Reductions of erythrocyte membrane viscoelastic coefficients reflect spectrin deficiencies in hereditary spherocytosis.
Hereditary spherocytosis is a common hemolytic anemia associated with deficiencies in spectrin, the principal structural protein of the erythrocyte membrane-skeleton. We have examined 20 different individuals from 10 spherocytosis kindreds and 2 elliptocytosis kindreds to determine the effects of different levels of spectrin deficiency on the viscoelastic properties of the erythrocyte membrane. Micropipettes were used to perform single-cell micromechanical measurements of approximately 1,000 individual cells to determine the membrane elastic shear modulus, the apparent membrane bending stiffness, and whole cell recovery time constant for the different cell populations. The membrane viscosity was calculated by the product of the shear modulus and the recovery time constant. Results show correlation between the fractional reduction in shear modulus and the fractional reduction in spectrin content (determined by spectrin radioimmunoassay) and spectrin density (determined by the ratios of spectrin to band 3 on electrophoresis gels) suggesting that membrane shear elasticity is directly proportional to the surface density of spectrin on the membrane (P less than 0.001). The apparent membrane bending stiffness is also reduced in proportion to the density of spectrin (P less than 0.001). The membrane viscosity is reduced relative to control (P less than 0.001), but the nature of the relationship between spectrin density and membrane viscosity is less clearly defined. These studies document striking relationships between partial deficiencies of erythrocyte spectrin and specific viscoelastic properties of the mutant membranes.


Contralateral suppression of transient evoked otoacoustic emissions in adults: A normative study.
Whilst otoacoustic emission (OAE) testing has proved to be valuable in revealing information about cochlear outer hair cell integrity, it does not provide insight into the afferent and efferent pathways once the stimulus has reached neural receptors. This information can be obtained objectively through contralateral acoustic stimulation (CAS) suppression. However, obtaining normative data is essential in the implementation of such tests. The primary aim was to undertake a small pilot study to collect the CAS suppression across a predefined frequency range in order to provide a preliminary normative data set to be used with the newly developed transient evoked otoacoustic emission (TEOAE)-CAS module (PATH MEDICAL, Germering, Germany). Secondary aims included the analysis of the relationships between left and right CAS suppression, between male and female CAS suppression and between TEOAE signal-to-noise ratio (SNR) and CAS suppression. The purpose of this study was to determine preliminary normative data for contralateral TEOAE suppression from 40 normal ears of 20 healthy young adults (10 males and 10 females). Subjects were recruited using purposive sampling. The CAS suppression responses were obtained automatically by means of the data-collection protocol on the device used. From the data obtained, correlations between TEOAE SNR and CAS suppression were made using Pearson's correlation coefficient. The data were statistically processed to form a normative database which possesses the potential of serving as a basis for further research aimed at determining the utility of CAS suppression testing when evaluating ear pathology. A mean CAS suppression of 0.8 decibels (dB) (0.61 SD) was obtained. There was no statistically significant relationship between TEOAE SNR and CAS suppression. There was no significant suppression difference in terms of laterality of ears or gender. Normative values for CAS suppression of TEOAEs in a group of normal-hearing individuals were obtained using the newly developed TEOAE-CAS module (PATH MEDICAL, Germering, Germany). The availability of normative data for contralateral TEOAE suppression using the studied module allows for it to become commercially available, which will enable researchers and audiologists to perform this measurement in different populations in the evaluation of ear pathology.


Hyperglycemic diet and training alter insulin sensitivity, intramyocellular lipid content but not UCP3 protein expression in rat skeletal muscles.
Intramyocellular lipids (IMCL) and mitochondrial uncoupling protein-3 (UCP3) have been implicated in the development of muscular insulin resistance. This study aimed to investigate the role of IMCL and UCP3 in the development of glucose intolerance and muscular insulin resistance during 12 weeks of an obesity-inducing 'cafeteria-style' diet alone (CAF), or in conjunction with exercise training from weeks 8-12 (CAFTR), in rats. At the end of the intervention period, gain in body weight was 20% higher in CAF (305+/-10 g) than controls (CON) (255+/-14 g; p<0.001) and CAFTR (253+/-7 g; p<0.001). Furthermore, compared with CON, the Matsuda insulin sensitivity index (ISI), assessed during a 2-h intravenous glucose tolerance test, was markedly lower in CAF (6.7+/-0.4) than in either CON (15.6+/-1.4; p<0.001) or CAFTR (11.2+/-1.1; p<0.001). Moreover, in CAF glucose transport at a submaximal insulin concentration (200 microU/ml) was reduced by approximately 60% (p<0.05) in both red and white gastrocnemius muscles, but not in m. soleus. However, glucose transport in CAFTR was similar to CON in red gastrocnemius. In CAF fiber-specific IMCL content determined in m. soleus and extensor digitorum longus (EDL), was higher than in CON (p<0.01) and CAFTR (p<0.001). Muscle UCP3 protein content was not changed by any of the interventions. Interestingly, within CAF and CAFTR, ISI closely negatively correlated with IMCL content in both type I (soleus, r=-0.93; EDL, r=-0.90; p<0.05) and IIa (EDL, r=-0.52, p<0.05) muscle fibers. These findings indicate that changes in IMCL content but not UCP3 content are implicated in short-term effects of cafeteria-style diet and exercise training on muscular insulin sensitivity in rats.


A Mechanistic Computational Model Reveals That Plasticity of CD4+ T Cell Differentiation Is a Function of Cytokine Composition and Dosage.
CD4+ T cells provide cell-mediated immunity in response to various antigens. During an immune response, naïve CD4+ T cells differentiate into specialized effector T helper (Th1, Th2, and Th17) cells and induced regulatory (iTreg) cells based on a cytokine milieu. In recent studies, complex phenotypes resembling more than one classical T cell lineage have been experimentally observed. Herein, we sought to characterize the capacity of T cell differentiation in response to the complex extracellular environment. We constructed a comprehensive mechanistic (logical) computational model of the signal transduction that regulates T cell differentiation. The model's dynamics were characterized and analyzed under 511 different environmental conditions. Under these conditions, the model predicted the classical as well as the novel complex (mixed) T cell phenotypes that can co-express transcription factors (TFs) related to multiple differentiated T cell lineages. Analyses of the model suggest that the lineage decision is regulated by both compositions and dosage of signals that constitute the extracellular environment. In this regard, we first characterized the specific patterns of extracellular environments that result in novel T cell phenotypes. Next, we predicted the inputs that can regulate the transition between the canonical and complex T cell phenotypes in a dose-dependent manner. Finally, we predicted the optimal levels of inputs that can simultaneously maximize the activity of multiple lineage-specifying TFs and that can drive a phenotype toward one of the co-expressed TFs. In conclusion, our study provides new insights into the plasticity of CD4+ T cell differentiation, and also acts as a tool to design testable hypotheses for the generation of complex T cell phenotypes by various input combinations and dosages.


Variable coordination geometries at the diiron(II) active site of ribonucleotide reductase R2.
The R2 subunit of Escherichia coli ribonucleotide reductase contains a dinuclear iron center that generates a catalytically essential stable tyrosyl radical by one electron oxidation of a nearby tyrosine residue. After acquisition of Fe(II) ions by the apo protein, the resulting diiron(II) center reacts with O(2) to initiate formation of the radical. Knowledge of the structure of the reactant diiron(II) form of R2 is a prerequisite for a detailed understanding of the O(2) activation mechanism. Whereas kinetic and spectroscopic studies of the reaction have generally been conducted at pH 7.6 with reactant produced by the addition of Fe(II) ions to the apo protein, the available crystal structures of diferrous R2 have been obtained by chemical or photoreduction of the oxidized diiron(III) protein at pH 5-6. To address this discrepancy, we have generated the diiron(II) states of wildtype R2 (R2-wt), R2-D84E, and R2-D84E/W48F by infusion of Fe(II) ions into crystals of the apo proteins at neutral pH. The structures of diferrous R2-wt and R2-D48E determined from these crystals reveal diiron(II) centers with active site geometries that differ significantly from those observed in either chemically or photoreduced crystals. Structures of R2-wt and R2-D48E/W48F determined at both neutral and low pH are very similar, suggesting that the differences are not due solely to pH effects. The structures of these "ferrous soaked" forms are more consistent with circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopic data and provide alternate starting points for consideration of possible O(2) activation mechanisms.


Strong Souls: development and validation of a culturally appropriate tool for assessment of social and emotional well-being in Indigenous youth.
The aim of the present study was to develop and validate an appropriate tool to assess the social and emotional well-being (SEWB) of Indigenous adolescents participating in the longitudinal Aboriginal Birth Cohort (ABC) Study. A range of tools was assessed as to the suitability of each for use in the ABC Study. Two existing tools and a newly developed one called 'Strong Souls' were piloted in a representative group (n = 67). Strong Souls was selected as the most appropriate for use in the ABC Study, and was completed by 361 participants. Exploratory factor analysis was used to explore construct validity. Cronbach alpha was used to assess the reliability of the latent constructs and the tool overall. Factor analysis produced a 25-item, four-factor model accounting for 34.5% of the variance. This model demonstrated sound construct validity and reliability. Factor structure was consistent with the epidemiological literature, identifying constructs of anxiety, resilience, depression and suicide risk. While these align with observations in mainstream populations, different relationships between distinct factors, and differences in symptomatology were found in this population. For example, two key findings were: feelings of sadness and low mood were linked with anxiety and not depression; and the expression of anger was verified as a unique symptom of depression for Indigenous people. Strong Souls demonstrated validity, reliability and cultural appropriateness as a tool for screening for SEWB among Indigenous young people in the Northern Territory.


