. This review aims to determine the relationship between HPA throughout life and LLMS above age 50. Using relevant databases and keywords, 70 studies that met the inclusion criteria were reviewed and where possible, a meta-analysis was performed. The main findings are: (1) the present level of HPA is positively related to LLMS; (2) HPA in the past has little effect on present LLMS; (3) HPA involving endurance have less influence on LLMS compared to HPA involving strength; (4) people with a stable habitually physically active life are able to delay a decline in LLMS. In conclusion, to obtain a high amount of LLMS during aging, it is important to achieve and maintain a high level of HPA with mainly muscle-strengthening activities.


Effect of Ca2+, cyclic GMP, and cyclic AMP added to artificial solution perfusing lingual artery on frog gustatory nerve responses.
The lingual artery of the bullfrog was perfused with artificial solution and the effects of Ca2+, Ca-channel blockers (MnCl2 and verapamil), cGMP, and cAMP added to the perfusing solution of the gustatory nerve responses were examined. The responses to chemical stimuli of group 1 (CaCl2, NaCl, distilled water, D-galactose, and L-threonine) applied to the tongue surface were greatly decreased by a decrease in Ca2+ concentration in the perfusing solution, suppressed by the Ca-channel blockers, enhanced by cGMP, and suppressed by cAMP. The responses to chemical stimuli of group 2 (quinine hydrochloride, theophylline, ethanol, and HCl) were practically not affected by a decrease in Ca2+ concentration, the Ca-channel blockers, cGMP, and cAMP. The responses to the stimuli of group 1 seem to be induced by Ca influx into a taste cell that is triggered by depolarization and modulated by the cyclic nucleotides in a taste cell. The responses to group 2 seem to be induced without accompanying Ca influx.


[A case of osteopathia striata].
We report a case of osteopathia striata, which is an osseous dysplasia with linear striations of the metaphyses of long bones. Our case is very unusual as there are no associated abnormalities of the bones. Osteopathia striata should not be mistaken for osteopoikilosis. Osteopathia striata is not only a peculiar roentgenologic feature: when it is diagnosed, other abnormalities should be looked for, especially cranial osteosclerosis, which may result in complications.


CSAR scoring challenge reveals the need for new concepts in estimating protein-ligand binding affinity.
The dG prediction accuracy by the Lead Finder docking software on the CSAR test set was characterized by R(2)=0.62 and rmsd=1.93 kcal/mol, and the method of preparation of the full-atom structures of the test set did not significantly affect the resulting accuracy of predictions. The primary factors determining the correlation between the predicted and experimental values were the van der Waals interactions and solvation effects. Those two factors alone accounted for R(2)=0.50. The other factors that affected the accuracy of predictions, listed in the order of decreasing importance, were the change of ligand's internal energy upon binding with protein, the electrostatic interactions, and the hydrogen bonds. It appears that those latter factors contributed to the independence of the prediction results from the method of full-atom structure preparation. Then, we turned our attention to the other factors that could potentially improve the scoring function in order to raise the accuracy of the dG prediction. It turned out that the ligand-centric factors, including Mw, cLogP, PSA, etc. or protein-centric factors, such as the functional class of protein, did not improve the prediction accuracy. Following that, we explored if the weak molecular interactions such as X-H...Ar, X-H...Hal, CO...Hal, C-H...X, stacking and π-cationic interactions (where X is N or O), that are generally of interest to the medicinal chemists despite their lack of proper molecular mechanical parametrization, could improve dG prediction. Our analysis revealed that out of these new interactions only CO...Hal is statistically significant for dG predictions using Lead FInder scoring function. Accounting for the CO...Hal interaction resulted in the reduction of the rmsd from 2.19 to 0.69 kcal/mol for the corresponding structures. The other weak interaction factors were not statistically significant and therefore irrelevant to the accuracy of dG prediction. On the basis of our findings from our participation in the CSAR scoring challenge we conclude that a significant increase of accuracy predictions necessitates breakthrough scoring approaches. We anticipate that the explicit accounting for water molecules, protein flexibility, and a more thermodynamically accurate method of dG calculation rather than single point energy calculation may lead to such breakthroughs.


