Infectiousness of pigs infected by the Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is time-dependent.
The time-dependent transmission rate of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and the correlation between infectiousness, virological parameters and antibody responses of the infected pigs were studied in experimental conditions. Seven successive transmission trials involving a total of 77 specific pathogen-free piglets were carried out from 7 to 63 days post-inoculation (dpi). A semi-quantitative real time RT-PCR was developed to assess the evolution of the viral genome load in blood and nasal swabs from inoculated and contact pigs, with time. Virus genome in blood was detectable in inoculated pigs from 7 to 77 dpi, whereas viral genome shedding was detectable from nasal swabs from 2 to 48 dpi. The infectiousness of inoculated pigs, assessed from the frequency of occurrence of infected pigs in susceptible groups in each contact trial, increased from 7 to 14 dpi and then decreased slowly until 42 dpi (3, 7, 2, 1 and 0 pigs infected at 7, 14, 21, 28 and 42 dpi, respectively). These data were used to model the time-dependent infectiousness by a lognormal-like function with a latency period of 1 day and led to an estimated basic reproduction ratio, R0 of 2.6 [1.8, 3.3]. The evolution of infectiousness was mainly correlated with the time-course of viral genome load in the blood whereas the decrease of infectiousness was strongly related to the increase in total antibodies.


Low-temperature oxidation of monobromobenzene: Bromine transformation and yields of phenolic species.
Brominated benzenes and phenols constitute direct precursors in the formation of bromine-bearing pollutants; most notably PBDD/Fs and other dioxin-type compounds. Elucidating accurate mechanisms and constructing robust kinetic models for the oxidative transformation of bromobenzenes and bromophenols into notorious Br-toxicants entail a comprehensive understanding of their initial oxidation steps. However, pertinent mechanistic studies, based on quantum chemical calculations, have only focused on secondary condensation reactions into PBDD/Fs and PBDEs. Literature provide kinetic parameters for these significant reactions, nonetheless, without attempting to compile the acquired Arrhenius coefficients into kinetic models. To fill in this gap, this study sets out to illustrate primary chemical phenomena underpinning the low-temperature combustion of a monobromobenzene molecule (MBZ) based on a detail chemical kinetic model. The main aim is to map out temperature-dependent profiles for major intermediates and products. The constructed kinetic model encompasses several sub-mechanisms (i.e, HBr and benzene oxidation, bromination of phenoxy radicals, and initial reaction of oxygen molecules with MBZ). In light of germane experimental observations, the formulated kinetic model herein offers an insight into bromine speciation, conversion profile of MBZ, and formation of higher brominated congeners of benzene and phenol. For instance, the model satisfactorily accounts for the yields of dibromophenols from oxidation of a 2-bromophenol (2-MBP) molecule, in reference to analogous experimental measurements. From an environmental perspective, the model reflects the accumulation of appreciable loads of 2-bromophenoxy radicals at intermediate temperatures (i.e., a bromine-containing environmental persistent free radical, EPFR) from combustion of MBZ and 2-MBP molecules. Acquired mechanistic/kinetic parameters shall be useful in comprehending the complex bromine transformation chemistry in real scenarios, most notably those prevailing in thermal recycling of brominated flame retardants (BFRs).


The function of Fcγ receptors in dendritic cells and macrophages.
Dendritic cells (DCs) and macrophages use various receptors to recognize foreign antigens and to receive feedback control from adaptive immune cells. Although it was long believed that all immunoglobulin Fc receptors are universally expressed by phagocytes, recent findings indicate that only monocyte-derived DCs and macrophages express high levels of activating Fc receptors for IgG (FcγRs), whereas conventional and plasmacytoid DCs express the inhibitory FcγR. In this Review, we discuss how the uptake, processing and presentation of antigens by DCs and macrophages is influenced by FcγR recognition of immunoglobulins and immune complexes in the steady state and during inflammation.


Revealing vascular abnormalities and measuring small vessel density in multiple sclerosis lesions using USPIO.
Multiple Sclerosis (MS) is a progressive, inflammatory, neuro-degenerative disease of the central nervous system (CNS) characterized by a wide range of histopathological features including vascular abnormalities. In this study, an ultra-small superparamagnetic iron oxide (USPIO) contrast agent, Ferumoxytol, was administered to induce an increase in susceptibility for both arteries and veins to help better reveal the cerebral microvasculature. The purpose of this work was to examine the presence of vascular abnormalities and vascular density in MS lesions using high-resolution susceptibility weighted imaging (SWI). Six subjects with relapsing remitting MS (RRMS, age = 47.3 ± 11.8 years with 3 females and 3 males) and fourteen age-matched healthy controls were scanned at 3 T with SWI acquired before and after the infusion of Ferumoxytol. Composite data was generated by registering the FLAIR data to the high resolution SWI data in order to highlight the vascular information in MS lesions. Both the central vein sign (CVS) and, a new measure, the multiple vessel sign (MVS) were identified, along with any vascular abnormalities, in the lesions on pre- and post-contrast SWI-FLAIR fusion data. The small vessel density within the periventricular normal-appearing white matter (NAWM) and the periventricular lesions were compared for all subjects. Averaged across two independent raters, a total of 530 lesions were identified across all patients. The total number of lesions with vascularity on pre- and post-contrast data were 287 and 488, respectively. The lesions with abnormal vascular behavior were broken up into following categories: small lesions appearing only at the vessel boundary; dilated vessels within the lesions; and developmental venous angiomas. These vessel abnormalities observed within lesions increased from 55 on pre-contrast data to 153 on post-contrast data. Finally, across all the patients, the periventricular lesional vessel density was significantly higher (p < 0.05) than that of the periventricular NAWM. By inducing a super-paramagnetic susceptibility in the blood using Ferumoxytol, the vascular abnormalities in the RRMS patients were revealed and small vessel densities were obtained. This approach has the potential to monitor the venous vasculature present in MS lesions, catalogue their characteristics and compare the vascular structures spatially to the presence of lesions. These enhanced vascular features may provide new insight into the pathophysiology of MS.


Bonded amalgam as a fissure sealant in low-income setting: A randomised controlled trial.
To compare one year retention rate of bonded amalgam sealants (BAS) with that of conventional resin-based sealant (RBS). It was a parallel group, equivalence, randomised controlled trial done during March 2018 to December 2019 at the dental clinics of Dow University of Health Sciences, and the Jinnah Sind Medical University, Karachi, Pakistan on children aged 12-16 years, who were randomly assigned to one of the two treatment groups (BAS vs. RBS). Complete retention of the sealant at one year follow-up was labeled as success. Multi-level mixed effect logistic regression model was employed. Study was registered at www.clinicaltrials.gov # NCT NCT03130725. There were 137 teeth (23 subjects) in the BAS and 128 teeth (15 subjects) in the RBS group that were evaluated for sealant retention at 12 months follow-up. Among the BAS group, 100/137 (73%) sealants were completely retained whereas 110/128 (86%) were fully retained in the RBS group. Nearly, 22/137 (16%) BAS and 10/128 (7.8%) RBS were completely dislodged. In multivariable analysis, subject age >15 years and male gender were found to be significantly associated with the dislodgement of sealants. At 12 months follow-up, the retention of bonded amalgam sealant (BAS) was significantly lower than that of the resin-based sealant (RBS). www.clinicaltrials.gov with # NCT NCT03130725.


Electrophysiological characterization of the mechanosensitive channel MscCG in Corynebacterium glutamicum.
Corynebacterium glutamicum MscCG, also referred to as NCgl1221, exports glutamate when biotin is limited in the culture medium. MscCG is a homolog of Escherichia coli MscS, which serves as an osmotic safety valve in E. coli cells. Patch-clamp experiments using heterogeneously expressed MscCG have shown that MscCG is a mechanosensitive channel gated by membrane stretch. Although the association of glutamate secretion with the mechanosensitive gating has been suggested, the electrophysiological characteristics of MscCG have not been well established. In this study, we analyzed the mechanosensitive gating properties of MscCG by expressing it in E. coli spheroplasts. MscCG is permeable to glutamate, but is also permeable to chloride and potassium. The tension at the midpoint of activation is 6.68 ± 0.63 mN/m, which is close to that of MscS. The opening rates at saturating tensions and closing rates at zero tension were at least one order of magnitude slower than those observed for MscS. This slow kinetics produced strong opening-closing hysteresis in response to triangular pressure ramps. Whereas MscS is inactivated under sustained stimulus, MscCG does not undergo inactivation. These results suggest that the mechanosensitive gating properties of MscCG are not suitable for the response to abrupt and harmful changes, such as osmotic downshock, but are tuned to execute slower processes, such as glutamate export.


Determination of the relative glomerular filtration rate of each kidney in man. Comparison between iohexol CT and 99mTc-DTPA scintigraphy.
Iohexol and 99mTc-DTPA were used in 43 patients to determine the relative glomerular filtration rate (GFR), i.e., the GFR of each kidney in percent of total GFR. The amount of any GFR marker accumulating in Bowman's space, tubuli and renal pelvis within a few minutes after i.v. injection, before any marker had left the kidney via the ureter, was defined as proportional to the GFR of that kidney. The renal accumulation of iohexol was determined by CT using 10 slices of 8-mm thickness 1 to 4 minutes after injection. The renal accumulation of 99mTc-DTPA was determined with a gamma camera within 2 minutes after injection. The correlation coefficient between the two methods was 0.98. Due to the higher radiation dose from CT than from 99mTc-DTPA injection, relative GFR determination with CT should be performed when there is also a diagnostic need to reveal morphology.