Graphical Tasks to Measure Upper Limb Function in Patients With Parkinson's Disease: Validity and Response to Dopaminergic Medication.
The most widely used method to assess motor functioning in Parkinson's disease (PD) patients is the unified Parkinson's disease rating scale-III (UPDRS-III). The UPDRS-III has limited ability to detect subtle changes in motor symptoms. Alternatively, graphical tasks can be used to provide objective measures of upper limb motor dysfunction. This study investigated the validity of such graphical tasks to assess upper limb function in PD patients and their ability to detect subtle changes in performance. Fourteen PD patients performed graphical tasks before and after taking dopaminergic medication. Graphical tasks included figure tracing, writing, and a modified Fitts' task. The Purdue pegboard test was performed to validate these graphical tasks. Movement time (MT), writing size, and the presence of tremor were assessed. MT on the graphical tasks correlated significantly with performance on the Purdue pegboard test (Spearman's ρ > 0.65; p < 0.05). MT decreased significantly after the intake of dopaminergic medication. Tremor power decreased after taking dopaminergic medication in most PD patients who suffered from tremor. Writing size did not correlate with performance on the Purdue pegboard test, nor did it change after taking medication. Our set of graphical tasks is valid to assess upper limb function in PD patients. MT proved to be the most useful measure for this purpose. The response on dopaminergic medication was optimally reflected by an improved MT on the graphical tasks in combination with a decreased tremor power, whereas writing size did not respond to dopaminergic treatment.


Quantum confinement effects in transferrable silicon nanomembranes and their applications on unusual substrates.
Two dimensional (2D) semiconductors have attracted attention for a range of electronic applications, such as transparent, flexible field effect transistors and sensors owing to their good optical transparency and mechanical flexibility. Efforts to exploit 2D semiconductors in electronics are hampered, however, by the lack of efficient methods for their synthesis at levels of quality, uniformity, and reliability needed for practical applications. Here, as an alternative 2D semiconductor, we study single crystal Si nanomembranes (NMs), formed in large area sheets with precisely defined thicknesses ranging from 1.4 to 10 nm. These Si NMs exhibit electronic properties of two-dimensional quantum wells and offer exceptionally high optical transparency and low flexural rigidity. Deterministic assembly techniques allow integration of these materials into unusual device architectures, including field effect transistors with total thicknesses of less than 12 nm, for potential use in transparent, flexible, and stretchable forms of electronics.


Nuclear PIM1 confers resistance to rapamycin-impaired endothelial proliferation.
The PIM serine/threonine kinases and the mTOR/AKT pathway integrate growth factor signaling and promote cell proliferation and survival. They both share phosphorylation targets and have overlapping functions, which can partially substitute for each other. In cancer cells PIM kinases have been reported to produce resistance to mTOR inhibition by rapamycin. Tumor growth depends highly on blood vessel infiltration into the malignant tissue and therefore on endothelial cell proliferation. We therefore investigated how the PIM1 kinase modulates growth inhibitory effects of rapamycin in mouse aortic endothelial cells (MAEC). We found that proliferation of MAEC lacking Pim1 was significantly more sensitive to rapamycin inhibition, compared to wildtype cells. Inhibition of mTOR and AKT in normal MAEC resulted in significantly elevated PIM1 protein levels in the cytosol and in the nucleus. We observed that truncation of the C-terminal part of Pim1 beyond Ser 276 resulted in almost exclusive nuclear localization of the protein. Re-expression of this Pim1 deletion mutant significantly increased the proliferation of Pim1(-/-) cells when compared to expression of the wildtype Pim1 cDNA. Finally, overexpression of the nuclear localization mutant and the wildtype Pim1 resulted in complete resistance to growth inhibition by rapamycin. Thus, mTOR inhibition-induced nuclear accumulation of PIM1 or expression of a nuclear C-terminal PIM1 truncation mutant is sufficient to increase endothelial cell proliferation, suggesting that nuclear localization of PIM1 is important for resistance of MAEC to rapamycin-mediated inhibition of proliferation.


Free and Bound Therapeutic Lithium in Brain Signaling.
Lithium, a first-line therapy for bipolar disorder, is effective in preventing suicide and new depressive/manic episodes. Yet, how this beguilingly simple monocation with only two electrons could yield such profound therapeutic effects remains unclear. An in-depth understanding of lithium's mechanisms of actions would help one to develop better treatments limiting its adverse side effects and repurpose lithium for treating traumatic brain injury and chronic neurodegenerative diseases. In this Account, we begin with a comparison of the physicochemical properties of Li+ and its key native rivals, Na+ and Mg2+, to provide physical grounds for their competition in protein binding sites. Next, we review the abnormal signaling pathways and proteins found in bipolar patients, who generally have abnormally high intracellular Na+ and Ca2+ concentrations, high G-protein levels, and hyperactive phosphatidylinositol signaling and glycogen synthase kinase-3β (GSK3β) activity. We briefly summarize experimental findings on how lithium, at therapeutic doses, modulates these abnormal signaling pathways and proteins. Following this survey, we address the following aspects of lithium's therapeutic actions: (1) Can Li+ displace Na+ from the allosteric Na+-binding sites in neurotransmitter transporters and G-protein coupled receptors (GPCRs); if so, how would this affect the host protein's function? (2) Why are certain Mg2+-dependent enzymes targeted by Li+? (3) How does Li+ binding to Mg2+-bound ATP/GTP (denoted as NTP) in solution affect the cofactor's conformation and subsequent recognition by the host protein? (4) How do NTP-Mg-Li complexes modulate the properties of the respective cellular receptors and signal-transducing proteins? We show that Li+ may displace Na+ from allosteric Na+-binding sites in certain GPCRs and stabilize inactive conformations, preventing these receptors from relaying signal to the respective G-proteins. It may also displace Mg2+ in enzymes containing highly cationic Mg2+-binding sites such as GSK3β, but not in enzymes containing Mg2+-binding sites with low or zero charge. We further show that Li+ binding to Mg2+-NTP in water does not alter the NTP conformation, which is locked by all three phosphates binding to Mg2+. However, bound lithium in the form of [NTP-Mg-Li]2- dianions can activate or inhibit the host protein depending on the NTP-binding pocket's shape, which determines the metal-binding mode: The ATP-binding pocket's shape in the P2X receptor is complementary to the native ATP-Mg solution conformation and nicely fits [ATP-Mg-Li]2-. However, since the ATP βγ phosphates bind Li+, bimetallic [ATP-Mg-Li]2- may be more resistant to hydrolysis than the native cofactor, enabling ATP to reside longer in the binding site and elicit a prolonged P2X response. In contrast, the elongated GTP-binding pockets in G-proteins allow only two GTP phosphates to bind Mg2+, so the GTP conformation is no longer "triply-locked". Consequently, Li+ binding to GTP-Mg can significantly alter the native cofactor's structure, lowering the activated G-protein level, thus attenuating hyperactive G-protein-mediated signaling in bipolar patients. In summary, we have presented a larger "connected" picture of lithium's diverse effects based on its competition as a free monocation with native cations or as a phosphate-bound polyanionic complex modulating the host protein function.


Fluid dynamics vascular theory of brain and inner-ear function in traumatic brain injury: a translational hypothesis for diagnosis and treatment.
It is hypothesized that in all traumatic brain injury (TBI) patients with a clinical history of closed or penetrating head injury, the initial head trauma is associated with a vibratory sensation and noise exposure, with resultant alteration in vascular supply to the structures and contents of the fluid compartments of brain and ear (i.e., the fluid dynamics vascular theory of brain-inner-ear function [FDVTBE]). The primary etiology-head trauma-results in an initial fluctuation, interference, or interaction in the normal fluid dynamics between brain and labyrinth of the inner ear, with a resultant clinical diversity of complaints varying in time of onset and severity. Normal function of the brain and ear is a reflection of a normal state of homeostasis between the fluid compartments in the brain of cerebrospinal fluid and perilymph-endolymph in the labyrinth of the ear. The normal homeostasis in the structures and contents between the two fluid compartment systems--intracerebral and intralabyrinthine--is controlled by mechanisms involved in the maintenance of normal pressures, water and electrolyte content, and neurotransmitter activities. The initial pathophysiology (a reflection of an alteration in the vascular supply to the brain-ear) is hypothesized to be an initial acute inflammatory response, persistence of which results in ischemia and an irreversible alteration in the involved neural substrates of brain-ear. Clinically, a chronic multisymptom complex becomes manifest. The multisymptom complex, individual for each TBI patient regardless of the diagnostic TBI category (i.e., mild, moderate, or severe), initially reflects processes of inflammation and ischemia which, in brain, result in brain volume loss identified as neurodegeneration and hydrocephalus ex vacuo or an alteration in cerebrospinal fluid production (i.e., pseudotumor cerebri) and, in ear, secondary endolymphatic hydrops with associated cochleovestibular complaints of hearing loss, tinnitus, vertigo, ear blockage, and hyperacusis. The FDVTBE integrates and translates a neurovascular hypothesis for Alzheimer's disease to TBI. This study presents an FDVTBE hypothesis of TBI to explain the clinical association of head trauma (TBI) and central nervous system neurodegeneration with multisensory complaints, highlighted by and focusing on cochleovestibular complaints. A clinical case report, previously published for demonstration of the cerebrovascular medical significance of a particular type of tinnitus, and evidence-based basic science and clinical medicine are cited to provide objective evidence in support and demonstration of the FDVTBE.