Microbe-Derived Indole Metabolite Demonstrates Potent Multidrug Efflux Pump Inhibition in Staphylococcus aureus.
Efflux pumps are always at the forefront of bacterial multidrug resistance and account for the failure of antibiotics. The present study explored the potential of 2-(2-Aminophenyl) indole (RP2), an efflux pump inhibitor (EPI) isolated from the soil bacterium, to overcome the efflux-mediated resistance in Staphylococcus aureus. The RP2/antibiotic combination was tested against efflux pump over-expressed S. aureus strains. The compound was further examined for the ethidium bromide (EtBr) uptake and efflux inhibition assay (a hallmark of EPI functionality) and cytoplasmic membrane depolarization. The safety profile of RP2 was investigated using in vitro cytotoxicity assay and Ca2+ channel inhibitory effect. The in vivo efficacy of RP2 was studied in an animal model in combination with ciprofloxacin. RP2 exhibited the synergistic activity with several antibiotics in efflux pump over-expressed strains of S. aureus. In the mechanistic experiments, RP2 increased the accumulation of EtBr, and demonstrated the inhibition of its efflux. The antibiotic-EPI combinations resulted in extended post antibiotic effects as well as a decrease in mutation prevention concentration of antibiotics. Additionally, the in silico docking studies suggested the binding of RP2 to the active site of modeled structure of NorA efflux pump. The compound displayed low mammalian cytotoxicity and had no Ca2+ channel inhibitory effect. In ex vivo experiments, RP2 reduced the intracellular invasion of S. aureus in macrophages. Furthermore, the RP2/ciprofloxacin combination demonstrated remarkable efficacy in a murine thigh infection model. In conclusion, RP2 represents a promising candidate as bacterial EPI, which can be used in the form of a novel therapeutic regimen along with existing and upcoming antibiotics, for the eradication of S. aureus infections.


Role of filamentous fungi in migration of radioactive cesium in the Fukushima forest soil environment.
The fate of radioactive Cs deposited after the Fukushima nuclear power plant accident and its associated radiological impacts are largely dependent on its mobility from surface soils to forest ecosystems. We measured the accumulation of radioactive Cs in the fruit bodies of wild fungi in a forest at Iitate, Fukushima, Japan. The transfer factors (TFs) of radioactive Cs from soil to the fruit bodies of wild fungi were between 10-2 and 102, a range similar to that reported for the fruit bodies collected in Europe after the Chernobyl accident and in parts of Japan contaminated by the nuclear bomb test fallout. Comparison of the TFs of wild mushroom and those of fungal hyphae of 704 stock strains grown on agar medium containing nutrients and 137Cs showed that the TFs of wild mushroom were lower. The TF was less than 0.1 after the addition of the minerals zeolite, vermiculite, phlogopite, smectite, or illite of 1.0% weight to the agar medium. These results indicate that the presence of minerals decreases Cs uptake by fungi grown on the agar medium, and filamentous fungi still accumulate radioactive Cs even when minerals are present in the medium.


The structure of raylike arrivals in a shallow-water waveguide.
Acoustic remote sensing of the oceans requires a detailed understanding of the acoustic forward problem. The results of a shallow-water transmission experiment between a vertical array of sources and a vertical array of receivers are reported. The source array is used to provide additional degrees of freedom to isolate and track raylike arrivals by beamforming over both source and receiver arrays. The coordinated source-receiver array processing procedure is presented and its effectiveness in an example of tracking raylike arrivals in a fluctuating ocean environment is shown. Many of these arrivals can be tracked over an hour or more and show slowly varying amplitude and phase. The use of a double-beamforming algorithm lays the foundation for shallow-water acoustic remote sensing using travel time and source and receive angles of selected eigenrays.


Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.
A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.


Heart rate variability in dilated cardiomyopathy.
Chronic heart failure is associated with excessive neurohormonal activation. Analysis of heart rate variability is considered a valid technique for assessment of the autonomic balance of the heart. Twenty symptomatic patients of dilated cardiomyopathy in NYHA class II-IV symptomatic status and as many normal controls were subjected to 24 hours Holter monitoring to assess the heart rate variability with both time domain and frequency domain analysis. Age of the patients ranged from 12 to 67 years (mean +/- SD 38.6 +/- 7 years), the male-female ratio was 4:1. The left ventricular ejection fraction of the patients was between 18-42 percent (mean +/- SD 30.2 +/- 9%) and all received diuretics, digoxin and angiotensin-converting enzyme inhibitors. Heart rate variability parameters measured included mean heart rate with standard deviation, hourly heart rate with SD and the mean of all normal RR intervals from the 24-hour recording. Time domain measures calculated were SD of all normal RR intervals, SD of 5 minute mean RR intervals and root mean square of difference of successive RR intervals. Using spectral plots, frequency domain subsets of low frequency and high frequency were analysed and expressed in normalised units. Total power was also measured. In the dilated cardiomyopathy patients, mean 24-hour heart rate in beats per minute was significantly higher in comparison to controls (82 +/- 13 vs 72 +/- 8; p < 0.001) whereas mean hourly heart rate with standard deviation (msec) was significantly lower (97 +/- 41 vs 232 +/- 25; p < 0.001), SD of all normal RR intervals (msec) was 85.5 +/- 26.3 vs 139.4 +/- 16.9 in controls (p < 0.001), SD of 5 minute mean RR intervals (msec) was also significantly less in patients in comparison to controls (75.8 +/- 39.6 vs 130.8 +/- 20.3; p < 0.001). However, although root mean square of difference of successive RR intervals (msec) was reduced in patients (30.1 +/- 9.3 vs 37.3 +/- 11.7; p < 0.05), the difference was non-significant. Low frequency power (0.05-0.15 Hz) (normalised units) was reduced in the dilated cardiomyopathy group (0.0721 +/- 0.003 vs 0.136 +/- 0.047 in the control group; p < 0.001). High frequency power (0.35-0.50 Hz) (normalised units) (0.08 +/- 0.05 in patients vs 0.09 +/- 0.02 in controls; p > 0.1) and total power frequency (0.02-0.50 Hz) (normalised units) (0.34 +/- 0.05 in patients vs 0.35 +/- 0.12 in controls; p > 0.1) was non-significantly different in the two groups. Regression analysis showed a significant decrease in SD of all normal RR intervals, SD of 5 minute mean RR intervals, low frequency, high frequency, total power and a non-significant decrease in root mean square of difference of successive RR intervals with a decrease in ejection fraction percent whereas there was a significant decrease in SD of all normal RR intervals, SD of 5 minute mean RR intervals, low frequency and total power and a less significant decrease in root mean square of difference of successive RR intervals and high frequency power with an increase in NYHA class. At 6 months duration, 6 patients were lost to follow-up, 3 patients were readmitted (2 for congestive cardiac failure, one of paroxysmal supraventricular tachycardia). One patient who was NYHA class IV at baseline was readmitted for congestive cardiac failure and showed much lower heart rate variability parameters compared to the average of the patients. We conclude that in symptomatic dilated cardiomyopathy patients, heart rate variability parameters are significantly reduced in comparison to control subjects.