Can breastfeeding promote child health equity? A comprehensive analysis of breastfeeding patterns across the developing world and what we can learn from them.
In 2010 more than 7.7 million children died before their fifth birthday. Over 98% of these deaths occurred in developing countries, and recent estimates have attributed hundreds of thousands of these deaths to suboptimal breastfeeding. This study estimated prevalence of suboptimal breastfeeding for 137 developing countries from 1990 to 2010. These estimates were compared against WHO infant feeding recommendations and combined with effect sizes from existing literature to estimate associated disease burden using a standard comparative risk assessment approach. These prevalence estimates were disaggregated by wealth quintile and linked with child mortality rates to assess how improved rates of breastfeeding may affect child health inequalities. In 2010, the prevalence of exclusive breastfeeding ranged from 3.5% in Djibouti to 77.3% in Rwanda. The proportion of child Disability Adjusted Life Years (DALYs) attributable to suboptimal breastfeeding is 7.6% at the global level and as high as 20.2% in Swaziland. Suboptimal breastfeeding is a leading childhood risk factor in all developing countries and consistently ranks higher than water and sanitation. Within most countries, breastfeeding prevalence rates do not vary considerably across wealth quintiles. Breastfeeding is an effective child health intervention that does not require extensive health system infrastructure. Improvements in rates of exclusive and continued breastfeeding can contribute to the reduction of child mortality inequalities in developing countries.


Single-cell sequencing reveals clonal expansions of pro-inflammatory synovial CD8 T cells expressing tissue-homing receptors in psoriatic arthritis.
Psoriatic arthritis (PsA) is a debilitating immune-mediated inflammatory arthritis of unknown pathogenesis commonly affecting patients with skin psoriasis. Here we use complementary single-cell approaches to study leukocytes from PsA joints. Mass cytometry demonstrates a 3-fold expansion of memory CD8 T cells in the joints of PsA patients compared to peripheral blood. Meanwhile, droplet-based and plate-based single-cell RNA sequencing of paired T cell receptor alpha and beta chain sequences show pronounced CD8 T cell clonal expansions within the joints. Transcriptome analyses find these expanded synovial CD8 T cells to express cycling, activation, tissue-homing and tissue residency markers. T cell receptor sequence comparison between patients identifies clonal convergence. Finally, chemokine receptor CXCR3 is upregulated in the expanded synovial CD8 T cells, while two CXCR3 ligands, CXCL9 and CXCL10, are elevated in PsA synovial fluid. Our data thus provide a quantitative molecular insight into the cellular immune landscape of psoriatic arthritis.


Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors.
Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming ciliary-directed vesicles, through organization of the vesicle fusion machinery at the periciliary membrane.


Electrodeposition of Pluronic F127 assisted rod-like EMD/carbon arrays for efficient energy storage.
In the traditional Duracell battery, the results obtained to date remain marginal in terms of cyclability. The development of the existing Zn-MnO2 with superior electrochemical performance for use in alkaline rechargeable batteries is reported. Electrolytic manganese dioxide (EMD) was synthesized from a conventional manganese sulphate bath but having a unique non-ionic surfactant (Pluronic F127), and activated carbon, in an electrolytic cell. The surface areas and morphologies of the as-prepared EMDs were influenced by the presence of these novel additives in the solution while the X-ray data revealed that there was no noticeable change in the crystal orientations thus all the EMDs were structurally similar. The synergistic effect of the optimal ratio of surfactant to carbon powder produced rod-like arrays exhibiting a larger surface area, which facilitates ion transport for better energy storage. It is interesting to note that EMD deposited in the presence of F127 showed better cyclability whereas in the presence of carbon, although it showed better storage capability, it was endowed with poor efficiency when compared with the surfactant added sample, nevertheless the results are better than the existing Zn-MnO2 technology (additive free EMD). Therefore, both the surfactant (50 mg dm(-3)) and the activated carbon (5 g dm(-3)) have been added together in the bath and the resultant EMD exhibits a high specific capacity and an excellent cycling stability. Moreover, the presence of surfactant and activated carbon improved the discharge capacity and its retention thus making this alkaline technology feasible for storing renewable energy for future use. The synergistic effect and the mechanism involved have been discussed.


p53 and stem cells: new developments and new concerns.
As the guardian of the genome, the tumor suppressor p53 prevents the accumulation of genetic mutations by inducing cell cycle arrest, apoptosis or senescence of somatic cells after genotoxic and oncogenic stresses. Recent studies have identified the roles of p53 in suppressing pluripotency and cellular dedifferentiation. In this context, p53 suppresses the self-renewal of embryonic stem cells after DNA damage and blocks the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). If the inactivation of p53 pathway is a prerequisite for successful reprogramming, these findings raise concerns for the genomic stability and tumorigenecity of iPSCs and their derivatives. Elucidation of the roles of p53 as a barrier to pluripotency and cellular dedifferentiation might also reveal the mechanisms by which p53 coordinates tumor suppression and aging.


Computed tomography and ultrasonography of the adrenal glands in paracoccidioidomycosis. Comparison with cortisol and aldosterone responses to ACTH stimulation.
Fifteen patients with proven disseminated paracoccidioidomycosis (PCM) had computed tomography (CT) and ultrasonography (US) performed to evaluate the form, shape, density and size of their adrenal glands. Plasma and urinary cortisol were determined and adrenal reserve assessed by measuring the cortisol and aldosterone responses to synthetic ACTH. The adrenal CT showed unilateral lesions in two cases and bilateral in another four. The US study showed more frequent alterations, unilateral in seven and bilateral in three subjects. Combining both methods increased the sensitivity to 85% of the cases. All patients had normal plasma cortisol concentrations and normal or increased urinary cortisol excretion. Plasma aldosterone concentration was also normal except in one patient with hypokalemia. Seven patients showed diminished cortisol responses, five had subnormal aldosterone responses and in five plasma aldosterone concentration increased more than normally after stimulation by ACTH. There was an incidence of limited adrenal reserve in 53% of the patients on ACTH stimulation. No correlation was evident between the disorders in adrenal steroid responses to ACTH and changes in morphology revealed by CT and/or US.


Scotopic electroretinogram in term infants born of mothers supplemented with docosahexaenoic acid during pregnancy.
To test the hypothesis that the supplementation of the diets of pregnant women with a fish oil rich in docosahexaenoic acid (DHA) enhances retinal development in their healthy term infants, as measured during the early postnatal period by the electroretinogram (ERG). One hundred pregnant women were randomized to receive either a fish oil (n = 50) or a placebo oleic acid dietary supplement (n = 50) from 15 weeks of pregnancy until delivery. Total fatty acids in red blood cells (RBCs) and plasma were measured in mothers at 15 and 28 weeks of pregnancy and at delivery and in their infants in umbilical cord blood. Infant retinal development was assessed within the first week of life with full-field ERGs that included a scotopic blue intensity series (n = 41) and a bright white flash (2.0 log cd-s/m(2); n = 44). Infants born of mothers who received supplements did not differ at birth in weight, gestational age, or any other standard variable. Infant DHA status at birth, as measured from umbilical cord blood, did not differ significantly between maternal supplementation groups. ERG implicit times, amplitudes, and parameters of the stimulus-response function did not differ significantly between infants in the maternal supplemented and placebo groups. There was, however, a relationship between infant DHA status and maturity of the retina at birth, regardless of maternal supplementation group. A measure of retinal sensitivity (log sigma) correlated significantly (P < 0.005) with DHA status (as a percentage of total fatty acid; TFA) in infant cord blood. Infants in the highest quartile for cord blood DHA had higher retinal sensitivity compared with infants in the lowest quartile. Infants in the highest quartile for plasma DHA, both as a percentage of TFA and concentration, were born at a significantly later gestational age than were infants in the lower quartiles. These findings demonstrate an association between the DHA status of term infants and retinal sensitivity, suggesting an essential role of this long-chain polyunsaturated fatty acid (LCPUFA) in the development and function of the retina. However, maternal DHA status was not significantly associated with infant retinal sensitivity and no direct effect of maternal supplementation was observed.


Developing the agenda for European Union collaboration on non-communicable diseases research in Sub-Saharan Africa.
Health research is increasing in Africa, but most resources are currently chanelled towards infectious diseases and health system development. While infectious diseases remain a heavy burden for some African countries, non-communicable diseases (NCDs) account for more than half of all deaths globally and WHO predicts 27% increase in NCDs in Africa over the next decade. We present findings of a European-Africa consultation on the research agenda for NCDs. A workshop was held in Yaoundé, Cameroon, organized by the Network for the Coordination and Advancement of Sub-Saharan Africa-European Union Science and Technology Cooperation (CAAST-Net). Drawing on initial presentations, a small expert group from academic, clinical, public-health and administrative positions considered research needs in Africa for cardiovascular disease, cancer and diabetes. Research in Africa can draw from different environmental and genetic characteristics to understand the causes of the disease, while economic and social factors are important in developing relevant strategies for prevention and treatment. The suggested research needs include better methods for description and recording, clinical studies, understanding cultural impacts, prevention strategies, and the integrated organisation of care. Specific fields proposed for research are listed. Our paper contributes to transparency in the process of priority-setting for health research in Africa. Although the European Union Seventh Framework Research Programme prioritises biomedical and clinical research, research for Africa should also address broader social and cultural research and intervention research for greatest impact. Research policy leaders in Africa must engage national governments and international agencies as well as service providers and research communities. None can act effectively alone. Bringing together the different stakeholders, and feeding the results through to the European Union research programme is a valuable contribution of CAAST-Net.


Drosophila Goosecoid requires a conserved heptapeptide for repression of paired-class homeoprotein activators.
Goosecoid (Gsc) is a homeodomain protein expressed in the organizer region of vertebrate embryos. Its Drosophila homologue, D-Gsc, has been implicated in the formation of the Stomatogastric Nervous System. Although there are no apparent similarities between the phenotypes of mutations in the gsc gene in flies and mice, all known Gsc proteins can rescue dorsoanterior structures in ventralized Xenopus embryos. We describe how D-Gsc behaves as a transcriptional repressor in Drosophila cells, acting through specific palindromic HD binding sites (P3K). D-Gsc is a 'passive repressor' of activator homeoproteins binding to the same sites and an 'active repressor' of activators binding to distinct sites. In addition, D-Gsc is able to strongly repress transcription activated by Paired-class homeoproteins through P3K, via specific protein-protein interactions in what we define as 'interactive repression'. This form of repression requires the short conserved GEH/eh-1 domain, also present in the Engrailed repressor. Although the GEH/eh-1 domain is necessary for rescue of UV-ventralized Xenopus embryos, it is dispensable for ectopic induction of Xlim-1 expression, demonstrating that this domain is not required for all Gsc functions in vivo. Interactive repression may represent specific interactions among Prd-class homeoproteins, several of which act early during development of invertebrate and vertebrate embryos.