The Effects of an Ecological Diversifying Experience on Creativity: An Experimental Study.
Sometimes, life houses rare and unexpected events, such as moving abroad or meeting a special person unexpectedly. Recently, these situations have been indicated as "diversifying experiences" (DEs), defined as unusual and unexpected events that drag people outside their daily routine and accustomed schemas. The core mechanism of DEs would entail the disruption of our mental schema, which can facilitate unexpected connections among even distant ideas, thus enhancing people's cognitive flexibility, that is, a key component of creative thinking. Despite both qualitative and lab-based studies have investigated the features of these experiences, an ecological assessment of their properties also in relation with creativity is still an open issue. The aim of this research is to study the DE-creativity link in a more ecological way, on the basis of a real-life disruptive experience of light deprivation. Specifically, we compared an ecological DE artistic established entertainment format (i.e., "dialogue in the dark," which is seeing people perform several daily life activities but in the absence of light) with an equivalent experience in which the same activities were acted in the sunlight. The absence of light played the role of violating mechanism, framed within the ecological experiential format of the "dialogue in the dark." We compared visitors' emotional profile [Positive and Negative Affect Schedule (PANAS), ad hoc Adjective Checklist], perceived impact of the experience [Centrality of Event Scale (CES)], and creative performance [Torrance Tests of Creative Thinking (TTCT)] in both groups of sighted people (in absence of light vs. in presence of light); and we also controlled for people's openness to experience and need for cognitive closure, as dispositions. Results showed that (vs. control group) "dialogue in the dark" (i) led to worse creative performances, (ii) produced more intense positive affect, and (iii) resulted as a more impacting experience. Intense short-term impact of DE could have been detrimental for participants' creativity. People may need more time to elaborate the DE and accommodate existing schema to generate more creative ideas. This is the first study proposing and succeeding in demonstrating the feasibility to investigate even real complex DEs in a controlled way, thus outlining how their link with creativity can take place in real life.


De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.).
In rubber tree, bark is one of important agricultural and biological organs. However, the molecular mechanism involved in the bark formation and development in rubber tree remains largely unknown, which is at least partially due to lack of bark transcriptomic and genomic information. Therefore, it is necessary to carried out high-throughput transcriptome sequencing of rubber tree bark to generate enormous transcript sequences for the functional characterization and molecular marker development. In this study, more than 30 million sequencing reads were generated using Illumina paired-end sequencing technology. In total, 22,756 unigenes with an average length of 485 bp were obtained with de novo assembly. The similarity search indicated that 16,520 and 12,558 unigenes showed significant similarities to known proteins from NCBI non-redundant and Swissprot protein databases, respectively. Among these annotated unigenes, 6,867 and 5,559 unigenes were separately assigned to Gene Ontology (GO) and Clusters of Orthologous Group (COG). When 22,756 unigenes searched against the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database, 12,097 unigenes were assigned to 5 main categories including 123 KEGG pathways. Among the main KEGG categories, metabolism was the biggest category (9,043, 74.75%), suggesting the active metabolic processes in rubber tree bark. In addition, a total of 39,257 EST-SSRs were identified from 22,756 unigenes, and the characterizations of EST-SSRs were further analyzed in rubber tree. 110 potential marker sites were randomly selected to validate the assembly quality and develop EST-SSR markers. Among 13 Hevea germplasms, PCR success rate and polymorphism rate of 110 markers were separately 96.36% and 55.45% in this study. By assembling and analyzing de novo transcriptome sequencing data, we reported the comprehensive functional characterization of rubber tree bark. This research generated a substantial fraction of rubber tree transcriptome sequences, which were very useful resources for gene annotation and discovery, molecular markers development, genome assembly and annotation, and microarrays development in rubber tree. The EST-SSR markers identified and developed in this study will facilitate marker-assisted selection breeding in rubber tree. Moreover, this study also supported that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for transcriptome characterization and molecular marker development in non-model species, especially those with large and complex genomes.


STAT6 links IL-4/IL-13 stimulation with pendrin expression in asthma and chronic obstructive pulmonary disease.
Signaling through the interleukin-4/interleukin-13 (IL-4/IL-13) receptor complex is a crucial mechanism in the development of bronchial asthma and chronic obstructive pulmonary disease (COPD). In bronchial epithelial cells, this signaling pathway leads to changes in the expression levels of several genes that are possibly involved in protection against and/or pathogenesis of these diseases. The expression of pendrin (SLC26A4), a candidate for the latter category, is upregulated by IL-4/IL-13 and leads to overproduction of mucus and increased viscosity of the airway surface liquid (ASL). Therefore, elucidating the transcriptional regulation of pendrin could aid in the development of new pharmacological leads for asthma and/or COPD therapy. Here we show that IL-4/IL-13 significantly increased human pendrin promoter activity in HEK-Blue cells but not in STAT6-deficient HEK293 Phoenix cells; that mutation of the STAT6 binding site (N(4) GAS motif) rendered the promoter insensitive to IL-4/IL-13; and that addition of the N(4) GAS motif to an IL-4/IL-13-unresponsive sequence of the human pendrin promoter conferred sensitivity to both ILs.


Functional genomics reveals the sole sulphate transporter of the Mycobacterium tuberculosis complex and its relevance to the acquisition of sulphur in vivo.
Sulphur is essential for some of the most vital biological activities such as translation initiation and redox maintenance, and genes involved in sulphur metabolism have been implicated in virulence. Mycobacterium tuberculosis has three predicted genes for the prototrophic acquisition of sulphur as sulphate: cysA, part of an ABC transporter, and cysA2 and A3, SseC sulphotransferases. Screening for amino acid auxotrophs of Mycobacterium bovis BCG, obtained by transposon mutagenesis, was used to select methionine auxotrophs requiring a sulphur-containing amino acid for growth. We have characterized one of these auxotrophs as being disrupted in cysA. Both the cysA mutant and a previously identified mutant in an upstream gene, subI, were functionally characterized as being completely unable to take up sulphate. Complementation of the cysA mutant with the wild-type gene from M. tuberculosis restored prototrophy and the ability to take up sulphate with the functional characteristics of an ABC transporter. Hence, it appears that this is the sole locus encoding inorganic sulphur transport in the M. tuberculosis complex.


Coagulation activity before and after acute psychosocial stress increases with age.
To assess whether stress further increases hypercoagulation in older individuals. We investigated whether acute stress-induced changes in coagulation parameters differ with age. It is known that hypercoagulation occurs in response to acute stress and that a shift in hemostasis toward a hypercoagulability state occurs with age. However, it is not yet known whether acute stress further increases hypercoagulation in older individuals, and thus may increase their risk for cardiovascular disease (CVD). A total of 63 medication-free nonsmoking men, aged between 20 and 65 years (mean +/- standard error of the mean = 36.7 +/- 1.7 years), underwent an acute standardized psychosocial stress task combining public speaking and mental arithmetic in front of an audience. We measured plasma clotting factor VII activity (FVII:C), fibrinogen, and D-dimer at rest, immediately, and 20 minutes after stress. Increased age predicted greater increases in fibrinogen (beta = 0.26, p = 0.041; DeltaR(2) = 0.05), FVII:C (beta = 0.40, p = .006; DeltaR(2) = 0.11), and D-dimer (beta = 0.51, p < .001; DeltaR(2) = 0.18) from rest to 20 minutes after stress independent of body mass index and mean arterial blood pressure. General linear models revealed significant effects of age and stress on fibrinogen, FVII:C, and D-dimer (main effects: p < .04), and greater D-dimer stress reactivity with older age (interaction age-by-stress: F(1.5/90.4) = 4.36, p = .024; f = 0.33). Our results suggest that acute stress might increase vulnerability in the elderly for hypercoagulability and subsequent hemostasis-associated diseases like CVD.


[Transcranial direct current stimulation--a new tool for human cognitive neuroscience].
Transcranial direct current stimulation (tDCS) is a non-invasive procedure of cortical stimulation, in which weak direct currents are used to polarize target brain regions. Depending on the polarity of the stimulation, tDCS can increase (anodal tDCS) or decrease (cathodal tDCS) cortical excitability in the stimulated brain regions and thereby enable the investigation of the causal relationships between brain activity and behavior. Recently, tDCS has been increasingly used to investigate human cognitive and motor functions in both healthy volunteers and neurological patients. Although tDCS generally produces diffuse cortical stimulation over a period of time, it has several advantages over other brain-stimulation tools such as transcranial magnetic stimulation (TMS). First, since tDCS produces less artifacts such as acoustic noise and muscle twitching, it is more suitable for double-blind, sham-controlled studies and clinical applications. Second, tDCS is not very expensive and can be performed with compact equipment, it can be easily combined with ongoing projects in neuroscience and psychology laboratories. Third, the facilitation of motor and cognitive functions by anodal tDCS may have great potential for cognitive and motor enhancement, for example, to support learning in healthy volunteers and to expedite the rehabilitation process in neurological patients. Finally, thus far, seizure incidents have not been reported in tDCS studies, tDCS has thus become a complementary tool to TMS and occupies a unique position in current cognitive neuroscience.


Abrogated RANKL expression in properdin-deficient mice is associated with better outcome from collagen-antibody-induced arthritis.
Properdin amplifies the alternative pathway of complement activation. In the present study, we evaluated its role in the development of collagen antibody-induced arthritis (CAIA). Arthritis was induced by intraperitoneal injection of a collagen antibody cocktail into properdin-deficient (KO) and wild-type (WT) C57BL/6 mice. Symptoms of disease were evaluated daily. The degree of joint damage was assessed histologically and with immunostaining for bone-resorption markers. Phenotypes of cell populations, their receptor expression, and intracellular cytokine production were determined with flow cytometry. Osteoclast differentiation of bone marrow (BM) precursors was evaluated by staining for tartrate-resistant acid phosphatase (TRAP). Properdin-deficient mice developed less severe CAIA than did WT mice. They showed significantly improved clinical scores and downregulated expression of bone-resorption markers in the joints at day 10 of disease. The frequencies of Ly6G⁺CD11b⁺ cells were fewer in BM, blood, and synovial fluid (SF) of KO than of WT CAIA mice. The receptor activator of nuclear factor κB ligand (RANKL) was downregulated on arthritic KO neutrophils from BM and the periphery. Decreased C5a amounts in KO SF contributed to lower frequencies of CD5aR⁺-bearing neutrophils. In blood, surface C5aR was detected on KO Ly6G⁺ cells as a result of low receptor engagement. Circulating CD4⁺ T cells had an altered ability to produce interleukin (IL)-17 and interferon (IFN)-γ and to express RANKL. In KO CAIA mice, decreased frequencies of CD4⁺ T cells in the spleen were related to low CD86 expression on Ly6GhighCD11b⁺ cells. Arthritic KO T cells spontaneously secreted IFN-γ but not IL-17 and IL-6, and responded to restimulation with less-vigorous cytokine production in comparison to WT cells. Fewer TRAP-positive mature osteoclasts were found in KO BM cell cultures. Our data show that the active involvement of properdin in arthritis is related to an increased proinflammatory cytokine production and RANKL expression on immune cells and to a stimulation of the RANKL-dependent osteoclast differentiation.


