Stimulation of fluid secretion of malpighian tubules of drosophila melanogaster meig. by cyclic nucleotides of inosine, cytidine, thymidine and uridine
External application of the 3',5'-cyclic monophosphates of inosine, cytidine, uridine and thymidine stimulated the fluid secretion rate (FSR) of Malpighian tubules isolated from Drosophila melanogaster. The evidence suggested that the cyclic nucleotides acted intracellularly in some capacity. Receptors of the 'purinergic' type appeared not to be major contributors to fluid secretion; of three purinergic agonists tried, adenosine, adenosine 5'-monophosphate (AMP) and adenosine 5'-triphosphate (ATP), only adenosine had an effect, but this was not observed consistently. None of the purinergic agonists interfered with the stimulation of the FSR by adenosine 3',5'-cyclic monophosphate (cAMP). The maximum stimulation of the fluid-secretion rate by any cyclic nucleotide was approximately double the unstimulated (control) rate. Tubules stimulated to less than maximal FSR by one cyclic nucleotide could be stimulated maximally by an appropriate concentration of another cyclic nucleotide. Malpighian tubules bathed in solutions that contained either [3H]cAMP or [3H]cGMP accumulated radioactivity to a level many times that in the medium. Accumulation of radioactivity by tubules bathed in 430 nmol l-1 [3H]cAMP was suppressed by 1 mmol l-1 non-radioactive cyclic nucleotides in the order cAMP>>cGMP>cIMP>cCMP; neither cTMP nor cUMP suppressed the accumulation of [3H]cAMP. Approximately 35 % of the [3H]cAMP and 80 % of the [3H]cGMP that entered the Malpighian tubule cells was metabolised to compounds that were not identified. It was concluded that cyclic nucleotides enter the Malpighian tubule cells by at least one transport mechanism which is particularly sensitive to purine-based nucleotides.


Pepducins as a potential treatment strategy for asthma and COPD.
Current therapies to treat asthma and other airway diseases primarily include anti-inflammatory agents and bronchodilators. Anti-inflammatory agents target trafficking and resident immunocytes and structural cells, while bronchodilators act to prevent or reverse shortening of airway smooth muscle (ASM), the pivotal tissue regulating bronchomotor tone. Advances in our understanding of the biology of G protein-coupled receptors (GPCRs) and biased agonism offers unique opportunities to modulate GPCR function that include the use of pepducins and allosteric modulators. Recent evidence suggests that small molecule inhibitors of Gαq as well as pepducins targeting Gq-coupled receptors can broadly inhibit contractile agonist-induced ASM function. Given these advances, new therapeutic approaches can be leveraged to diminish the global rise in morbidity and mortality associated with asthma and chronic obstructive pulmonary disease.


Halothane effect on formalin-induced paw edema and flinching in rat.
The formalin test is a model of injury-produced inflammatory pain. Anesthetics, in clinically relevant concentrations, affect neutrophils and immune suppression. This study was to determine whether halothane reliably inhibits inflammatory reaction and formalin induced pain behavior or does not. Rats were exposed to 100% oxygen (control) or halothane, respectively for 30 min and then 24 hr later five percent formalin test was assessed. The base values of the paw's diameter were obtained earlier, and then formalin induced edema was assessed by measuring diameters of the injected paws at 5 min, 1 hr, 4 hr and 24 hr after the injection. Nociceptive behavior was quantified by counting the number of times with the paw flinched at 5 min intervals for 60 min. The diameters of edema in the halothane group lessened more than those in the oxygen group at 1 and 24 hr in each following of the injection (p<0.05). The rats pre-administered with oxygen or halothane were similar appearances in nociceptive behaviors. It suggests that halothane anesthesia might inhibit slightly the inflammatory reaction with the formalin-induced edema but might not inhibit the formalin-induced pain behavior in the event of pre-administration halothane 24 hr earlier before the formalin test of rat.