"Inclusive Working Life" in Norway--experience from "Models of Good Practice" enterprises.
To determine whether enterprises belonging to the Bank of Models of Good Practice were more successful than average Norwegian enterprises in the reduction of sickness absence, promotion of early return to work, and prevention of early retirement. In 2004 we selected 86 enterprises with a total of approximately 90000 employees from the Inclusive Working Life (IWL) Bank of Models of Good Practice. One representative of workers and one of management from each enterprise received a questionnaire on the aims, organization, and the results of the IWL program by mail. Data on sickness absence, use of early retirement, and disability retirement in the 2000-2004 period were collected from the National Insurance Registry. Data on comparable enterprises were obtained from the National Bureau of Statistics. The response rate was 65%. Although the IWL campaign was directed at reducing sickness absence, preventing early retirement, and promoting employment of the functionally impaired, most attention was paid to reducing sickness absence. Sickness absence rate in Models of Good Practice enterprises (8.2%) was higher than in comparable enterprises that were not part of the Models of Good Practice (6.9%). Implementation of many IWL activities, empowerment and involvement of employees, and good cooperation with the occupational health service were associated with a lower rate of sickness absence. On average, 0.7% new employees per year received disability pension, which is a significantly lower percentage than expected on the basis of the rate of 1.3% per year in comparable enterprises. Frequent use of disability pensioning was associated with high rate of sickness absence and having many employees older than 50 years. On average, 0.4% employees per year received early retirement compensation, which was expected on the basis of national estimates. Frequent use of early retirement was associated with having many employees older than 50 years. Models of Good Practice enterprises had a higher than expected sickness absence rate. This indicates that it is difficult to identify Models of Good Practice enterprises and that they cannot be treated as role model enterprises. Good cooperation with the occupational health service and the empowerment and involvement of the employees is associated with a low sickness absence rate.


A process for analysis of microarray comparative genomics hybridisation studies for bacterial genomes.
Microarray based comparative genomic hybridisation (CGH) experiments have been used to study numerous biological problems including understanding genome plasticity in pathogenic bacteria. Typically such experiments produce large data sets that are difficult for biologists to handle. Although there are some programmes available for interpretation of bacterial transcriptomics data and CGH microarray data for looking at genetic stability in oncogenes, there are none specifically to understand the mosaic nature of bacterial genomes. Consequently a bottle neck still persists in accurate processing and mathematical analysis of these data. To address this shortfall we have produced a simple and robust CGH microarray data analysis process that may be automated in the future to understand bacterial genomic diversity. The process involves five steps: cleaning, normalisation, estimating gene presence and absence or divergence, validation, and analysis of data from test against three reference strains simultaneously. Each stage of the process is described and we have compared a number of methods available for characterising bacterial genomic diversity, for calculating the cut-off between gene presence and absence or divergence, and shown that a simple dynamic approach using a kernel density estimator performed better than both established, as well as a more sophisticated mixture modelling technique. We have also shown that current methods commonly used for CGH microarray analysis in tumour and cancer cell lines are not appropriate for analysing our data. After carrying out the analysis and validation for three sequenced Escherichia coli strains, CGH microarray data from 19 E. coli O157 pathogenic test strains were used to demonstrate the benefits of applying this simple and robust process to CGH microarray studies using bacterial genomes.


Localization of DNA damage and its role in altered antigen-presenting cell function in ultraviolet-irradiated mice.
Prior ultraviolet (UV) irradiation of the site of application of hapten on murine skin reduces contact sensitization, impairs the ability of dendritic cells in the draining lymph nodes (DLN) to present antigen, and leads to development of hapten-specific suppressor T lymphocytes. We tested the hypothesis that UV-induced DNA damage plays a role in the impaired antigen-presenting activity of DLN cells. First, we assessed the location and persistence of cells containing DNA damage. A monoclonal antibody specific for cyclobutyl pyrimidine dimers (CPD) was used to identify UV-damaged cells in the skin and DLN of C3H mice exposed to UV radiation. Cells containing CPD were present in the epidermis, dermis, and DLN and persisted, particularly in the dermis, for at least 4 d after UV irradiation. When fluorescein isothiocyanate (FITC) was applied to UV-exposed skin, the DLN contained cells that were Ia+, FITC+, and CPD+; such cells from mice sensitized 3 d after UV irradiation exhibited reduced antigen-presenting function in vivo. We then assessed the role of DNA damage in UV-induced modulation of antigen-presenting cell (APC) function by using a novel method of increasing DNA repair in mouse skin in vivo. Liposomes containing T4 endonuclease V (T4N5) were applied to the site of UV exposure immediately after irradiation. This treatment prevented the impairment in APC function and reduced the number of CPD+ cells in the DLN of UV-irradiated mice. Treatment of unirradiated skin with T4N5 in liposomes or treatment of UV-irradiated skin with liposomes containing heat-inactivated T4N5 did not restore immune function. These studies demonstrate that cutaneous immune cells sustain DNA damage in vivo that persists for several days, and that FITC sensitization causes the migration of these to the DLN, which exhibits impaired APC function. Further, they support the hypothesis that DNA damage is an essential initiator of one or more of the steps involved in impaired APC function after UV irradiation.


Fluorescence of the DNA double helices (dAdT)n.(dAdT)n studied by femtosecond spectroscopy.
Polymeric and oligomeric DNA helices, poly(dAdT).poly(dAdT) and (dAdT)(10).(dAdT)(10), composed of 200-400 and 20 adenine-thymine base pairs, respectively, are studied by fluorescence upconversion. Fluorescence decays, anisotropy decays and time-resolved spectra, obtained for this alternating base sequence, are compared with those determined previously for the homopolymeric sequence (dA)(n).(dT)(n). It is shown that identical fluorescence decays may correspond to quite different anisotropy decays and vice versa, both varying with the emission wavelength, the base sequence and the duplex size. Our observations cannot be explained in terms of monomer and excimer emission exclusively, as concluded in the past on the basis of steady-state measurements. Excitons also contribute to the fluorescence. These are rapidly trapped by excimers, characterized by long-lived weak emission.


The mechanical properties of the human L4-5 functional spinal unit during cyclic loading. The structural effects of the posterior elements.
Cyclic axial compression-tension tests and cyclic torsional tests were performed on ten fresh human L4-5 functional spinal units to investigate the structural effects of the posterior elements on the mechanical properties of L4-5 functional spinal units. The stiffness of the functional spinal unit increased with the increase of displacement under every loading. This was same in the intact functional spinal units and the functional spinal units after removal of each posterior element, respectively. All the posterior elements contributed to the compressive, tensile, and torsional stiffness of L4-5 functional spinal units. The apophyseal joints had a significant effect on the compressive and torsional stiffness. The effect of the apophyseal joints on the torsional stiffness became greater according to the extent of displacement, whereas their effect on the compressive stiffness was constant. The posterior ligaments (supraspinous and interspinous ligaments) had a significant effect on the tensile stiffness.


Complete Genome Sequence of Cd(II)-Resistant Arthrobacter sp. PGP41, a Plant Growth-Promoting Bacterium with Potential in Microbe-Assisted Phytoremediation.
Microbe-assisted phytoremediation has great potential for practical applications. Plant growth-promoting bacteria (PGPB) with heavy metal (HM) resistance are important for the implementation of PGPB-assisted phytoremediation of HM-contaminated environments. Arthrobacter sp. PGP41 is a Cd(II)-resistant bacterium isolated from the rhizosphere soils of a Cd(II) hyperaccumulator plant, Solanum nigrum. Strain PGP41 can significantly improve plant seedling and root growth under Cd(II) stress conditions. This bacterium exhibited the ability to produce high levels of indole-3-acetic acid (IAA), as well as the ability to fix nitrogen and solubilize phosphate, and it possessed 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Here, we present the complete genome sequence of strain PGP41. The genome consists of a single chromosome with a G+C content of 65.38% and no plasmids. The genome encodes 3898 genes and contains 49 tRNA and 12 rRNA genes. Multiple genes associated with plant growth promotion were identified in the genome. The whole genome sequence of PGP41 provides information useful for further clarifying the molecular mechanisms behind plant growth promotion by PGPB and facilitates its potential use as an inoculum in the bioremediation of HM-contaminated environments.


Development of a rapid enzyme immunoassay for the detection of retinol-binding protein.
Retinol-binding protein (RBP) was chosen as a surrogate marker for retinol because of the close correspondence between retinol and RBP. To meet the need for rapid, cost-effective determination of vitamin A status in populations, a quantitative enzyme immunoassay (EIA) for detection of RBP was developed. The resulting RBP EIA, a competitive assay, uses RBP adsorbed to microtest strip wells to compete with RBP in serum. The assay takes approximately 40 min. With a reference panel of sera, test accuracy was found to be within 4% of expected values through the calibrated range of 0.48-1.92 micro mol RBP/L (10-40 micro g RBP/mL). Intraassay and interassay variability averaged 6.7% and 8.9%, respectively. Specificity testing showed no interference from other serum proteins, prealbumin, rheumatoid factor, bilirubin, estrogen, or C-reactive protein. The RBP EIA provided linear results between 0.43 and 1.80 micro mol RBP/L (9 and 38 micro g RBP/mL). Preliminary laboratory evaluations indicated that the RBP EIA correlates well with radial immunodiffusion for RBP and with HPLC for retinol, the current reference standard. A field evaluation in a population at risk for vitamin A deficiency (VAD) resulted in close correlation between RBP EIA measures and retinol measures by HPLC (R(2) = 0.82). The RBP EIA is as reliable in estimating VAD as is HPLC retinol. After successful validations, the test should enable public health authorities to rapidly monitor VAD and track vitamin A status in populations.