Learning what matters for patients: qualitative evaluation of a health promotion program for those with serious mental illness.
Sedentary lifestyle, poor dietary behaviors and metabolic alterations associated with psychiatric medications contribute to poor health and high rates of obesity among individuals with serious mental illness (SMI). Interventions that increase engagement in physical exercise, dietary modifications, lifestyle changes and preventive health care can provide health benefits across the lifespan. These interventions have led to substantial physical improvements in some persons with SMI, while others have not improved or have experienced worsening physical health. We set out to identify characteristics of a health promotion program that persons with SMI associated with physical health improvements. Interviews were conducted with eight participants from the In SHAPE health-promotion program who lost at least 10 pounds or diminished their waist circumference by at least 10 cm. Interviews aimed to determine which aspects of the program were perceived to be most helpful in promoting physical health improvement. Among successful participants, three themes emerged, highlighting the importance of: (i) individualized interventions promoting engagement in the program; (ii) relationships with health-promotion program employees and (iii) self-confidence resulting from program participation. Health-promotion programs that target these areas may have better success in achieving health benefits for persons with SMI.


Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid.
In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA-protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by Western blotting, mass spectrometry, immunofluorescence staining, and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and those with OA. Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA.


Field performance of a low-cost sensor in the monitoring of particulate matter in Santiago, Chile.
Integration of low-cost air quality sensors with the internet of things (IoT) has become a feasible approach towards the development of smart cities. Several studies have assessed the performance of low-cost air quality sensors by comparing their measurements with reference instruments. We examined the performance of a low-cost IoT particulate matter (PM10 and PM2.5) sensor in the urban environment of Santiago, Chile. The prototype was assembled from a PM10-PM2.5 sensor (SDS011), a temperature and relative humidity sensor (BME280) and an IoT board (ESP8266/Node MCU). Field tests were conducted at three regulatory monitoring stations during the 2018 austral winter and spring seasons. The sensors at each site were operated in parallel with continuous reference air quality monitors (BAM 1020 and TEOM 1400) and a filter-based sampler (Partisol 2000i). Variability between sensor units (n = 7) and the correlation between the sensor and reference instruments were examined. Moderate inter-unit variability was observed between sensors for PM2.5 (normalized root-mean-square error 9-24%) and PM10 (10-37%). The correlations between the 1-h average concentrations reported by the sensors and continuous monitors were higher for PM2.5 (R2 0.47-0.86) than PM10 (0.24-0.56). The correlations (R2) between the 24-h PM2.5 averages from the sensors and reference instruments were 0.63-0.87 for continuous monitoring and 0.69-0.93 for filter-based samplers. Correlation analysis revealed that sensors tended to overestimate PM concentrations in high relative humidity (RH > 75%) and underestimate when RH was below 50%. Overall, the prototype evaluated exhibited adequate performance and may be potentially suitable for monitoring daily PM2.5 averages after correcting for RH.


Synthesis of surfactants based on pentaerythritol. I. Cationic and zwitterionic gemini surfactants.
Simple strategies for the synthesis of five series of cationic gemini surfactants and one series of zwitterionic gemini surfactants from pentaerythritol have been developed. Two lipophilic groups were introduced onto pentaerythritol by alkylation of the known compound, O-benzylidenepentaerythritol, with 1-bromooctane, 1-bromodecane, 1-bromododecane, and 1-bromotetradecane. Hydrogenolysis of the benzylidene acetals gave diols which were converted into three different series of trimethylammonium derivatives. The diiodides derived from the diols could be displaced by dimethylamine, even though they are adjacent to a quaternary carbon atom. Alkylation with methyl iodide gave the first series. The iodides were easily displaced by cyanide ion and the resulting dinitriles were hydrolyzed, converted to N,N-dimethylamides, and reduced to give a second series. Oxidation of the diols to dialdehydes under Swern conditions followed by Horner-Wadsworth-Emmons reactions with diethyl N,N-dimethylcarbamoylmethylphosphonate followed by two-stage reduction gave a third series. The dialkoxides derived from the four di-O-alkylpentaerthritol diols were reacted with 2-dimethylaminoethyl chloride and 3-dimethylaminopropyl chloride in neighboring-group assisted double Williamson ether syntheses to give precursors to two more series. As expected because the neighboring group participation occurs through a four-membered-ring intermediate, considerably more vigorous conditions were required for the reactions with 3-dimethylaminopropyl chloride. 2-Diethylaminoethyl bromide was found to be less reactive than 2-dimethylaminoethyl chloride. The products were alkylated with methyl iodide and, in some cases, other alkyl halides to give cationic gemini surfactants. Alkylation of one series with ethyl bromoacetate followed by anion exchange resin catalyzed ester hydrolysis gave zwitterionic gemini surfactants. The members of all series have superior surfactant properties.


Catastrophic health payments: does the equivalence scale matter?
We present a revised method for estimating equivalence scales. Such scales are used to adjust household welfare to account for the size of the household, and are used extensively in the application of the World Health Organization's (WHO) methodology for the evaluation of catastrophic health payments. Applications of the WHO method are underpinned by early estimates that do not control for household income, and, therefore, are likely to overstate equivalence. Thus, in addition to revising the method, we update the scale estimates for one country, South Africa, using more recent data. South Africa is considered, because the end of Apartheid has led to extensive social and economic changes that have influenced household structure and, presumably, equivalence. We also present information on the possible degree to which earlier estimates are overstated, as well as the effect that has on other components of the WHO method, especially the determinants of out-of-pocket expenditures and catastrophic health payments. We find that, in the worst case, initial estimates could be overstated by as much 46%, leading to the understatement of poverty lines by as much as 17%. Despite these large differences, the average incidence of catastrophe in health expenditure was largely unaffected. Instead, differences in scales affect conclusions related to the determinants of out-of-pocket payments and catastrophic health expenditures, as well as the distribution of catastrophe across household size. Given that South Africa has low levels of catastrophic health expenditure, the effect could be even larger in other countries, and, therefore, we recommend that researchers consider a range of scales, when examining catastrophic health expenditures.


SENEQUE: a multi-scaling GIS interface to the Riverstrahler model of the biogeochemical functioning of river systems.
The Riverstrahler model describes the biogeochemical functioning of an entire river system, from 100 to 100,000 km(2) or more, taking into account the constraints set by the morphology of the drainage network, the meteorological/hydrological conditions, and the inputs of material from point and non-point sources in the watershed. This tool has been applied for research purpose to several river systems differing in terms of hydrological regime and anthropogenic influences. In order to improve its capabilities and its generic dimension, as well as to develop a user-friendly interface allowing its transfer to non-specialist users including managers, the model has been coupled to a GIS interface. This gives the user the possibility to visualize the available geospatial database, to select the best geographical representation of the drainage network, to automatically prepare the corresponding input files required for the model, to pilot the model calculation and to visualize the results. The coupling with a GIS interface has considerably improved the capabilities of the Riverstrahler model. The code of the model is now entirely generic and can be run on any river system for which a suitable database is available. Its spatial resolution can be adapted to the requirement of the relevant problem, from the highest level, where each elementary watershed is individualized, to the lower level, where the whole basin is idealized as one basin with tributaries of each order having the same characteristics. As an illustration of the new potentialities offered by the coupling of Riverstrahler with a GIS through the SENEQUE interface, the results of a same modeling scenario are compared at different spatial resolutions. For the first time, with on-line coupling to a geodatabase, the effect of increasing the spatial resolution of the drainage network representation on the performance of the Riverstrahler model has been examined. At the outlet of the basin, the water quality results were found invariant to a large degree, whatever the details of its representation in the calculations. This result justifies the use of a low resolution representation of the upstream watershed when results are required only at the outlet of the basin.


Plasma extracellular vesicles as a source of biomarkers in traumatic brain injury.
The objective of this study was to isolate extracellular vesicles (EVs) from plasma in a cohort of patients with traumatic brain injury (TBI) and analyze their contents for novel biomarkers that could prove useful for rapid diagnosis and classification of brain injury during initial evaluation. Plasma EVs were isolated by serial ultracentrifugation from patients with TBI (n = 15) and healthy controls (n = 5). Samples were obtained from the TRACK-TBI biorepository (2010-present). Size and concentration were determined by nanoparticle tracking. Glial fibrillary acidic protein (GFAP) concentration was determined in EV protein. EV RNA was isolated and deep sequencing of short noncoding RNA was performed. Plasma EVs are physically similar but contained approximately 10 times more GFAP in TBI patients with altered consciousness than patients and controls with normal consciousness. Eleven highly differentially expressed microRNAs (miRNAs) were identified between these groups. Genes targeted by these miRNAs are highly associated with biologically relevant cellular pathways, including organismal injury, cellular development, and organismal development. Multiple additional coding and noncoding RNA species with potential biomarker utility were identified. Isolating plasma EVs in patients with TBI is feasible. Increased GFAP concentration-a validated plasma TBI marker-in EVs from TBI patients with altered consciousness, along with differential expression of multiple miRNAs targeting TBI-relevant pathways, suggests that EVs may be a useful source of TBI biomarkers. Additional evaluation in larger patient cohorts is indicated.