Potential underestimation of pesticide suicide and its impact on secular trends in South Korea, 1991-2012.
Pesticide ingestion is among the most commonly used suicide methods globally. Underestimation of deaths by pesticide self-poisoning is thought to be common but under-researched. We investigated potential misclassification and underestimation of suicide by pesticide poisoning and their impact on trends in pesticide suicide in South Korea. We compared sociodemographic profiles between deaths certified as suicide, undetermined deaths and injuries by poisoning using pesticides, unspecified chemicals and medicines/alcohol in South Korea (1991-2012), and calculated similarity scores. Joinpoint regression analysis was used to examine time trends. The profiles of deaths classified as pesticide suicides showed generally similar patterns to those for undetermined deaths/injuries by poisoning using pesticides and suicides/undetermined deaths/injuries by poisoning using unspecified chemicals. Over the study period, the number of pesticide suicides (on average 2033 per year; range 623-3530) would increase by 15%, 23% and 31%, when assuming that 20%, 30% and 40% of deaths in alternative cause-of-death categories were misclassified pesticide suicides respectively. Age-standardised rates of pesticide suicide increased more than four times in 1991-2003 but fell by around 50% in 2003-2012. This trend did not change when the assumed proportion of misclassification was 20%, whereas the rapid increase in 2000-2003 would be lessened if misclassification occurred by 30% or more. Pesticide suicides may be underestimated in South Korea as some are possibly misclassified as deaths due to other causes; however, such underestimation would not substantially affect estimated secular trends.


Safety of an aqueous ethanolic extract of Labisia pumila as a novel food pursuant to Regulation (EU) 2015/2283.
Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on aqueous extract of Labisia pumila as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is a standardised hydroalcoholic extract from a dried whole plant (including roots) of L. pumila, mixed with maltodextrin (as a drying aid), and proposed by the applicant to be used as a food supplement in amounts up to 750 mg/day. The target population is the general adult population, except pregnant and lactating women. The major constituents of this NF are carbohydrates (up to 85.5%), with a smaller amount of proteins (up to 6.5%), gallic acid (up to 3.7%) and fats (up to 1.6%). The Panel considers that taking into account the composition of the NF and the proposed conditions of use, consumption of the NF is not nutritionally disadvantageous. The provided genotoxicity studies do not raise concerns about the genotoxicity of the NF. Based on the available toxicological data, the Panel considers an intake of up to 5 mg/kg body weight per day as safe. For the target population, this level corresponds to 350 mg/day, which is lower than the use level proposed by the applicant. The Panel concludes that the NF is safe for the target population up to 350 mg/day.


Cloning and characterization of a fourth human lysyl oxidase isoenzyme.
We report here the complete cDNA sequence and exon-intron organization of the human lysyl oxidase-like (LOXL)3 gene, a new member of the lysyl oxidase (LO) gene family. The predicted polypeptide is 753 amino acids in length, including a signal peptide of 25 residues. The C-terminal region, residues 529-729, contains a LO domain similar to those in the LOX (the first characterized LO isoenzyme), LOXL and LOXL2 polypeptides. It possesses the putative copper binding sequence, and the lysine and tyrosine residues that form the lysyltyrosyl quinone cofactor. The N-terminal region, which is similar to that in LOXL2 but not those in LOX and LOXL, contains four subregions similar to scavenger receptor cysteine-rich domains and a putative nuclear localization signal. Recombinant LOXL3, expressed in HT-1080 cells, was secreted into the culture medium but was not detected by immunofluorescence staining in nuclei. The LOXL3 mRNA is 3.1 kb in size and is expressed in many tissues, the highest levels among the tissues studied being seen in the placenta, heart, ovary, testis, small intestine and spleen.