Differential expression of CD40 and CD95 in ovarian carcinoma.
The role of CD95 (Fas) as a mediator of apoptosis has been well documented. CD40 ligation has been recently shown to initiate apoptosis and modulate CD95 mediated apoptosis in normal and some neoplastic tissues. Here we report the expression of CD95 and CD40 in cryopreserved cell suspensions from ovarian cancer associated ascites, fresh primary and recurrent ovarian carcinoma (OVCA) specimens, and ten established ovarian cancer cell lines. The effect of CD95 and CD40 receptor binding on apoptosis is described in two cell lines. Ascites specimens, fresh primary and recurrent OVCA specimens were dissociated to single cell suspensions. Expression of CD95 and CD40 was analyzed using flow cytometry. Apoptosis was determined via annexin uptake by flow cytometry following incubation with anti-CD95 antibody, CH11 and trimeric CD40L. Ascites showed the highest expression of both CD95 and CD40. Recurrent OVCA, in contrast, expressed low levels of CD95 and CD40. Primary OVCA showed moderate expression of both receptors. CD40 expression in ascites was significantly greater when compared to solid specimens (p < 0.05). Both CD40 and CD95 were strongly expressed in eight of ten cell lines studied. Binding of CD40L did not influence CD95 mediated apoptosis. CD40 is ubiquitously expressed in ovarian carcinomas and expression differs between ascites and solid tumor. There may be differential expression of both CD40 and CD95 in recurrent vs primary ovarian carcinoma, which may contribute to increased clinical malignancy of recurrent disease. In contrast to other epithelial malignancies, CD40 ligation does not appear to modulate CD95 mediated apoptosis.


Omega-3 fatty acids enhance phagocytosis of Alzheimer's disease-related amyloid-β42 by human microglia and decrease inflammatory markers.
The use of supplements with omega-3 (ω3) fatty acids (FAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is widespread due to proposed beneficial effects on the nervous and cardiovascular systems. Many effects of ω3 FAs are believed to be caused by down-regulation and resolution of inflammation. Alzheimer's disease (AD) is associated with inflammation mediated by microglia and astrocytes, and ω3 FAs have been proposed as potential treatments for AD. The focus of the present study is on the effects of DHA and EPA on microglial phagocytosis of the AD pathogen amyloid-β (Aβ), on secreted and cellular markers of immune activity, and on production of brain-derived neurotrophic factor (BDNF). Human CHME3 microglial cells were exposed to DHA or EPA, with or without the presence of Aβ42. Phagocytosis of Aβ42 was analyzed by flow cytometry in conjunction with immunocytochemistry using antibodies to cellular proteins. Secreted proteins were analyzed by ELISA. Both DHA and EPA were found to stimulate microglial phagocytosis of Aβ42. Phagocytosis of Aβ42 was performed by microglia with a predominance of M2 markers. EPA increased the levels of BDNF in the culture medium. The levels of TNF-α were decreased by DHA. Both DHA and EPA decreased the pro-inflammatory M1 markers CD40 and CD86, and DHA had a stimulatory effect on the anti-inflammatory M2 marker CD206. DHA and EPA can be beneficial in AD by enhancing removal of Aβ42, increasing neurotrophin production, decreasing pro-inflammatory cytokine production, and by inducing a shift in phenotype away from pro-inflammatory M1 activation.


Experimental demonstration of a novel bio-sensing platform via plasmonic band gap formation in gold nano-patch arrays.
In this paper we discuss the possibility of implementing a novel bio-sensing platform based on the observation of the shift of the leaky surface plasmon mode that occurs at the edge of the plasmonic band gap of metal gratings, when an analyte is deposited on top of the metallic structure. We report numerical calculations, fabrication and experimental measurements to prove the sensing capability of a two-dimensional array of gold nano-patches in the detection of a small quantity of Isopropyl Alcohol (IPA) deposited on top of sensor surface. The calculated sensitivity of our device approaches a value of 1000 nm/RIU with a corresponding Figure of Merit (FOM) of 222 RIU(-1). The presence of IPA can also be visually estimated by observing a color variation in the diffracted field. We show that color brightness and intensity variations can be ascribed to a change in the aperture size, keeping the periodicity constant, and to different types of analyte deposited on the sample, respectively. Moreover, we demonstrate that unavoidable fabrication imperfections revealed by the presence of rounded corners and surface roughness do not significantly affect device performance.


Blind Flight? A New Troglobiotic Orthoclad (Diptera, Chironomidae) from the Lukina Jama - Trojama Cave in Croatia.
The genus Troglocladius Andersen, Baranov et Hagenlund, gen. n. is erected based on T. hajdi Andersen, Baranov et Hagenlund, sp. n. collected at 980 m depth in the Lukina jama-Trojama cave system in Croatia. Morphological features such as pale color, strongly reduced eyes and very long legs make it a typical cave animal. Surprisingly, it has also retained large wings and appears to be capable of flight which would make T. hajdi the first flying troglobiont worldwide, disproving previous beliefs that bats are the only animals capable of flying in complete darkness. Morphologically the new species does not readily fit within any described genus, but shares characteristics with genera both in the tribes "Metriocnemini" and "Orthocladiini". Bayesian molecular phylogenetic analysis using the markers COI, 18S rDNAs, 28S rDNA, CADI, and CADIV groups it with the genera Tvetenia, Cardiocladius and Eukiefferiella in the tribe "Metriocnemini". Troglocladius hajdi may be parthenogenetic, as only females were collected. The discovery confirms the position of the Dinaric arch as a highly important hotspot of subterranean biodiversity.


Effects of myofascial trigger point dry needling in patients with sleep bruxism and temporomandibular disorders: a prospective case series.
To investigate the effects of deep dry needling (DN) of myofascial trigger points (MTrPs) of the masseter and temporalis on pain, pressure pain threshold (PPT), pain-free maximal jaw opening and temporomandibular disorder (TMD)-related disability in patients with sleep bruxism (SB) and myofascial TMD. Seventeen subjects (11 women, 6 men) aged 39±13 years (range 23-66) diagnosed with SB and myofascial TMD were invited to participate in this prospective case series study. Each subject received a deep DN intervention in the masseter and temporalis MTrPs. Pain intensity, PPT, pain-free maximal jaw opening and TMD-related disability were assessed before treatment, immediately after treatment and at 1-week follow-up. Jaw disability was assessed using the jaw disability checklist (JDC) at baseline and 1 week post-treatment only. One-way analyses of variance showed significant improvements in pain intensity, PPT and jaw opening (p<0.001). Post-hoc analysis revealed significant differences between baseline and post-intervention follow-up time points in pain (immediate: Cohen's d=1.72, p<0.001; 1 week: d=3.24, p<0.001), jaw opening (immediate: d=0.77, p<0.001; 1 week: d=1.02, p<0.001) and PPT in the masseter (immediate: d=1.02, p<0.001; 1 week: d=1.64, p<0.001) and temporalis (immediate: d=0.91, p=0.006; 1 week: d=1.8, p<0.001). A dependent t-test showed a significant improvement in jaw functioning, reflected by a large reduction in 1-week JDC scores relative to baseline (d=3.15, p<0.001). Deep DN of active MTrPs in the masseter and temporalis in patients with myofascial TMD and SB was associated with immediate and 1-week improvements in pain, sensitivity, jaw opening and TMD-related disability. NCT02587182; Results.


Results of the AANS membership survey of adult spinal deformity knowledge: impact of training, practice experience, and assessment of potential areas for improved education: Clinical article.
Adult spinal deformity (ASD) surgery is increasing in the spinal neurosurgeon's practice. A survey of neurosurgeon AANS membership assessed the deformity knowledge base and impact of current training, education, and practice experience to identify opportunities for improved education. Eleven questions developed and agreed upon by experienced spinal deformity surgeons tested ASD knowledge and were subgrouped into 5 categories: (1) radiology/spinopelvic alignment, (2) health-related quality of life, (3) surgical indications, (4) operative technique, and (5) clinical evaluation. Chi-square analysis was used to compare differences based on participant demographic characteristics (years of practice, spinal surgery fellowship training, percentage of practice comprising spinal surgery). Responses were received from 1456 neurosurgeons. Of these respondents, 57% had practiced less than 10 years, 20% had completed a spine fellowship, and 32% devoted more than 75% of their practice to spine. The overall correct answer percentage was 42%. Radiology/spinal pelvic alignment questions had the lowest percentage of correct answers (38%), while clinical evaluation and surgical indications questions had the highest percentage (44%). More than 10 years in practice, completion of a spine fellowship, and more than 75% spine practice were associated with greater overall percentage correct (p < 0.001). More than 10 years in practice was significantly associated with increased percentage of correct answers in 4 of 5 categories. Spine fellowship and more than 75% spine practice were significantly associated with increased percentage correct in all categories. Interestingly, the highest error was seen in risk for postoperative coronal imbalance, with a very low rate of correct responses (15%) and not significantly improved with fellowship (18%, p = 0.08). The results of this survey suggest that ASD knowledge could be improved in neurosurgery. Knowledge may be augmented with neurosurgical experience, spinal surgery fellowships, and spinal specialization. Neurosurgical education should particularly focus on radiology/spinal pelvic alignment, especially pelvic obliquity and coronal imbalance and operative techniques for ASD.


LC-HRMS Metabolomics for Untargeted Diagnostic Screening in Clinical Laboratories: A Feasibility Study.
Today's high-resolution mass spectrometers (HRMS) allow bioanalysts to perform untargeted/global determinations that can reveal unexpected compounds or concentrations in a patient's sample. This could be performed for preliminary diagnosis attempts when usual diagnostic processes and targeted determinations fail. We have evaluated an untargeted diagnostic screening (UDS) procedure. UDS is a metabolome analysis that compares one sample (e.g., a patient) with control samples (a healthy population). Using liquid chromatography (LC)-HRMS full-scan analysis of human serum extracts and unsupervised data treatment, we have compared individual samples that were spiked with one xenobiotic or a higher level of one endogenous compound with control samples. After the use of different filters that drastically reduced the number of metabolites detected, the spiked compound was eventually revealed in each test sample and ranked. The proposed UDS procedure appears feasible and reliable to reveal unexpected xenobiotics (toxicology) or higher concentrations of endogenous metabolites. HRMS-based untargeted approaches could be useful as preliminary diagnostic screening when canonical processes do not reveal disease etiology nor establish a clear diagnosis and could reduce misdiagnosis. On the other hand, the risk of overdiagnosis of this approach should be reduced with mandatory biomedical interpretation of the patient's UDS results and with confirmatory targeted and quantitative determinations.