Genetics of progression from MDS to secondary leukemia.
Our understanding of the genetics of acute myeloid leukemia (AML) development from myelodysplastic syndrome (MDS) has advanced significantly as a result of next-generation sequencing technology. Although differences in cell biology and maturation exist between MDS and AML secondary to MDS, these 2 diseases are genetically related. MDS and secondary AML cells harbor mutations in many of the same genes and functional categories, including chromatin modification, DNA methylation, RNA splicing, cohesin complex, transcription factors, cell signaling, and DNA damage, confirming that they are a disease continuum. Differences in the frequency of mutated genes in MDS and secondary AML indicate that the order of mutation acquisition is not random during progression. In almost every case, disease progression is associated with clonal evolution, typically defined by the expansion or emergence of a subclone with a unique set of mutations. Monitoring tumor burden and clonal evolution using sequencing provides advantages over using the blast count, which underestimates tumor burden, and could allow for early detection of disease progression prior to clinical deterioration. In this review, we outline advances in the study of MDS to secondary AML progression, with a focus on the genetics of progression, and discuss the advantages of incorporating molecular genetic data in the diagnosis, classification, and monitoring of MDS to secondary AML progression. Because sequencing is becoming routine in the clinic, ongoing research is needed to define the optimal assay to use in different clinical situations and how the data can be used to improve outcomes for patients with MDS and secondary AML.


Conditional abrogation of Atm in osteoclasts extends osteoclast lifespan and results in reduced bone mass.
Ataxia-telangiectasia mutated (ATM) kinase is a central component involved in the signal transduction of the DNA damage response (DDR) and thus plays a critical role in the maintenance of genomic integrity. Although the primary functions of ATM are associated with the DDR, emerging data suggest that ATM has many additional roles that are not directly related to the DDR, including the regulation of oxidative stress signaling, insulin sensitivity, mitochondrial homeostasis, and lymphocyte development. Patients and mice lacking ATM exhibit growth retardation and lower bone mass; however, the mechanisms underlying the skeletal defects are not fully understood. In the present study, we generated mutant mice in which ATM is specifically inactivated in osteoclasts. The mutant mice did not exhibit apparent developmental defects but showed reduced bone mass due to increased osteoclastic bone resorption. Osteoclasts lacking ATM were more resistant to apoptosis and showed a prolonged lifespan compared to the controls. Notably, the inactivation of ATM in osteoclasts resulted in enhanced NF-κB signaling and an increase in the expression of NF-κB-targeted genes. The present study reveals a novel function for ATM in regulating bone metabolism by suppressing the lifespan of osteoclasts and osteoclast-mediated bone resorption.


Accurate determination of microparticle size using Fourier transform of light scattering spectrum over wavenumber.
We discovered that the far-field light scattering spectra of microparticles over wavenumber at a certain angle could be decomposed into periodic components, with an oscillation frequency linearly dependent on the particle size at each angle. Based on this observation, we propose and experimentally demonstrate a new Fourier transform technique for microparticle size determination. This technique is simple, fast, robust in its data processing algorithm, and flexible in its detection system.


Cigarette smoking in relation to risk of ductal carcinoma in situ of the breast in a cohort of postmenopausal women.
In numerous studies, investigators have examined the association of active smoking with risk of invasive breast cancer, but to the authors' knowledge, no cohort study has assessed smoking in relation to the risk of in situ breast cancer, the postulated penultimate stage preceding invasive breast cancer. The authors examined the latter association using data collected at baseline from 63,393 women in the Women's Health Initiative Clinical Trial. A total of 486 cases of ductal carcinoma in situ (DCIS) of the breast were identified during 8 years of follow-up between 1993 and 2005. Cox proportional hazards models were used to estimate hazard ratios and 95% confidence intervals. For the primary analysis, invasive breast cancer was treated as a competing risk. After adjustment for covariates, associations with smoking status, smoking intensity, duration, pack-years, and age at quitting were all close to the null value and showed few meaningful trends. Sensitivity analyses performed to address different possibilities with respect to the natural history of breast cancer also did not provide consistent evidence of an association of smoking with DCIS. The results of this large cohort study provide little support for an association of cigarette smoking with risk of DCIS in postmenopausal women.


Elucidating the specificity of binding of sulfonylurea herbicides to acetohydroxyacid synthase.
Acetohydroxyacid synthase (AHAS, EC 220.127.116.11) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulfometuron methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 A), sulfometuron methyl (2.34 A), and two other sulfonylureas, metsulfuron methyl (2.29 A) and tribenuron methyl (2.58 A). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethyl-ThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme.


[Chronotoxicity of 1.8 GHz radio-frequency radiation on plasma stress hormones and immune factors in mice].
To study the chronotoxicity of radio-frequency radiation(RF) on the plasma stress hormones and immune factors in mice. A total of 72 healthy C57 BL mice with circadian rhythm were divided into twelve groups: 6 Sham group and 6 RF groups. RF groups were exposed to 1.8 GHz RF at 226 μW/cm~2 for 60 days with 2 h/day respectively at corresponding zeitgeber time(ZT 0:00, ZT 4:00, ZT 8:00, ZT 12:00, ZT 16:00, ZT 20:00). The Sham group mice were exposed to the same condition without electromagnetic signal. At the end of last RF exposure, blood samples were collected from each animal. The concentrations of plasma stress hormones(ACTH, CORT) and immune factors(GM-CSF, TNF-α) were determined by enzyme linked immunosorbent assay(ELISA) method. The daily average levels of ACTH, CORT, GM-CSF and TNF-α were 84.12, 60.14, 1112.02 and 594.49 ng/L, which were decreased to 62.07, 41.21, 84.18 and 305.08 ng/L after 60 days of RF exposure. Compared to sham-exposed animals, the daily average levels of ACTH, CORT, GM-CSF and TNF-α were all significantly decreased(P<0.05). Circadian rhythms in the secreting of CORT, GM-CSF, TNF-α were disappeared(P>0.05), circadian rhythms of ACTH was shifted in RF-exposed mice, with the amplitude reduced from 12.45 to 4.88 and peak time postponed from 1:39 to 5:29. 1.8 GHz RF may weaken the function of stress and immune, and disturb their circadian rhythmicities.


Proposals for enhanced health risk assessment and stratification in an integrated care scenario.
Population-based health risk assessment and stratification are considered highly relevant for large-scale implementation of integrated care by facilitating services design and case identification. The principal objective of the study was to analyse five health-risk assessment strategies and health indicators used in the five regions participating in the Advancing Care Coordination and Telehealth Deployment (ACT) programme (http://www.act-programme.eu). The second purpose was to elaborate on strategies toward enhanced health risk predictive modelling in the clinical scenario. The five ACT regions: Scotland (UK), Basque Country (ES), Catalonia (ES), Lombardy (I) and Groningen (NL). Responsible teams for regional data management in the five ACT regions. We characterised and compared risk assessment strategies among ACT regions by analysing operational health risk predictive modelling tools for population-based stratification, as well as available health indicators at regional level. The analysis of the risk assessment tool deployed in Catalonia in 2015 (GMAs, Adjusted Morbidity Groups) was used as a basis to propose how population-based analytics could contribute to clinical risk prediction. There was consensus on the need for a population health approach to generate health risk predictive modelling. However, this strategy was fully in place only in two ACT regions: Basque Country and Catalonia. We found marked differences among regions in health risk predictive modelling tools and health indicators, and identified key factors constraining their comparability. The research proposes means to overcome current limitations and the use of population-based health risk prediction for enhanced clinical risk assessment. The results indicate the need for further efforts to improve both comparability and flexibility of current population-based health risk predictive modelling approaches. Applicability and impact of the proposals for enhanced clinical risk assessment require prospective evaluation.


Arthroscopic labral reconstruction of the hip using local capsular autograft.
Labral reconstruction is becoming an important treatment modality for hips with nonsalvageable labra. Nonsalvageable labra can be present in cases of intrasubstance damage, revision surgery after debridement, labral calcification, and hypoplasia. Previous methods of reconstruction have been performed in an open manner and arthroscopically using ligamentum teres, iliotibial band, and gracilis autograft. We present an alternate method of arthroscopic labral reconstruction using capsular autograft. The technique uses readily available capsular tissue during arthroscopy with no donor-site morbidity. This technique may be valuable in appropriately selected patients with labral deficiency.


Tear function alterations in patients with polycystic ovary syndrome.
Complaints of dry eye are prevalent worldwide and are known to be associated with insulin resistance (IR) and hyperandrogenism. However, dry eye is often overlooked in the context of polycystic ovary syndrome (PCOS). The aim of the present study was to evaluate whether or not there is any relation between tear functions and PCOS, which is a multifaceted disorder associated with IR or hyperandrogenism. A total of 35 women with polycystic ovary syndrome (PCOS) were enrolled in this study, along with 27 healthy controls. Body mass index (BMI), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and free testosterone levels on the third day of menstruation were recorded, as well as hirsutism score (using the Ferriman Gallwey scoring system), insulin resistance (homeostasis model assessment), neutrophil-to- lymphocyte ratios (NLR), platelet-to-lymphocyte ratios (PLR), and mean platelet volumes (MPV). A complete ocular examination was followed by administration of the ocular surface disease index (OSDI) questionnaire and Schirmer and tear break-up time (TBUT) tests. Schirmer and OSDI results were similar between groups, but TBUT was significantly lower in the PCOS group (p = 0.002). There were negative correlations between FG score and TBUT test (r = -0.406, p = 0.001) and between NLR and Schirmer test (r = -0.294, p = 0.025). Although subjective dry eye symptoms do not present in all patients, these results confirm that tear reduction, which can cause further complications in patients with PCOS, can be detected by careful examination and sensitive tests.