Novel cell-free regeneration of bone using stem cell-derived growth factors.
Tissue engineering and regenerative medicine may now be used for the treatment of maxillofacial defects, but the related procedures have several limitations, including high capital investment, expensive cell culture techniques, and complicated safety and quality management issues. Stem cells secrete many cytokines that can affect cell mobilization and differentiation that accumulate in conditioned media. This study investigated the effects of stem cell-conditioned media from human bone marrow-derived mesenchymal stem cells (MSCs) on bone regeneration and its ability to induce endogeneous stem cell mobilization and bone regeneration. Human MSCs that were 70% to 80% confluent were refed with serum-free Dulbecco's modified Eagle medium, and the cell-cultured conditioned media were collected after 48 hours of incubation. The collected media were defined as cultured conditioned media from MSCs (MSC-CM). Rat bone-marrow-derived stem cells (rMSCs) were cultured with MSC-CM for 48 hours; then, cell mobilization and the expression of osteogenic-related genes were investigated. The presence of cytokines in MSC-CM was determined by enzyme-linked immunosorbent assay. Collagen sponge was then soaked in MSC-CM and grafted into rat calvarial bone defects. At 2 or 4 weeks after implantation of graft materials, implanted sections were evaluated by microcomputed tomography and histologic analysis. MSC-CM enhanced the migration, proliferation, and expression of osteogenic marker genes, such as alkaline phosphatase, osteocalcin, and Runx2, of rMSCs in vitro. Cytokines such as insulinlike growth factor-1, vascular endothelial growth factor, transforming growth factor-β1, and hepatocyte growth factor were present in the MSC-CM. MSC-CM significantly increased the migration and expression of osteogenic-related genes of rMSCs. Early bone regeneration in rat calvaria was also observed. These results suggest that MSC-CM may have potential for novel cell-free regeneration of bone.


Regulation of B versus T lymphoid lineage fate decision by the proto-oncogene LRF.
Hematopoietic stem cells in the bone marrow give rise to lymphoid progenitors, which subsequently differentiate into B and T lymphocytes. Here we show that the proto-oncogene LRF plays an essential role in the B versus T lymphoid cell-fate decision. We demonstrate that LRF is key for instructing early lymphoid progenitors in mice to develop into B lineage cells by repressing T cell-instructive signals produced by the cell-fate signal protein, Notch. We propose a new model for lymphoid lineage commitment, in which LRF acts as a master regulator of the cell's determination of B versus T lineage.


High intimal flap mobility assessed by intravascular ultrasound is associated with better short-term results after TEVAR in chronic aortic dissection.
Thoracic endovascular aortic repair (TEVAR) in chronic aortic dissection remains controversial. We analysed whether a high intimal flap mobility (IFM) of the dissection membrane has an impact on aortic remodelling after TEVAR in chronic Type B aortic dissection. Patients undergoing TEVAR with intravascular ultrasound (IVUS) were analysed and IFM was calculated. High IFM was defined as maximum flap amplitude >3 mm. For determining aortic remodelling, the degree of true lumen (TL) expansion was analysed in the last available follow-up CT. Fifty-two patients (63.6 ± 15.4 years) with a mean follow-up of 26.6 ± 20.7 months were analysed. The mobile flap group (n = 29) showed higher absolute TL expansion at the distal stent-graft (5.9 ± 3.1 vs. 3.3 ± 5.4 mm; p = 0.036) and a higher increase in TL diameter (18 ± 10 vs. 9 ± 15%; p = 0.017) compared to the non-mobile group (n = 23). Basic TEVAR-related outcome characteristics were comparable, but the mobile intimal flap group showed a lower re-intervention rate (3 vs. 8pts.; p = 0.032) in chronic dissections. High IFM in chronic Type B aortic dissection is linked to improved aortic remodelling and is associated with a lower re-intervention rate over time. IVUS assessment of IFM in chronic Type B aortic dissection might be helpful in identifying patients with better remodelling after TEVAR.


Selective separation of cadmium(ii) from zinc(ii) by a novel hydrophobic ionic liquid including an N,N,N',N'-tetrakis(2-methylpyridyl)-1,2-phenylenediamine-4-amido structure: a hard-soft donor combined method.
A novel hydrophobic ionic liquid including an N,N,N',N'-tetrakis(2-methylpyridyl)-1,2-phenylenediamine-4-amido structure ((IL-1,2-tpbd)+NTf2-) was successfully synthesized. (IL-1,2-tpbd)+NTf2- combined one amido (O-hard donor) and four pyridine (N-soft donor) groups. Its Cd2+ and Zn2+ separation behavior in nitric acid solution was investigated as a function of the extraction time, effect of pH etc. by dissolving (IL-1,2-tpbd)+NTf2- in a room temperature ionic liquid, 1-hexyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ((C6mim)+NTf2-). The extraction kinetics were fairly fast and could reach equilibrium within 4 h. When pHeq ≥ 1.8, the extraction percentage of Cd2+ and Zn2+ remained constant and the maximum separation factor was calculated as 12.78 at pHeq = 3.1; when pHeq < 1.8, the extraction percentage of Cd2+ and Zn2+ decreased drastically due to the protonation of the pyridine groups. Complete stripping of the extracted Cd2+ and Zn2+ from the ionic liquid phase into an aqueous phase was successfully achieved under highly acidic conditions ([HNO3] = 2 M) without adding any other metal complex forming agents. The extraction mechanism was summarized as a cation exchange due to the independence of nitrate ions in the extraction process. Additionally, the results of the slope analysis and UV-vis titration revealed the formation of a 1 : 2 complex. Furthermore, (IL-1,2-tpbd)+NTf2- showed a higher preference for Cd2+ even under the interference of various co-existing metal ions.