Electroporation: a general phenomenon for manipulating cells and tissues.
Electroporation is a fascinating cell membrane phenomenon with several existing biological applications and others likely. Although DNA introduction is the most common use, electroporation of isolated cells has also been used for: (1) introduction of enzymes, antibodies, and other biochemical reagents for intracellular assays; (2) selective biochemical loading of one size cell in the presence of many smaller cells; (3) introduction of virus and other particles; (4) cell killing under nontoxic conditions; and (5) insertion of membrane macromolecules into the cell membrane. More recently, tissue electroporation has begun to be explored, with potential applications including: (1) enhanced cancer tumor chemotherapy, (2) gene therapy, (3) transdermal drug delivery, and (4) noninvasive sampling for biochemical measurement. As presently understood, electroporation is an essentially universal membrane phenomenon that occurs in cell and artificial planar bilayer membranes. For short pulses (microsecond to ms), electroporation occurs if the transmembrane voltage, U(t), reaches 0.5-1.5 V. In the case of isolated cells, the pulse magnitude is 10(3)-10(4) V/cm. These pulses cause reversible electrical breakdown (REB), accompanied by a tremendous increase molecular transport across the membrane. REB results in a rapid membrane discharge, with the elevated U(t) returning to low values within a few microseconds of the pulse. However, membrane recovery can be orders of magnitude slower. An associated cell stress commonly occurs, probably because of chemical influxes and effluxes leading to chemical imbalances, which also contribute to eventual survival or death. Basic phenomena, present understanding of mechanism, and the existing and potential applications are briefly reviewed.


A time transect of exomes from a Native American population before and after European contact.
A major factor for the population decline of Native Americans after European contact has been attributed to infectious disease susceptibility. To investigate whether a pre-existing genetic component contributed to this phenomenon, here we analyse 50 exomes of a continuous population from the Northwest Coast of North America, dating from before and after European contact. We model the population collapse after European contact, inferring a 57% reduction in effective population size. We also identify signatures of positive selection on immune-related genes in the ancient but not the modern group, with the strongest signal deriving from the human leucocyte antigen (HLA) gene HLA-DQA1. The modern individuals show a marked frequency decrease in the same alleles, likely due to the environmental change associated with European colonization, whereby negative selection may have acted on the same gene after contact. The evident shift in selection pressures correlates to the regional European-borne epidemics of the 1800s.


Menopause, Reproductive Life, Hormone Replacement Therapy, and Bone Phenotype at Age 60-64 Years: A British Birth Cohort.
Previous studies of menopausal age and length of reproductive life on bone are limited by retrospective reproductive histories, being cross-sectional, or lacking gold standard bone technologies or information on hormone replacement therapy (HRT) or surgical treatment. The objective of the study was to investigate age at menopause, length of reproductive life, and HRT use in relation to volumetric and areal bone mineral density (vBMD, aBMD), bone size, and strength in women aged 60-64 years. This was a birth cohort study that followed up for 64 years with prospective measures of age at menarche and menopause and monthly HRT histories. The study was conducted in England, Scotland, and Wales. Participants included 848 women with a known type of menopause and bone measures at 60-64 years. Peripheral quantitative computed tomography measurements of the distal radius total and trabecular vBMD were measured. Diaphyseal radius total and medullary cross-sectional area, cortical vBMD, and polar strength strain index (SSI); dual-energy x-ray absorptiometry measurements of aBMD at the lumbar spine and total hip were also measured. A 10-year increase in age at natural (but not surgical) menopause was associated with 8.2% (95% confidence interval [CI] 1.3%-15.1%, P = .02) greater trabecular vBMD and a 6.0% (95% CI 0.51%-11.5%, P = .03) greater total vBMD; findings were similar for length of reproductive life. A 10-year difference in HRT use was associated with a 6.0% (95% CI 2.6%-9.3%, P < .001) greater polar SSI and a 0.9% (95% CI 0.4%-1.5%, P = .001) greater cortical vBMD. These estimates changed little on adjustment. Estimates for aBMD were consistent with those for peripheral quantitative computed tomography. The positive effects on trabecular vBMD of later natural menopause and longer reproductive life persisted into early old age. HRT use was associated with greater radius cortical vBMD and polar SSI and aBMD.


Manipulation of quadratic cascading processes in a locally quasi-periodic χ(²) medium.
We theoretically and numerically investigate the quadratic cascading effect of third-harmonic (TH) generation in a locally quasi-periodic nonlinear photonic structure. We study the effect of structure parameters on the acceptance bandwidth and conversion efficiency of the cascading process. We demonstrate that the conversion efficiency of the cascading process can be enhanced by using a longer locally quasi-periodic nonlinear photonic crystal, without adversely affecting the acceptance bandwidth of the emitted radiation.


Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes.
Monocytes were maintained in tissue culture for greater than 3 mo in media supplemented with rCSF-1. These cultures provided susceptible target cells for isolation and propagation of virus from PBMC of HIV-infected patients. HIV isolated into monocytes readily infected other rCSF-1-treated monocytes but only inefficiently infected PHA-stimulated lymphoblasts. Similarly, laboratory HIV strains passaged in T cell lines or virus isolated from patients' leukocytes into PHA-stimulated lymphoblasts inefficiently infected rCSF-1-treated monocytes. Persistent, low-level virion production was detected in macrophage culture fluids by reverse transcriptase activity or HIV antigen capture through 6-7 wk. Marked changes in cell morphology with cell death, syncytia, and giant cell formation were observed in monocyte cultures 2 wk after infection, but at 4-6 wk, all cells appeared morphologically normal. However, the frequency of infected cells in these cultures at 6 wk was 60-90% as quantified by in situ hybridization with HIV RNA probes or by immunofluorescence with AIDS patients' sera. Ultrastructural analysis by EM also showed a high frequency of infected cells; virtually all HIV budded into and accumulated within cytoplasmic vacuoles and virus particles were only infrequently associated with the plasma membrane. Retention of virus within macrophages and the macrophage tropism of HIV variants may explain mechanisms of both virus persistence and dissemination during disease.


Dynamic causal models and physiological inference: a validation study using isoflurane anaesthesia in rodents.
Generative models of neuroimaging and electrophysiological data present new opportunities for accessing hidden or latent brain states. Dynamic causal modeling (DCM) uses Bayesian model inversion and selection to infer the synaptic mechanisms underlying empirically observed brain responses. DCM for electrophysiological data, in particular, aims to estimate the relative strength of synaptic transmission at different cell types and via specific neurotransmitters. Here, we report a DCM validation study concerning inference on excitatory and inhibitory synaptic transmission, using different doses of a volatile anaesthetic agent (isoflurane) to parametrically modify excitatory and inhibitory synaptic processing while recording local field potentials (LFPs) from primary auditory cortex (A1) and the posterior auditory field (PAF) in the auditory belt region in rodents. We test whether DCM can infer, from the LFP measurements, the expected drug-induced changes in synaptic transmission mediated via fast ionotropic receptors; i.e., excitatory (glutamatergic) AMPA and inhibitory GABA(A) receptors. Cross- and auto-spectra from the two regions were used to optimise three DCMs based on biologically plausible neural mass models and specific network architectures. Consistent with known extrinsic connectivity patterns in sensory hierarchies, we found that a model comprising forward connections from A1 to PAF and backward connections from PAF to A1 outperformed a model with forward connections from PAF to A1 and backward connections from A1 to PAF and a model with reciprocal lateral connections. The parameter estimates from the most plausible model indicated that the amplitude of fast glutamatergic excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) behaved as predicted by previous neurophysiological studies. Specifically, with increasing levels of anaesthesia, glutamatergic EPSPs decreased linearly, whereas fast GABAergic IPSPs displayed a nonlinear (saturating) increase. The consistency of our model-based in vivo results with experimental in vitro results lends further validity to the capacity of DCM to infer on synaptic processes using macroscopic neurophysiological data.


The (un)suitability of modern liquid crystal displays (LCDs) for vision research.
Psychophysical and physiological studies of vision have traditionally used cathode ray tube (CRT) monitors to present stimuli. These monitors are no longer easily available, and liquid crystal display (LCD) technology is continually improving; therefore, we characterized a number of LCD monitors to determine if newer models are suitable replacements for CRTs in the laboratory. We compared the spatial and temporal characteristics of a CRT with five LCDs, including monitors designed with vision science in mind (ViewPixx and Display++), "prosumer" gaming monitors, and a consumer-grade LCD. All monitors had sufficient contrast, luminance range and reliability to support basic vision experiments with static images. However, the luminance of all LCDs depended strongly on viewing angle, which in combination with the poor spatial uniformity of all monitors except the VPixx, caused up to 80% drops in effective luminance in the periphery during central fixation. Further, all monitors showed significant spatial dependence, as the luminance of one area was modulated by the luminance of other areas. These spatial imperfections are most pronounced for experiments that use large or peripheral visual stimuli. In the temporal domain, the gaming LCDs were unable to generate reliable luminance patterns; one was unable to reach the requested luminance within a single frame whereas in the other the luminance of one frame affected the luminance of the next frame. The VPixx and Display++ were less affected by these problems, and had good temporal properties provided stimuli were presented for 2 or more frames. Of the consumer-grade and gaming displays tested, and if problems with spatial uniformity are taken into account, the Eizo FG2421 is the most suitable alternative to CRTs. The specialized ViewPixx performed best among all the tested LCDs, followed closely by the Display++; both are good replacements for a CRT, provided their spatial imperfections are considered.