Corticospinal Motor Neurons Are Susceptible to Increased ER Stress and Display Profound Degeneration in the Absence of UCHL1 Function.
Corticospinal motor neurons (CSMN) receive, integrate, and relay cerebral cortex's input toward spinal targets to initiate and modulate voluntary movement. CSMN degeneration is central for numerous motor neuron disorders and neurodegenerative diseases. Previously, 5 patients with mutations in the ubiquitin carboxy-terminal hydrolase-L1 (UCHL1) gene were reported to have neurodegeneration and motor neuron dysfunction with upper motor neuron involvement. To investigate the role of UCHL1 on CSMN health and stability, we used both in vivo and in vitro approaches, and took advantage of the Uchl1(nm3419) (UCHL1(-/-)) mice, which lack all UCHL1 function. We report a unique role of UCHL1 in maintaining CSMN viability and cellular integrity. CSMN show early, selective, progressive, and profound cell loss in the absence of UCHL1. CSMN degeneration, evident even at pre-symptomatic stages by disintegration of the apical dendrite and spine loss, is mediated via increased ER stress. These findings bring a novel understanding to the basis of CSMN vulnerability, and suggest UCHL1(-/-) mice as a tool to study CSMN pathology.


Therapeutics to block autoantibody initiation and propagation in systemic lupus erythematosus and rheumatoid arthritis.
Most current therapies for the autoimmune diseases systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), as well as many of the drugs in the therapeutic pipeline, reduce the autoimmune inflammatory process but lead to a general immunosuppression. The goal of the next generation of therapies should be to reduce autoimmunity while at the same time better maintain immunocompetence. We propose three approaches for accomplishing this goal: (i) modulate antigen presentation to the adaptive immune system, (ii) alter B cell selection in the germinal center, and (iii) use decoy antigens to prevent the formation of proinflammatory immune complexes. These approaches are based on recent advances in the field: We now appreciate the role of dendritic cell function in autoimmune disease and the importance of citrullinated proteins as neoantigens in RA. There is also new recognition that most pathogenic autoantibodies are produced by B cells that have matured within the germinal center and that immune complexes in both diseases contain ligands for Toll-like receptors. We propose that treatments that target these newly revealed aspects of RA and SLE will decrease systemic inflammation with less immunocompromise.


Medical applications of cold atmospheric plasma: state of the science.
Cold atmospheric plasmas (CAP) have been used in multiple medical fields and have become a promising medical technology. CAP-generating devices are safe and easy to operate and can now be manufactured at a low cost due to advancements in electronics and microchips. A primary application of CAP is as a broad-spectrum antimicrobial technology. With the high incidence of infections caused by drug-resistant microorganisms, a non-antibiotic based treatment modality such as CAP holds great therapeutic promise, particularly in the wound care field. In addition to its antimicrobial properties, CAP treatment enhances wound healing by increasing cutaneous microcirculation, monocyte stimulation, and keratinocyte proliferation. CAP has been used by dentists for disinfection of teeth, enhancing gingival fibroblast activity, and even teeth whitening. CAP can combat tumour growth by increasing the efficacy of antitumour therapeutic agents, reactivating apoptotic pathways, or down-regulating growth-related gene sites. Most of the health-care related research on CAP has occurred in the past 15 years; the field is relatively young and needs additional research, as well as confirmation of the existing supporting literature. The purpose of this report is to provide the reader with an overview of the therapeutic application of the cold plasma technology.


Factors influencing the development of early- or late-onset Parkinson's disease in a cohort of South African patients.
Neurodegenerative disorders such as Parkinson's disease (PD) contribute significantly to global disease burden. PD can be categorised into early-onset PD (EOPD) with an age at onset (AAO) of ≤50 years and late-onset PD (LOPD) with an AAO of 50 years. To identify factors influencing EOPD and LOPD development in a group of patients in South Africa (SA). A total of 397 unrelated PD patients were recruited from the Movement Disorders Clinic at Tygerberg Hospital and via the Parkinson's Association of SA. Patient demographic and environmental data were recorded and associations with PD onset (EOPD v. LOPD) were analysed with a Pearson's Chi-squared test. The English- and Afrikaans-speaking (Afrikaner) white patients were analysed separately. Logistic regression analysis showed that ethnicity (p<0.001) and family history (p=0.004) were independently associated with AAO of PD. Average AAO was younger in black, coloured and Afrikaner patients than English-speaking white patients. A positive family history of PD, seen in 31.1% of LOPD patients, was associated with a younger AAO in the study population. These associations may be attributed to specific genetic and/or environmental risk factors that increase PD susceptibility and influence the clinical course of the disorder. More studies on PD in the unique SA populations are required to provide novel insights into mechanisms underlying this debilitating condition.


Better decisions through science: exercise testing scores.
Statistical tools can be used to create scores for assisting in the diagnosis of coronary artery disease and assessing prognosis. General practitioners and internists frequently function as gatekeepers, deciding which patients must be referred to the cardiologist. Therefore, they need to use the basic tools they have available (ie, history, physical examination and the exercise test) in an optimal fashion. Scores derived from multivariable statistical techniques considering clinical and exercise data have demonstrated superior discriminating power compared with diagnosis only using the ST segment response. In addition, by stratifying patients as to probability of disease and prognosis, they provide a more practical management strategy than a response of normal or abnormal. Although computers, as part of information management systems, can calculate complicated equations and derive these scores, physicians are reluctant to trust them. However, when represented as nomograms or simple additive discrete pieces of information, scores are more readily accepted. The scores have been compared with physician judgment and have been found to estimate the presence of coronary disease and prognosis as well as expert cardiologists and often better than nonspecialists. However, the discriminating power of specific variables from the medical history and exercise test remains unclear because of inadequate study design and differences in study populations. Should expired gases be substituted for estimated METs? Should ST/heart rate index be used instead of putting ST depression and heart rate separately into the models? Should right-sided chest leads and heart rate in recovery be considered? There is a need for further evaluation of these easily obtained variables to improve the accuracy of prediction algorithms, especially in women. The portability and reliability of scores must be ensured because access to specialized care must be safeguarded. Assessment of the clinical and exercise test data and application of the newer scores can empower the clinician to assure the cardiac patient access to appropriate and cost-effective cardiologic care.


Cyclic 3', 5'-AMP relay in Dictyostelium discoideum: adaptation is independent of activation of adenylate cyclase.
In Dictyostelium discoideum, binding of cAMP to high affinity surface receptors leads to a rapid activation of adenylate cyclase followed by subsequent adaptation within several minutes. The rate of secretion of [ 3H ]cAMP, which reflects the state of activation of the enzyme, was measured. Caffeine noncompetitively inhibited the response to cAMP. Inhibition was rapidly reversible and pretreatment of cells with caffeine for up to 22 min had little effect on the subsequent responsiveness to cAMP. However, cells pretreated with caffeine plus cAMP for greater than or equal to 8 min did not respond when caffeine was removed and the same concentration of cAMP was applied. The following observations indicate that both adaptation and deadaptation to cAMP occurred to the same extent and at the same rate whether or not cAMP synthesis was inhibited. First, when cells were pretreated with 10(-9)-10(-6) M cAMP in the presence or absence of caffeine and the stimulus was switched to a saturating dose of cAMP, the response to the increment was the same whether or not the initial response was blocked. Second, cells progressively lost responsiveness to 10(-6) M cAMP as pretreatment with 10(-6) M cAMP plus caffeine was extended from 0 to 8 min with the same time course as for those pretreated with 10(-6) M cAMP alone. Third, cells which were adapted in the presence of caffeine and cAMP deadapted within the same time period as controls when cAMP was removed. These observations demonstrate that while some part of the activation process is inhibited by caffeine the adaptation process is unaffected. Our conclusion is that adaptation does not depend on the activation of adenylate cyclase.


The presence of ascorbate induces expression of brain derived neurotrophic factor in SH-SY5Y neuroblastoma cells after peroxide insult, which is associated with increased survival.
Oxidative stress and free radical production have been implicated in Alzheimer's disease, where low levels of the antioxidant vitamin C (ascorbate) have been shown to be associated with the disease. In this study, neuroblastoma SH-SY5Y cells were treated with hydrogen peroxide in the presence of ascorbate in order to elucidate the mechanism(s) of protection against oxidative stress afforded by ascorbate. Protein oxidation, glutathione levels, cell viability and the effects on the proteome and its oxidized counterpart were monitored. SH-SY5Y cells treated with ascorbate prior to co-incubation with peroxide showed increased viability in comparison to cells treated with peroxide alone. This dual treatment also caused an increase in protein carbonyl content and a decrease in glutathione levels within the cells. Proteins, extracted from SH-SY5Y cells that were treated with either ascorbate or peroxide alone or with ascorbate prior to peroxide, were separated by two-dimensional gel electrophoresis and analyzed for oxidation. Co-incubation for 24 hours decreased the number of oxidised proteins (e.g. acyl CoA oxidase 3) and induced brain derived neurotrophic factor (BDNF) expression. Enhanced expression of BDNF may contribute to the protective effects of ascorbate against oxidative stress in neuronal cells.


Computational modeling of the neural representation of object shape in the primate ventral visual system.
Neurons in successive stages of the primate ventral visual pathway encode the spatial structure of visual objects. In this paper, we investigate through computer simulation how these cell firing properties may develop through unsupervised visually-guided learning. Individual neurons in the model are shown to exploit statistical regularity and temporal continuity of the visual inputs during training to learn firing properties that are similar to neurons in V4 and TEO. Neurons in V4 encode the conformation of boundary contour elements at a particular position within an object regardless of the location of the object on the retina, while neurons in TEO integrate information from multiple boundary contour elements. This representation goes beyond mere object recognition, in which neurons simply respond to the presence of a whole object, but provides an essential foundation from which the brain is subsequently able to recognize the whole object.