A Novel lncRNA ENST00000512916 Facilitates Cell Proliferation, Migration and Cell Cycle Progression in Ameloblastoma.
Our purpose was to identify up-regulated long noncoding RNA ENST00000512916 in ameloblastoma (AB) and explore its role in the progression of AB. We analyzed lncRNA microarray expression profile between six paired AB and normal oral mucosa (NOM) tissues. An up-regulated lncRNA, ENST00000512916 was identified and validated by real-time qPCR. Cell proliferation, migration and cell cycle were detected by CCK-8 assay, transwell chamber and flow cytometry, respectively. Western blotting analysis was used to measure the expression of cell-cycle-related proteins including CyclinD1 and Cyclin-dependent kinase (CDK) 2/4/6. In addition, Xenograft tumor model was constructed to investigate tumor growth. Real-time qPCR confirmed that lncRNA ENST00000512916 was up-regulated in AB tissues. ENST00000512916 knockdown significantly inhibited cell proliferation, migration and the expression of CDK2/4/6 in AM-1 cells. Moreover, ENST00000512916 knockdown suppressed tumor growth in vivo. We also found that ENST00000512916 overexpression significantly promoted the expression of HOXC13 in AM-1 cells. Overexpression of ENST00000512916 promoted cell cycle progression in AM-1 cells, which was reversed by HOXC13 knockdown. Our findings reveal that lncRNA ENST00000512916 promotes cell proliferation, migration and cell cycle progression of AB.


Effects of culturally specific cessation messages on theoretical antecedents of behavior among low-income african american smokers.
Previous research has highlighted the importance of cultural relevance in health risk communications, including tobacco interventions. However, few studies have examined the active components of smoking cessation messages targeting low-income African American smokers. This study tested the influence of message content and culturally specific framing in a sample of adult smokers. In a 2 x 2 factorial experiment, 243 African American smokers (M = 19 cigarettes/day) recruited from the community (55% women; mean age = 43 years) were randomly assigned to 1 of 4 conditions: culturally specific smoking messages, standard smoking messages, culturally specific exercise/weight messages, or standard exercise/weight messages. The primary outcome measures were theoretical antecedents to behavior change, including risk perceptions (general, personal, and culturally specific), readiness to quit smoking, and smoking-related knowledge. The results showed that the smoking messages produced greater culturally specific risk perceptions, readiness to quit smoking, and smoking-related knowledge. The culturally specific messages produced greater personal risk perceptions and intentions to quit. Culturally specific risk perceptions were most affected by culturally specific smoking messages. Findings support the roles of message content and culturally specific framing in the efficacy of brief written interventions for smoking cessation in this population. Future research is needed to examine the influence of these constructs on behavior change.


Dynamic changes in mitochondrial DNA, distribution and activity within cat oocytes during folliculogenesis.
Mitochondria play fundamental roles during oocyte development. The accumulation and spatial redistribution of these energy-producing organelles have been linked to the developmental competence of mammalian oocytes. Here, we assessed the copy number, distribution and activity of mitochondria within cat oocytes during folliculogenesis. In Experiment 1, oocytes were recovered from primordial (n = 152), primary (112), secondary (95), early (131), small (118), antral (86) and advanced antral (5) stages follicles, and mitochondria DNA extracted and quantified using qPCR. In Experiment 2, oocytes from pre-antral (n = 44), early antral (n = 66), small antral (n = 59), antral (n = 41) and advanced antral (n = 21) follicles were isolated and stained with CMXRos MitoTracker dye to assess mitochondrial distribution pattern and activity levels. Oocyte's mitochondria DNA (mtDNA) copy numbers gradually increased as folliculogenesis progressed, with a significant shift at the small antral stage (0.5 to <1 mm in diameter). The location of mitochondria gradually shifted from a homogeneous distribution throughout the cytoplasm in pre-antral oocytes to a pericortical concentration in the advanced antral stage. Quantification of CMXRos fluorescent intensity revealed a progressive increase in mitochondrial activity in oocytes from the pre-antral to the large antral follicles. Taken together, these findings demonstrated that cat oocytes undergo dynamic changes in mitochondrial copy number, distribution and activity during folliculogenesis. These significant modifications to this crucial cytoplasmic organelle are likely associated with the acquisition of developmental competency by cat oocytes.