A high-throughput method to deliver targeted optogenetic stimulation to moving C. elegans populations.
We present a high-throughput optogenetic illumination system capable of simultaneous closed-loop light delivery to specified targets in populations of moving Caenorhabditis elegans. The instrument addresses three technical challenges: It delivers targeted illumination to specified regions of the animal's body such as its head or tail; it automatically delivers stimuli triggered upon the animal's behavior; and it achieves high throughput by targeting many animals simultaneously. The instrument was used to optogenetically probe the animal's behavioral response to competing mechanosensory stimuli in the the anterior and posterior gentle touch receptor neurons. Responses to more than 43,418 stimulus events from a range of anterior-posterior intensity combinations were measured. The animal's probability of sprinting forward in response to a mechanosensory stimulus depended on both the anterior and posterior stimulation intensity, while the probability of reversing depended primarily on the anterior stimulation intensity. We also probed the animal's response to mechanosensory stimulation during the onset of turning, a relatively rare behavioral event, by delivering stimuli automatically when the animal began to turn. Using this closed-loop approach, over 9,700 stimulus events were delivered during turning onset at a rate of 9.2 events per worm hour, a greater than 25-fold increase in throughput compared to previous investigations. These measurements validate with greater statistical power previous findings that turning acts to gate mechanosensory evoked reversals. Compared to previous approaches, the current system offers targeted optogenetic stimulation to specific body regions or behaviors with many fold increases in throughput to better constrain quantitative models of sensorimotor processing.


Photoluminescence imaging of Zn(2+) in living systems.
Zn(2+) plays essential roles in various physiological processes in living systems, and the investigation of Zn(2+) related physiology and pathology has attracted considerable interest. Because photoluminescence (PL) imaging possesses distinct advantages, such as high sensitivity and non-invasiveness, and excellent temporal and spatial resolution, it has become a powerful tool for the real time monitoring of Zn(2+) distribution, uptake, and trafficking. Over the last two decades, great efforts have been devoted to PL Zn(2+) imaging in living systems, which proved the Zn(2+) fluctuations in physiological processes and the temporal-spatial distribution of labile Zn(2+) as well as the localization of labile Zn(2+) pools. Advances in PL techniques, such as fluorescence microscopy, confocal fluorescence microscopy, two photon fluorescence microscopy, lifetime based techniques and luminescence optical imaging systems, have made remarkable contributions in tackling major challenges in Zn(2+) PL imaging. With the rational design and proper use of fluorescent sensors, Zn(2+) imaging in various cell lines, organelles, tissues, organs and living animals has been realized, which was shown to be crucial in elucidating the biological and physiological roles of labile Zn(2+).


Vasotocin mRNA expression is sensitive to testosterone and oestradiol in the bed nucleus of the stria terminalis in female Japanese quail.
Vasotocin-producing parvocellular neurones in the medial part of the bed nucleus of the stria terminalis (BSTM) of many species of birds and mammals show sexual dimorphism and great plasticity in response to hormonal and environmental stimuli. In the BSTM of Japanese quail, vasotocin-immunoreactive neurones are visible and sensitive to testosterone exclusively in males. In males, gonadectomy decreases and testosterone restores vasotocin-immunoreactive cells and fibres by acting on vasotocin mRNA transcription. The insensitivity of female vasotocin-immunoreactive neurones to the activating effects of testosterone is the result of organisational effects of early exposure to oestradiol. Female quail also show vasotocin mRNA-expressing neurones in the BSTM, although it is not known whether the insensitivity of the vasotocinergic neurones to testosterone originates at the level of vasotocin gene transcription in this sex. Therefore, initially, the present study analysed the effects of acute treatment with testosterone on vasotocin mRNA expression in the BSTM of gonadectomised male and female quail using in situ hybridisation. Gonadectomy decreased (and a single injection of testosterone increased) the number of vasotocin mRNA-expressing neurones and intensity of the vasotocin mRNA hybridisation signal similarly in both sexes. Notably, testosterone increased vasotocin mRNA expression in ovariectomised females over that shown by intact quail. However, this treatment had no effect on vasotocin immunoreactivity. A second experiment analysed the effects of testosterone metabolites, oestradiol and 5α-dihydrotestosterone, on vasotocin mRNA expression in female quail. Oestradiol (but not 5α-dihydrotestosterone) fully mimicked the effects of testosterone on the number of vasotocin mRNA-expressing neurones and the intensity of the vasotocin mRNA hybridisation signal. Taken together, these results show, for the first time, that gonadal steroids strongly activate vasotocin mRNA expression in the BSTM of female quail.


Reactivation of fear memory renders consolidated amygdala synapses labile.
It is believed that memory reactivation transiently renders consolidated memory labile and that this labile or deconsolidated memory is reconsolidated in a protein synthesis-dependent manner. The synaptic correlate of memory deconsolidation upon reactivation, however, has not been fully characterized. Here, we show that 3,5-dihydroxyphenylglycine (DHPG), an agonist for group I metabotropic glutamate receptors (mGluRI), induces synaptic depotentiation only at thalamic input synapses onto the lateral amygdala (T-LA synapses) where synaptic potentiation is consolidated, but not at synapses where synaptic potentiation is not consolidated. Using this mGluRI-induced synaptic depotentiation (mGluRI-depotentiation) as a marker of consolidated synapses, we found that mGluRI-depotentiation correlated well with the state of memory deconsolidation and reconsolidation in a predictable manner. DHPG failed to induce mGluRI-depotentiation in slices prepared immediately after reactivation when the reactivated memory was deconsolidated. DHPG induced mGluRI-depotentiation 1 h after reactivation when the reactivated memory was reconsolidated, but it failed to do so when reconsolidation was blocked by a protein synthesis inhibitor. To test the memory-specificity of mGluRI-depotentiation, conditioned fear was acquired twice using two discriminative tones (2.8 and 20 kHz). Under this condition, mGluRI-depotentiation was fully impaired in slices prepared immediately after reactivation with both tones, whereas mGluRI-depotentiation was partially impaired immediately after reactivation with the 20 kHz tone. Consistently, microinjection of DHPG into the LA 1 h after reactivation reduced fear memory retention, whereas DHPG injection immediately after reactivation failed to do so. Our findings suggest that, upon memory reactivation, consolidated T-LA synapses enter a temporary labile state, displaying insensitivity to mGluRI-depotentiation.


CAST: randomised placebo-controlled trial of early aspirin use in 20,000 patients with acute ischaemic stroke. CAST (Chinese Acute Stroke Trial) Collaborative Group.
Aspirin is effective in the treatment of acute myocardial infarction and in the long-term prevention of serious vascular events in survivors of stroke and myocardial infarction. There is, however, no reliable evidence on the effectiveness of early aspirin use in acute ischaemic stroke. The Chinese Acute Stroke Trial (CAST) was a large randomised, placebo-controlled trial of the effects in hospital of aspirin treatment (160 mg/day) started within 48 h of the onset of suspected acute ischaemic stroke and continued in hospital for up to 4 weeks. The primary endpoints were death from any cause during the 4-week treatment period and death or dependence at discharge, and the analyses were by intention to treat. 21,106 patients with acute ischaemic stroke were enrolled in 413 Chinese hospitals at a mean of 25 h after the onset of symptoms (10,554 aspirin, 10,552 placebo). 87% had a CT scan before randomisation. It was prospectively planned that the results would be analysed in parallel with those of the concurrent. International Stroke Trial (IST) of 20,000 patients with acute stroke from other countries. There was a significant 14% (SD 7) proportional reduction in mortality during the scheduled treatment period (343 [3.3%] deaths among aspirin-allocated patients vs 398 [3.9%] deaths among placebo-allocated patients; 2p = 0.04). There were significantly fewer recurrent ischaemic strokes in the aspirin-allocated than in the placebo-allocated group (167 [1.6%] vs 215 [2.1%]; 2p = 0.01) but slightly more haemorrhagic strokes (115 [1.1%] vs 93 [0.9%]; 2p > 0.1). For the combined in-hospital endpoint of death or non-fatal stroke at 4 weeks, there was a 12% (6) proportional risk reduction with aspirin (545 [5.3%] vs 614 [5.9%]; 2p = 0.03), an absolute difference of 6.8 (3.2) fewer cases per 1000. At discharge, 3153 (30.5%) aspirin-allocated patients and 3266 (31.6%) placebo-allocated patients were dead or dependent, corresponding to 11.4 (6.4) fewer per 1000 in favour of aspirin (2p = 0.08). There are two major trials of aspirin in acute ischaemic stroke. Taken together, CAST and the similarly large IST show reliably that aspirin started early in hospital produces a small but definite net benefit, with about 9 (SD 3) fewer deaths or non-fatal strokes per 1000 in the first few weeks (2p = 0.001), and with 13 (5) fewer dead or dependent per 1000 after some weeks or months of follow-up (2p < 0.01).