Clinical spectrum of 4H leukodystrophy caused by POLR3A and POLR3B mutations.
To study the clinical and radiologic spectrum and genotype-phenotype correlation of 4H (hypomyelination, hypodontia, hypogonadotropic hypogonadism) leukodystrophy caused by mutations in POLR3A or POLR3B. We performed a multinational cross-sectional observational study of the clinical, radiologic, and molecular characteristics of 105 mutation-proven cases. The majority of patients presented before 6 years with gross motor delay or regression. Ten percent had an onset beyond 10 years. The disease course was milder in patients with POLR3B than in patients with POLR3A mutations. Other than the typical neurologic, dental, and endocrine features, myopia was seen in almost all and short stature in 50%. Dental and hormonal findings were not invariably present. Mutations in POLR3A and POLR3B were distributed throughout the genes. Except for French Canadian patients, patients from European backgrounds were more likely to have POLR3B mutations than other populations. Most patients carried the common c.1568T>A POLR3B mutation on one allele, homozygosity for which causes a mild phenotype. Systematic MRI review revealed that the combination of hypomyelination with relative T2 hypointensity of the ventrolateral thalamus, optic radiation, globus pallidus, and dentate nucleus, cerebellar atrophy, and thinning of the corpus callosum suggests the diagnosis. 4H is a well-recognizable clinical entity if all features are present. Mutations in POLR3A are associated with a more severe clinical course. MRI characteristics are helpful in addressing the diagnosis, especially if patients lack the cardinal non-neurologic features.


Using Health Information Technology to Bring Social Determinants of Health into Primary Care: A Conceptual Framework to Guide Research.
Several recent national initiatives have called for increased efforts to integrate social determinants of health (SDH) into health care settings using health information technology (HIT). However, there is limited evidence to guide the implementation of these recommendations in practice. Research is needed to understand what SDH information is most important to collect, how SDH information can be used to inform clinical care and referrals, and ultimately, whether and how integrating SDH screening and action into primary care affects individual and population health. We recently proposed a conceptual framework to illustrate how HIT can be used to bring SDH information into primary care. In this paper, we describe how we are putting this conceptual model into practice within the OCHIN network of community health centers by highlighting examples of ongoing research, identifying knowledge gaps, and outlining a roadmap of future research to move the field forward.


Patterns of recreational drug use at dance events in Edinburgh, Scotland.
To describe the patterns of drug use at dance (rave) events in terms of prevalence, frequency, type of drugs used, patterns of use, access and risk-associated behaviours. Self-selecting participant-completed survey. Three dance events in Edinburgh, Scotland, UK. One hundred and twenty-two drug users (57% males, 43% females), 90% of whom were in employment or education, with an age range of 16-47, 80% between 18 and 23 years. Participants who answered 'yes' to the question 'Have you used drugs for dance events in the past year' reported (i) the prevalence, types and frequency of drugs used; (ii) prevalence and contents of mixing drugs; (iii) accessing drugs; and (iv) engagement with drug-associated risk behaviours. Over 80% of the participants had used ecstasy and amphetamine, over 30% cocaine and LSD; over 10% nitrites, psilocybin and ketamine and less than 5% had used crack or tranquillizers. Participants reported regular consumption of ecstasy and amphetamine (e.g. 35% used ecstasy and 25% amphetamine on a weekly basis) often taken in combination, with the occasional use of cocaine, LSD, ketamine and psilocybin. Poly- and mixing-drug behaviours were significantly more likely than monodrug usage. Drugs were accessed through friends than from any other source. Eighty-five per cent reported mixing drugs and/or alcohol, 35% driving on drugs, 36% having a bad experience on drugs; 30% unprotected sex; and 0.9% injecting drugs. Women in the sample reported higher consumption than men. Dance-drug use has a characteristic pattern that has implications for health promotion and criminal policy.


Vitamin D is a determinant of mouse intestinal Lgr5 stem cell functions.
Lgr5+ intestinal crypt base columnar cells function as stem cells whose progeny populate the villi, and Lgr5+ cells in which Apc is inactivated can give rise to tumors. Surprisingly, these Lgr5+ stem cell properties were abrogated by the lower dietary vitamin D and calcium in a semi-purified diet that promotes both genetically initiated and sporadic intestinal tumors. Inactivation of the vitamin D receptor in Lgr5+ cells established that compromise of Lgr5 stem cell function was a rapid, cell autonomous effect of signaling through the vitamin D receptor. The loss of Lgr5 stem cell function was associated with presence of Ki67 negative Lgr5+ cells at the crypt base. Therefore, vitamin D, a common nutrient and inducer of intestinal cell maturation, is an environmental factor that is a determinant of Lgr5+ stem cell functions in vivo. Since diets used in reports that establish and dissect mouse Lgr5+ stem cell activity likely provided vitamin D levels well above the range documented for human populations, the contribution of Lgr5+ cells to intestinal homeostasis and tumor formation in humans may be significantly more limited, and variable in the population, then suggested by published rodent studies.


Cost-utility analysis comparing meropenem with imipenem plus cilastatin in the treatment of severe infections in intensive care.
This study compared the cost-effectiveness of meropenem with that of imipenem plus cilastatin in the treatment of severe infections in hospital intensive care in the UK. A Markov model was constructed to model lifetime costs and quality-adjusted life years (QALYs) of using meropenem and imipenem plus cilastatin for the treatment of severe infections in intensive care. Estimates of effectiveness, utility weights and costs were obtained from the published literature. Probabilistic sensitivity analysis was conducted to assess the robustness of the results. Estimated treatment costs for the patient cohort were pound 14,938 with meropenem and pound 15,585 with imipenem plus cilastatin. QALYs gained were 7,495 with meropenem and 7,413 with imipenem plus cilastatin. Probabilistic sensitivity analysis showed meropenem to be significantly less costly (-pound 636.47, 95% CI -pound 132.33 to -pound 1,140.62) and more effective (0.084, 95% CI 0.023 to 0.144). Meropenem thus appears significantly more effective and less expensive than imipenem plus cilastatin and should therefore be considered the dominant treatment strategy.


Too much of a good thing: sea ice extent may have forced emperor penguins into refugia during the last glacial maximum.
The relationship between population structure and demographic history is critical to understanding microevolution and for predicting the resilience of species to environmental change. Using mitochondrial DNA from extant colonies and radiocarbon-dated subfossils, we present the first microevolutionary analysis of emperor penguins (Aptenodytes forsteri) and show their population trends throughout the last glacial maximum (LGM, 19.5-16 kya) and during the subsequent period of warming and sea ice retreat. We found evidence for three mitochondrial clades within emperor penguins, suggesting that they were isolated within three glacial refugia during the LGM. One of these clades has remained largely isolated within the Ross Sea, while the two other clades have intermixed around the coast of Antarctica from Adélie Land to the Weddell Sea. The differentiation of the Ross Sea population has been preserved despite rapid population growth and opportunities for migration. Low effective population sizes during the LGM, followed by a rapid expansion around the beginning of the Holocene, suggest that an optimum set of sea ice conditions exist for emperor penguins, corresponding to available foraging area.


Decreased transferrin receptor expression by neuromelanin cells in restless legs syndrome.
Restless legs syndrome (RLS) is a sensory-movement disorder affecting 5 to 10% of the population. Its etiology is unknown, but MRI analyses and immunohistochemical studies on autopsy tissue suggest the substantia nigra (SN) of patients with RLS has subnormal amounts of iron. Neuromelanin cells from the SN of four RLS and four control brains were isolated by laser capture microdissection, and a profile of iron-management protein expression was obtained by immunoblot analysis. Binding assays for iron regulatory protein activity were performed on cell homogenates. Ferritin, divalent metal transporter 1, ferroportin, and transferrin receptor (TfR) were decreased in RLS neuromelanin cells compared with control. Transferrin was increased in RLS neuromelanin cells. This protein profile in RLS neuromelanin cells is consistent with iron deficiency with the exception that TfR expression was decreased rather than increased. The concentration and activity of the iron regulatory proteins (IRP1 and IRP2) were analyzed to determine whether there was a functional deficit in the post-transcriptional regulatory mechanism for TfR expression. Total IRP activity, IRP1 activity, and IRP1 protein levels were decreased in RLS, but total IRP2 protein levels were not decreased in RLS. Restless legs syndrome may result from a defect in iron regulatory protein 1 in neuromelanin cells that promotes destabilization of the transferrin receptor mRNA, leading to cellular iron deficiency.


Experience seeking abortion among unmarried young women in Bihar and Jharkhand, India: delays and disadvantages.
Studies suggest that the experiences of unmarried young women seeking abortion in India differ from those of their married counterparts, but the evidence is limited. Research was undertaken among nulliparous young women aged 15-24 who had abortions at the clinics of a leading NGO in Bihar and Jharkhand. Over a 14-month period in 2007-08, 246 married and 549 unmarried young abortion seekers were surveyed and 26 who were unmarried were interviewed in depth. Those who were unmarried were far more likely to report non-consensual sexual relations. As many as 25% of unmarried young women, compared to only 9% of married young women, had had a second trimester abortion. The unmarried were far more likely to report non-consensual sexual relations leading to pregnancy. They were also more likely to report such obstacles to timely abortion as failure to recognise the pregnancy promptly, exclusion from abortion-related decision-making, seeking confidentiality as paramount in selection of abortion facility, unsuccessful previous attempts to terminate the pregnancy, and lack of partner support. After controlling for background factors, findings suggest that unmarried young women who also experienced these obstacles were, compared to married young women, most likely to experience second trimester abortion. Programmes need to take steps to improve access to safe and timely abortion for unmarried young women.