Ethylene-Related Gene Expression Networks in Wood Formation.
Thickening of tree stems is the result of secondary growth, accomplished by the meristematic activity of the vascular cambium. Secondary growth of the stem entails developmental cascades resulting in the formation of secondary phloem outwards and secondary xylem (i.e., wood) inwards of the stem. Signaling and transcriptional reprogramming by the phytohormone ethylene modifies cambial growth and cell differentiation, but the molecular link between ethylene and secondary growth remains unknown. We addressed this shortcoming by analyzing expression profiles and co-expression networks of ethylene pathway genes using the AspWood transcriptome database which covers all stages of secondary growth in aspen (Populus tremula) stems. ACC synthase expression suggests that the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is synthesized during xylem expansion and xylem cell maturation. Ethylene-mediated transcriptional reprogramming occurs during all stages of secondary growth, as deduced from AspWood expression profiles of ethylene-responsive genes. A network centrality analysis of the AspWood dataset identified EIN3D and 11 ERFs as hubs. No overlap was found between the co-expressed genes of the EIN3 and ERF hubs, suggesting target diversification and hence independent roles for these transcription factor families during normal wood formation. The EIN3D hub was part of a large co-expression gene module, which contained 16 transcription factors, among them several new candidates that have not been earlier connected to wood formation and a VND-INTERACTING 2 (VNI2) homolog. We experimentally demonstrated Populus EIN3D function in ethylene signaling in Arabidopsis thaliana. The ERF hubs ERF118 and ERF119 were connected on the basis of their expression pattern and gene co-expression module composition to xylem cell expansion and secondary cell wall formation, respectively. We hereby establish data resources for ethylene-responsive genes and potential targets for EIN3D and ERF transcription factors in Populus stem tissues, which can help to understand the range of ethylene targeted biological processes during secondary growth.


Successful efforts toward elimination iodine deficiency disorders in India.
Iodine deficiency (ID) is the world's single most important preventable cause of brain damage and mental retardation. Iodine deficiency disorders (IDDs) is a public health problem in 130 countries, affecting 13% of the world population. The simplest solution to prevent the IDD is to consume iodized common salt every day. In India, significant progress has been achieved toward elimination of IDD, in the last 30 years. Satisfactory levels of urinary iodine excretion and iodine content of salt have been documented by the research surveys conducted by research scientists. The results indicate that we are progressing toward elimination of IDD. IDD is due to a nutritional deficiency, which is prima-rily that of iodine, in soil and water. IDD is known to re-appear if the IDD Control Program is not sustained. To ensure that the population continues to have intake of adequate amount of iodine, there is a need of i) periodic surveys to assess the magnitude of the IDD with respect to impact of iodized salt (IS) intervention; ii) strengthening the health and nutrition education activities to create demand for IS and iii) development of a monitoring information system (MIS) for ensuring that the adequately IS is available to the beneficiaries.


Orientia tsutsugamushi: comprehensive analysis of the mobilome of a highly fragmented and repetitive genome reveals the capacity for ongoing lateral gene transfer in an obligate intracellular bacterium.
Obligate intracellular bacteria, or those only capable of growth inside other living cells, have limited opportunities for horizontal gene transfer with other microbes due to their isolated replicative niche. The human pathogen Ot, an obligate intracellular bacterium causing scrub typhus, encodes an unusually high copy number of a ~40 gene mobile genetic element that typically facilitates genetic transfer across microbes. This proliferated element is heavily degraded in Ot and previously assumed to be inactive. Here, we conducted a detailed analysis of this element in eight Ot strains and discovered two strains with at least one intact copy. This implies that the element is still capable of moving across Ot populations and suggests that the genome of this bacterium may be even more dynamic than previously appreciated. Our work raises questions about intracellular microbial evolution and sounds an alarm for gene-based efforts focused on diagnosing and combatting scrub typhus.