Dermatologic health literacy in underserved communities: a case report of south Los Angeles middle schools.
The incidence of melanoma and other skin cancers has risen drastically in the United States. As with most types of cancer, the prognosis and survival rates are significantly improved with early diagnosis, but dismal for patients who present with advanced disease. It remains a fact that although melanoma is most common in Caucasian populations, ethnic minorities have a worse prognosis. Our hypothesis in this dermatologic health literacy study was that before necessary education, the required fund of knowledge with respect to skin cancer risk is lacking in several ethnic communities, but that intended compliance occurs when educational intervention occurs. Three middle schools in South Los Angeles with predominantly Latino and African American youth were surveyed. Permission was obtained from the principals of the middle schools for the multi-day educational initiative. A total of 150 students were ultimately recruited and a pre-intervention survey administered. After preliminary review of the pre-intervention dermatologic health literacy results, a set of "core" learning concepts about sun safety were summarized and solidified for incorporation into the adolescent-appropriate sun safety protection pamphlet that was designed by designers at UCLA/Johnson & Johnson Health Care Institute. A full day of education on skin disease and the importance of sun protection from an early age was executed, followed three months later by a post-intervention visit that assessed compliance with the sun protection products and intended future use. Results from the pre- and post-intervention surveys/questionnaires were analyzed and interpreted. Of 150 pre-intervention surveys that were distributed, 54 identified as African American and 96 of whom identified as Latino. Of these, 75% of Latino students reported having a sunburn in the last year, whereas only 38.9% of African American students reported a sunburn. A total of 80% of the students reported as least some use of sunscreen in the 3 months prior to the post-intervention survey. Only 8% of African American students reported "everyday" use, whereas 24% of Latino students reported "everyday" use (P < 0.05). A total of 94% of the students intend to wear sunscreen in the future (89% of African American students and 97% of Latino students, with a P < 0.05 calculated using a two-sample t test). However, it should be noted that more than half (54%) of the total students reported that although they planned to apply the sunscreen daily, they deemed it too expensive, which might prevent consistent future use. Our hypothesis in this dermatologic health literacy study was that before necessary education, the required fund of knowledge with respect to skin cancer risk is lacking in several ethnic communities, but that intended compliance occurs when educational intervention occurs. The data, both quantitative and qualitative, demonstrate that our hypothesis is substantiated.


Rapid, laser-induced conversion of 20-hydroxyecdysone and its diacetonide -- experimental set-up of a system for photochemical transformation of bioactive substances.
Photochemical transformation of certain bioactive compounds for the purpose of obtaining derivatives with increased bioactivity is a prospective area of synthetic chemistry. Ecdysteroids, analogs of the insect molting hormone, which can also exert several beneficial effects in mammals including humans, contain an enone moiety in their B ring, and, as such, are good candidates for photochemical transformations. 20-hydroxyecdysone (20E), the most common ecdysteroid in Nature, and the easily obtained derivative 20-hydroxyecdysone 2,3;20,22-diacetonide (20ED), at different concentrations, were exposed to a 266 nm laser beam at an energy level of 6.5 mJ for different periods of time and evaluated for fluorescence emission during the process of irradiation. The products of irradiation were scanned from 200 to 1500 nm and then subjected to one-dimensional and two-dimensional thin layer chromatography. During irradiation, progressive significant changes in the fluorescence emission spectra were noted for both compounds with time that were accompanied by changes in their UV-Vis spectra. Full conversion of both compounds was reached within 14 minutes, and both compounds yielded several major products and several minor ones representing a wide polarity range. The photo-transformation system described here was proven to be a useful and flexibly adjustable tool for the laser-catalyzed conversion of bioactive compounds. Due to the multi-drug resistance reversal activity of the less polar ecdysteroids, several new products are promising for being tested against various cancer cell lines. Fractionation, isolation and characterization of the irradiated products are currently in process.


Analysis of published studies on the detection of extrathoracic metastases in patients presumed to have operable non-small cell lung cancer.
A study was undertaken to determine the proportion of patients with potentially operable non-small cell lung cancer that could be spared thoracotomy by a systematic search for extrathoracic metastases. An English language literature search was carried out using MEDLINE (1966-91) and bibliographic reviews of textbooks, review articles, and key articles. Studies were included in which at least 90% of the patients had histologically proven non-small cell cancer of the lung, were presumed otherwise operable, and for which the results of computed tomography of the head or abdomen, ultrasonography of the abdomen, or radionuclide imaging (scan) of bone or liver were available. Study quality and specific descriptive information concerning population, intervention, and outcome measurements were assessed. Of approximately 1500 citations which were screened, 100 were reviewed in detail and data abstracted from 16. The number of patients (total number, followed in square brackets by number of asymptomatic patients) shown to be inoperable directly as a result of the investigation and thus spared unnecessary thoracotomy was: computed tomography of the head: 26/785 (3.3%), 95% confidence interval (CI) 2.1% to 4.4% [14/353 (4.0%), 95% CI 2% to 6%], computed tomography of the adrenal glands: 30/632 (4.7%), 95% CI 3.0% to 6.4% [number asymptomatic indeterminate], bone scan: 45/480 (9.3%), 95% CI 6.7% to 12% [9/301 (3.0%), 95% CI 1.1% to 4.9%], liver imaging: 12/529 (2.3%), 95% CI 0.9% to 3.3% [4/268 (1.5%), 95% CI 0.1% to 3%]. A study with a large sample size and preferably incorporating thoracic computed tomography is required to narrow the confidence intervals around each test. All tests may play an important part in the preoperative evaluation of patients with non-small cell carcinoma of the lung who are presumed to be operable, including asymptomatic patients. Limitations of present data preclude definitive recommendations for asymptomatic patients.


Inhibition of hematopoietic cell kinase dysregulates microglial function and accelerates early stage Alzheimer's disease-like neuropathology.
Emerging evidence have posited that dysregulated microglia impair clearance and containment of amyloid-β (Aβ) species in the brain, resulting in aberrant buildup of Aβ and onset of Alzheimer's disease (AD). Hematopoietic cell kinase (Hck) is one of the key regulators of phagocytosis among the Src family tyrosine kinases (SFKs) in myeloid cells, and its expression is found to be significantly altered in AD brains. However, the role of Hck signaling in AD pathogenesis is unknown. We employed pharmacological inhibition and genetic ablation of Hck in BV2 microglial cells and J20 mouse model of AD, respectively, to evaluate the impact of Hck deficiency on Aβ-stimulated microglial phagocytosis, Aβ clearance, and resultant AD-like neuropathology. Our in vitro data reveal that pharmacological inhibition of SFKs/Hck in BV2 cells and genetic ablation of their downstream kinase, spleen tyrosine kinase (Syk), in primary microglia significantly attenuate Aβ oligomers-stimulated microglial phagocytosis. Whereas in Hck-deficient J20 mice, we observed exacerbated Aβ plaque burden, reduced microglial coverage, containment, and phagocytosis of Aβ plaques, and induced iNOS expression in plaque-associated microglial clusters. These multifactorial changes in microglial activities led to attenuated PSD95 levels in hippocampal DG and CA3 regions, but did not alter the postsynaptic dendritic spine morphology at the CA1 region nor cognitive function of the mice. Hck inhibition thus accelerates early stage AD-like neuropathology by dysregulating microglial function and inducing neuroinflammation. Our data implicate that Hck pathway plays a prominent role in regulating microglial neuroprotective function during the early stage of AD development.


The GENOTEND chip: a new tool to analyse gene expression in muscles of beef cattle for beef quality prediction.
Previous research programmes have described muscle biochemical traits and gene expression levels associated with beef tenderness. One of our results concerning the DNAJA1 gene (an Hsp40) was patented. This study aims to confirm the relationships previously identified between two gene families (heat shock proteins and energy metabolism) and beef quality. We developed an Agilent chip with specific probes for bovine muscular genes. More than 3000 genes involved in muscle biology or meat quality were selected from genetic, proteomic or transcriptomic studies, or from scientific publications. As far as possible, several probes were used for each gene (e.g. 17 probes for DNAJA1). RNA from Longissimus thoracis muscle samples was hybridised on the chips. Muscles samples were from four groups of Charolais cattle: two groups of young bulls and two groups of steers slaughtered in two different years. Principal component analysis, simple correlation of gene expression levels with tenderness scores, and then multiple regression analysis provided the means to detect the genes within two families (heat shock proteins and energy metabolism) which were the most associated with beef tenderness. For the 25 Charolais young bulls slaughtered in year 1, expression levels of DNAJA1 and other genes of the HSP family were related to the initial or overall beef tenderness. Similarly, expression levels of genes involved in fat or energy metabolism were related with the initial or overall beef tenderness but in the year 1 and year 2 groups of young bulls only. Generally, the genes individually correlated with tenderness are not consistent across genders and years indicating the strong influence of rearing conditions on muscle characteristics related to beef quality. However, a group of HSP genes, which explained about 40% of the variability in tenderness in the group of 25 young bulls slaughtered in year 1 (considered as the reference group), was validated in the groups of 30 Charolais young bulls slaughtered in year 2, and in the 21 Charolais steers slaughtered in year 1, but not in the group of 19 steers slaughtered in year 2 which differ from the reference group by two factors (gender and year). When the first three groups of animals were analysed together, this subset of genes explained a 4-fold higher proportion of the variability in tenderness than muscle biochemical traits. This study underlined the relevance of the GENOTEND chip to identify markers of beef quality, mainly by confirming previous results and by detecting other genes of the heat shock family as potential markers of beef quality. However, it was not always possible to extrapolate the relevance of these markers to all animal groups which differ by several factors (such as gender or environmental conditions of production) from the initial population of reference in which these markers were identified.


Translating evidence into practice: Implementing culturally safe continuity of midwifery care for First Nations women in three maternity services in Victoria, Australia.
Strategies to improve outcomes for Australian First Nations mothers and babies are urgently needed. Caseload midwifery, where women have midwife-led continuity throughout pregnancy, labour, birth and the early postnatal period, is associated with substantially better perinatal health outcomes, but few First Nations women receive it. We assessed the capacity of four maternity services in Victoria, Australia, to implement, embed, and sustain a culturally responsive caseload midwifery service. A prospective, non-randomised research translational study design was used. Site specific culturally responsive caseload models were developed by site working groups in partnership with their First Nations health units and the Victorian Aboriginal Community Controlled Health Organisation. The primary outcome was to increase the proportion of women having a First Nations baby proactively offered and receiving caseload midwifery as measured before and after programme implementation. The study was conducted in Melbourne, Australia. Data collection commenced at the Royal Women's Hospital on 06/03/2017, Joan Kirner Women's and Children's Hospital 01/10/2017 and Mercy Hospital for Women 16/04/2018, with data collection completed at all sites on 31/12/2020. The model was successfully implemented in three major metropolitan maternity services between 2017 and 2020. Prior to this, over a similar timeframe, only 5.8% of First Nations women (n = 34) had ever received caseload midwifery at the three sites combined. Of 844 women offered the model, 90% (n = 758) accepted it, of whom 89% (n = 663) received it. Another 40 women received standard caseload. Factors including ongoing staffing crises, prevented the fourth site, in regional Victoria, implementing the model. Key enablers included co-design of the study and programme implementation with First Nations people, staff cultural competency training, identification of First Nations women (and babies), and regular engagement between caseload midwives and First Nations hospital and community teams. Further work should include a focus on addressing cultural and workforce barriers to implementation of culturally responsive caseload midwifery in regional areas. Partnership Grant (# 1110640), Australian National Health and Medical Research Council and La Trobe University.