Neuroprotection in glaucoma: recent advances and clinical translation.
Intraocular pressure (IOP) reduction is currently the only evidence-based treatment strategy for glaucoma. However, IOP control in some individuals is challenging. Despite optimal treatment, a significant proportion of individuals will progress, with loss of visual field, loss of driving vision and impaired quality of life. A new modality that could augment current treatment and reduce the rate of neurodegeneration to preserve vision throughout life would be a major breakthrough. A vast number of studies have reported effective neuroprotection in animal models of glaucoma; however, translation to the clinic remains a major hurdle. Herein, we explore the therapeutic advancements in non-IOP-dependent neuroprotection research based upon potential pathogenic mechanisms and propose strategies to improve the clinical translation of neuroprotective research in glaucoma.


Comparative physical mapping reveals features of microsynteny between Glycine max, Medicago truncatula, and Arabidopsis thaliana.
To gain insight into genomic relationships between soybean (Glycine max) and Medicago truncatula, eight groups of bacterial artificial chromosome (BAC) contigs, together spanning 2.60 million base pairs (Mb) in G. max and 1.56 Mb in M. truncatula, were compared through high-resolution physical mapping combined with sequence and hybridization analysis of low-copy BAC ends. Cross-hybridization among G. max and M. truncatula contigs uncovered microsynteny in six of the contig groups and extensive microsynteny in three. Between G. max homoeologous (within genome duplicate) contigs, 85% of coding and 75% of noncoding sequences were conserved at the level of cross-hybridization. By contrast, only 29% of sequences were conserved between G. max and M. truncatula, and some kilobase-scale rearrangements were also observed. Detailed restriction maps were constructed for 11 contigs from the three highly microsyntenic groups, and these maps suggested that sequence order was highly conserved between G. max duplicates and generally conserved between G. max and M. truncatula. One instance of homoeologous BAC contigs in M. truncatula was also observed and examined in detail. A sequence similarity search against the Arabidopsis thaliana genome sequence identified up to three microsyntenic regions in A. thaliana for each of two of the legume BAC contig groups. Together, these results confirm previous predictions of one recent genome-wide duplication in G. max and suggest that M. truncatula also experienced ancient large-scale genome duplications.


Development of a Tool to Evaluate Asthma Preparedness and Management in Child-Care Centers.
Introduction: Asthma is a common condition affecting many children in child-care centers. The National Asthma Education and Prevention Program offers recommendations about creating an asthma-friendly child-care setting. However, no studies have investigated the extent to which child-care centers adhere to these recommendations. This study describes the development of a novel instrument to determine the ability of child-care centers to meet national recommendations for asthma. Methods: The Preparing for Asthma in Child Care (PACC) Instrument was developed using information from existing recommendations and standards, the peer-reviewed literature, site visits, and expert interviews. The survey questions were pilot-tested at 36 child-care centers throughout San Francisco. Results: The instrument is composed of 43 items across seven domains: smoking exposure, presence of a medical consultant and policies, management of ventilation and triggers, access to medication, presence of asthma action plans, staff training, and encouragement of physical activity. Discussion: The PACC Instrument is an evidence-based and comprehensive tool designed to identify areas to target to improve asthma care for children in child-care centers.


Synthesis of carboxylated chitosan and its adsorption properties for cadmium (II), lead (II) and copper (II) from aqueous solutions.
Carboxylated chitosan (CKCTS) was prepared for the removal of Cd(II), Pb(II), and Cu(II) from aqueous solutions. The effects of experimental parameters such as pH value, initial concentration, contact time and temperature on the adsorption were studied. From the results we can see that the adsorption capacities of Cd(II), Pb(II), and Cu(II) increase with increasing pH of the solution. The kinetic rates were best fitted to the pseudo-second-order model. The adsorption equilibrium data were fitted well with the Langmuir isotherm, which revealed that the maximum adsorption capacities for monolayer saturation of Cd(II), Pb(II), and Cu(II) were 0.555, 0.733 and 0.827 mmol/g, respectively. The adsorption was an exothermic process.


XYZeq: Spatially resolved single-cell RNA sequencing reveals expression heterogeneity in the tumor microenvironment.
Single-cell RNA sequencing (scRNA-seq) of tissues has revealed remarkable heterogeneity of cell types and states but does not provide information on the spatial organization of cells. To better understand how individual cells function within an anatomical space, we developed XYZeq, a workflow that encodes spatial metadata into scRNA-seq libraries. We used XYZeq to profile mouse tumor models to capture spatially barcoded transcriptomes from tens of thousands of cells. Analyses of these data revealed the spatial distribution of distinct cell types and a cell migration-associated transcriptomic program in tumor-associated mesenchymal stem cells (MSCs). Furthermore, we identify localized expression of tumor suppressor genes by MSCs that vary with proximity to the tumor core. We demonstrate that XYZeq can be used to map the transcriptome and spatial localization of individual cells in situ to reveal how cell composition and cell states can be affected by location within complex pathological tissue.


Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain.
Proteases that cleave protease-activated receptor-2 (PAR(2)) at Arg(36)↓Ser(37) reveal a tethered ligand that binds to the cleaved receptor. PAR(2) activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR(2) at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR(2)-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR(2), causes inflammation and pain by activating PAR2 and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR(2) at Ala(66)↓Ser(67) and Ser(67)↓Val(68). Elastase stimulated PAR(2)-dependent cAMP accumulation and ERK1/2 activation, but not Ca(2+) mobilization, in KNRK cells. Elastase induced PAR(2) coupling to Gαs but not Gαq in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK(2)) or β-arrestin to PAR(2), consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR(2)-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR(2)-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR(2)- and TRPV4-mediated influx of extracellular Ca(2+) in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR(2)- and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR(2) causes Gαs-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR(2) as a mediator of protease-driven inflammation and pain.


Periodic heat shock accelerated the chondrogenic differentiation of human mesenchymal stem cells in pellet culture.
Osteoarthritis (OA) is one of diseases that seriously affect elderly people's quality of life. Human mesenchymal stem cells (hMSCs) offer a potential promise for the joint repair in OA patients. However, chondrogenic differentiation from hMSCs in vitro takes a long time (∼ 6 weeks) and differentiated cells are still not as functionally mature as primary isolated chondrocytes, though chemical stimulations and mechanical loading have been intensively studied to enhance the hMSC differentiation. On the other hand, thermal stimulations of hMSC chondrogenesis have not been well explored. In this study, the direct effects of mild heat shock (HS) on the differentiation of hMSCs into chondrocytes in 3D pellet culture were investigated. Periodic HS at 41 °C for 1 hr significantly increased sulfated glycosaminoglycan in 3D pellet culture at Day 10 of chondrogenesis. Immunohistochemical and Western Blot analyses revealed an increased expression of collagen type II and aggrecan in heat-shocked pellets than non heat-shocked pellets on Day 17 of chondrogenesis. In addition, HS also upregulated the expression of collagen type I and X as well as heat shock protein 70 on Day 17 and 24 of differentiation. These results demonstrate that HS accelerated the chondrogenic differentiation of hMSCs and induced an early maturation of chondrocytes differentiated from hMSCs. The results of this study will guide the design of future protocols using thermal treatments to facilitate cartilage regeneration with human mesenchymal stem cells.


Neuraxial morphine and respiratory depression: finding the right balance.
Morphine is a drug commonly administered via the epidural or intrathecal route, and is regarded by many as the 'gold-standard' single-dose neuraxial opioid due to its postoperative analgesic efficacy and prolonged duration of action. However, respiratory depression is a recognized side effect of neuraxial morphine administered in the perioperative setting. We conducted an extensive review of articles published since 1945 that examine respiratory depression or failure associated with perioperative intrathecal or epidural morphine use. Respiratory depression was previously thought to result from the interaction of opioid in the cerebrospinal fluid with ventral medullary opioid receptors. More recently, the preBötzinger complex located in the medulla has been identified as the site responsible for the decrease in respiratory rate following systemic administration of opioids. Neurons in the preBötzinger complex expressing neurokinin-1 receptors are selectively inhibited by opioids, and therefore are the mediators of opioid-induced respiratory depression. Epidural, intrathecal and plasma pharmacokinetics of opioids are complex, vary between neuraxial compartments, and can even differ within the epidural space itself depending upon level of insertion. Caution should be exercised when prescribing systemic opioids (intravenous or oral) in addition to neuraxial morphine as this can compound the potential for early or delayed respiratory depression. There is a wide range of incidences for respiratory depression following neuraxial morphine in a perioperative setting. Disparity of definitions used for the diagnosis of respiratory depression in the literature precludes identification of the exact incidence of this rare event. The optimal neuraxial opioid dose is a balance between the conflicting demands of providing optimal analgesia while minimizing dose-related adverse effects. Dose-response studies show that neuraxial morphine appears to have an analgesic efficacy 'ceiling'. The optimal 'single-shot' intrathecal dose appears to be 0.075-0.15 mg and the ideal 'single-shot' epidural morphine dose is 2.5-3.75 mg. Analgesic efficacy studies have not been adequately powered to show differences in the incidence of clinically significant respiratory depression. Opioid antagonists such as naloxone to prevent or treat opioid-induced respiratory depression have a number of limitations. Researchers have recently focused on non-opioid drugs such as serotonin receptor agonists. Early evidence suggests that ampakine (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid [AMPA]) receptor modulators may be effective at reducing opioid-induced respiratory depression while maintaining analgesia. Sodium/proton exchanger type 3 (NHE3) inhibitors, which act centrally on respiratory pathways, also warrant further study.