The role of alternative sugars on endurance capacity in balb/c mice.
Alternative sugars containing isomaltulose were investigated to confirm the hypothesis that isomaltulose ingestion affects endurance capacity due to slow rates of hydrolysis and absorption rate at the intestine. A swimming time of the control group tends to decrease, but the group administrated with low glycemic index (GI) sweeteners tend to increase gradually. Fructo-oligosaccharide (FOS) and inverted sugar (IS), contained isomaltulose, groups showed a significant difference of change in blood glucose and lactic acid level than control group (p < .05). Serum creatine phosphokinase (CPK) and serum lactate dehydrogenases (LDH) of PAL100 were significantly lower than that of the control group (p < .05). IS showed a significant difference in glutathione peroxidase (GSH-Px) compared to Con and PAL20 (20% isomaltulose) group (p < .05). Consuming FOS seems to increase an endurance capacity since fructose and FOS based in isomaltulose contained syrup showed low absorption rate and GI level. PRACTICAL APPLICATIONS: Sugar is a major energy source for exercise, but it causes excessive intake because of the short duration of sweetness, which causes diseases such as obesity, diabetes, and skin aging. An alternative sugar complex containing isomatulose was found to be a sugar substitute for athletic performance. Athletic performance is not just for athletes or active people. In general, elderly people with low muscle mass have low mobility. Alternative sugars can be a good source of supplements to help them perform smoothly with less intake. Therefore, an endurance test with alternative sugars is and important study for energy supplements industry.


Species- and Strain-Specific Adaptation of the HSP70 Super Family in Pathogenic Trypanosomatids.
All eukaryotic genomes encode multiple members of the heat shock protein 70 (HSP70) family, which evolved distinctive structural and functional features in response to specific environmental constraints. Phylogenetic analysis of this protein family thus can inform on genetic and molecular mechanisms that drive species-specific environmental adaptation. Here we use the eukaryotic pathogen Leishmania spp. as a model system to investigate the evolution of the HSP70 protein family in an early-branching eukaryote that is prone to gene amplification and adapts to cytotoxic host environments by stress-induced and chaperone-dependent stage differentiation. Combining phylogenetic and comparative analyses of trypanosomatid genomes, draft genome of Paratrypanosoma and recently published genome sequences of 204 L. donovani field isolates, we gained unique insight into the evolutionary dynamics of the Leishmania HSP70 protein family. We provide evidence for (i) significant evolutionary expansion of this protein family in Leishmania through gene amplification and functional specialization of highly conserved canonical HSP70 members, (ii) evolution of trypanosomatid-specific, non-canonical family members that likely gained ATPase-independent functions, and (iii) loss of one atypical HSP70 member in the Trypanosoma genus. Finally, we reveal considerable copy number variation of canonical cytoplasmic HSP70 in highly related L. donovani field isolates, thus identifying this locus as a potential hot spot of environment-genotype interaction. Our data draw a complex picture of the genetic history of HSP70 in trypanosomatids that is driven by the remarkable plasticity of the Leishmania genome to undergo massive intra-chromosomal gene amplification to compensate for the absence of regulated transcriptional control in these parasites.


A method for concentrating lipid peptide DNA and siRNA nanocomplexes that retains their structure and transfection efficiency.
Nonviral gene and small interfering RNA (siRNA) delivery formulations are extensively used for biological and therapeutic research in cell culture experiments, but less so in in vivo and clinical research. Difficulties with formulating the nanoparticles for uniformity and stability at concentrations required for in vivo and clinical use are limiting their progression in these areas. Here, we report a simple but effective method of formulating monodisperse nanocomplexes from a ternary formulation of lipids, targeting peptides, and nucleic acids at a low starting concentration of 0.2 mg/mL of DNA, and we then increase their concentration up to 4.5 mg/mL by reverse dialysis against a concentrated polymer solution at room temperature. The nanocomplexes did not aggregate and they had maintained their biophysical properties, but, importantly, they also mediated DNA transfection and siRNA silencing in cultured cells. Moreover, concentrated anionic nanocomplexes administered by convection-enhanced delivery in the striatum showed efficient silencing of the β-secretase gene BACE1. This method of preparing nanocomplexes could probably be used to concentrate other nonviral formulations and may enable more widespread use of nanoparticles in vivo.


Does adipose tissue thermogenesis play a role in metabolic health?
The function ascribed to brown adipose tissue in humans has long been confined to thermoregulation in neonates, where this thermogenic capacity was thought lost with maturation. Recently, brown adipose tissue depots have been identified in adult humans. The significant oxidative capacity of brown adipocytes and the ability of their mitochondria to respire independently of ATP production, has led to renewed interest in the role that these adipocytes play in human energy metabolism. In our view, there is a need for robust physiological studies determining the relationship between molecular signatures of brown adipose tissue, adipose tissue mitochondrial function, and whole body energy metabolism, in order to elucidate the significance of thermogenic adipose tissue in humans. Until such information is available, the role of thermogenic adipose tissue in human metabolism and the potential that these adipocytes may prevent or treat obesity and metabolic diseases in humans will remain unknown. In this article, we summarize the recent literature pertaining to brown adipose tissue function with the aims of drawing the readers' attention to the lack of data concerning the role of brown adipocytes in human physiology, and to the potential limitations of current research strategies.


Nucleotide sequence and expression of a deep-sea ribulose-1,5-bisphosphate carboxylase gene cloned from a chemoautotrophic bacterial endosymbiont.
The gene coding for ribulose-1,5-bisphosphate carboxylase [RuBisCO; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 184.108.40.206] was cloned from a sulfur-oxidizing chemoautotrophic bacterium that resides as an endosymbiont within the gill tissues of Alvinoconcha hessleri, a gastropod inhabiting deep-sea hydrothermal vents. Nucleotide sequence analysis of the cloned fragment demonstrated that the genes encoding the large (RbcL) and small (RbcS) subunits of the symbiont RuBisCO were organized similarly to the RuBisCO operons of free-living photo- and chemoautotrophic prokaryotes. The symbiont rbcL gene shared the highest degree of nucleotide sequence identity with the cyanobacterium Anabaena (69%) while the rbcS nucleotide sequence shared 61% identity with that of the green alga Chlamydomonas reinhardtii. Comparison with a 153-nucleotide partial rbcL sequence from a symbiont of the bivalve Solemya reidi indicated that the two symbiont sequences shared 85% sequence identity at the nucleotide level and 93% at the amino acid level, suggesting a relatively recent common origin. Escherichia coli transformed with a plasmid carrying the RuBisCO operon of the gastropod symbiont in the proper orientation for transcription from the plasmid lac promoter expressed catalytically active RuBisCO. The presence of enzyme activity suggests the proper assembly of the subunits of this deep-sea RuBisCO into the holoenzyme.


Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes.
Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O-methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O-methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o-and p-vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o- and p-isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p-vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo. This work expands the known aromatic O-demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.


Insulin-like growth factor-I (IGF-I) and its association with lymphocyte homeostasis in the ageing cat.
Ageing affects feline lymphocyte homeostasis in a similar pattern to that observed in other long-lived mammalian species, contributing to increased levels of morbidity and mortality in the ageing cat. Insulin-like growth factor-I (IGF-I) is now recognised as an important endocrine regulator of immunity and has been shown to decline with age in humans and rodent species. Analysis of plasma IGF-I in adult and senior cats confirmed that the older cats had significantly lower circulating levels of IGF-I. In order to determine whether an association existed between lymphocyte subpopulations and IGF-I levels in the cat, each parameter was measured and subjected to regression analysis. A highly significant association was found in vivo between plasma IGF-I and CD4(+) T-cell values in the senior group, but no such association was observed in the adult group. In order that this relationship could be examined further, in vitro studies were undertaken to investigate the effects of physiologically relevant concentrations of recombinant human IGF-I (rhIGF-l) on peripheral blood lymphocyte (PBL) cultures from adult and senior cats. While rhlGF-I induced low-level thymidine incorporation in the lymphocytes isolated from the senior group, it did not enhance the proliferative response to T-cell mitogens, Con A and PHA in either group, nor did it rescue cells from oxidatively induced apoptosis. Furthermore, the proliferative response of PBL from seniors did not attain the magnitude of that from the adults at any concentration of rhIGF-l. We propose that the observed association is not a direct effect of IGF-I on PBL, but may be mediated through an effect of IGF-I on the thymus.


Genetic components of root architecture and anatomy adjustments to water-deficit stress in spring barley.
Roots perform vital roles for adaptation and productivity under water-deficit stress, even though their specific functions are poorly understood. In this study, the genetic control of the nodal-root architectural and anatomical response to water deficit were investigated among diverse spring barley accessions. Water deficit induced substantial variations in the nodal root traits. The cortical, stele, and total root cross-sectional areas of the main-shoot nodal roots decreased under water deficit, but increased in the tiller nodal roots. Root xylem density and arrested nodal roots increased under water deficit, with the formation of root suberization/lignification and large cortical aerenchyma. Genome-wide association study implicated 11 QTL intervals in the architectural and anatomical nodal root response to water deficit. Among them, three and four QTL intervals had strong effects across seasons and on both root architectural and anatomical traits, respectively. Genome-wide epistasis analysis revealed 44 epistatically interacting SNP loci. Further analyses showed that these QTL intervals contain important candidate genes, including ZIFL2, MATE, and PPIB, whose functions are shown to be related to the root adaptive response to water deprivation in plants. These results give novel insight into the genetic architectures of barley nodal root response to soil water deficit stress in the fields, and thus offer useful resources for root-targeted marker-assisted selection.