Mechanism of action of HTX-011: a novel, extended-release, dual-acting local anesthetic formulation for postoperative pain.
Obtaining consistent efficacy beyond 12-24 hours with local anesthetics, including extended-release formulations, has been a challenging goal. Inflammation resulting from surgery lowers the pH of affected tissues, reducing neuronal penetration of local anesthetics. HTX-011, an investigational, nonopioid, extended-release dual-acting local anesthetic combining bupivacaine and low-dose meloxicam, was developed to reduce postsurgical pain through 72 hours using novel extended-release polymer technology. Preclinical studies and a phase II clinical trial were conducted to confirm the mechanism of action of HTX-011. In a validated postoperative pain pig model and a phase II bunionectomy trial, the analgesic effects of HTX-011, oral meloxicam (preclinical only), liposomal bupivacaine (preclinical only) and saline placebo were evaluated. The optimal meloxicam:bupivacaine ratio for HTX-011 and the effect of HTX-011 on incisional tissue pH were also evaluated preclinically. Preclinical data demonstrate the ability of HTX-011 to address local tissue inflammation as demonstrated by a less acidic tissue pH, which was associated with potentiated and prolonged analgesic activity. In the phase II bunionectomy study, HTX-011 achieved superior and sustained pain relief through 72 hours after surgery compared with each component in the polymer. Preclinical animal and clinical results confirm that the low-dose meloxicam in HTX-011 normalizes the local pH in the incision, resulting in superior and synergistic analgesic activity compared with extended-release bupivacaine. HTX-011 represents an extended-release local anesthetic with a dual-acting mechanism of action that may provide an important advancement in the treatment of postoperative pain. NCT02762929.


Liposome formulations of hydrophobic drugs.
Here, we report methods of preparation for liposome formulations containing lipophilic drugs. In contrast to the encapsulation of water-soluble compounds into the entrapped aqueous volume of a liposome, drugs with lipophilic properties are incorporated into the phospholipid bilayer membrane. Water-soluble molecules, for example, cytotoxic or antiviral nucleosides can be transformed into lipophilic compounds by attachment of long alkyl chains, allowing their stable incorporation into liposome membranes and taking advantage of the high loading capacity lipid bilayers provide for lipophilic molecules. We created a new class of cytotoxic drugs by chemical transformation of the hydrophilic drugs cytosine-arabinoside (ara-C), 5-fluoro-deoxyuridine (5-FdU) and ethinylcytidine (ETC) into lipophilic compounds and their formulation in liposomes. The concept of chemical modification of water-soluble molecules by attachment of long alkyl chains and their stable incorporation into liposome bilayer membranes represent a very promising method for the development of new drugs not only for the treatment of tumors or infections, but also for many other diseases.


Proteomics technology in systems biology.
It has now become apparent that a full understanding of a biological process (e.g. a disease state) is only possible if all biomolecular interactions are taken into account. Systems biology works towards understanding the intricacies of cellular life through the collaborative efforts of biologists, chemists, mathematicians and computer scientists and recently, a number of laboratories around the world have embarked upon such research agendas. The fields of genomics and proteomics are foundational in systems biology studies and a great deal of research is currently being conducted in each worldwide. Moreover, many technological advances (particularly in mass spectrometry) have led to a dramatic rise in the number of proteomic studies over the past two decades. This short review summarizes a selection of technological innovations in proteomics that contribute to systems biology studies.


External monitor for buried free flaps in head and neck reconstructions.
Head and neck defects following oncological surgery must often be repaired with soft tissue and/or bone from other areas of the body. Significant loss of soft tissue requires flaps of sufficient bulk to adequately reconstruct the defect. Microvascular free tissue transfer is a good method for reconstructing even large defects following oncological surgery for head and neck cancer. Continuous post-operative monitoring of the perfusion of a free flap is vitally important to achieve not only a favourable outcome but also to decrease morbidity. Microvascular thrombosis occurs in 4% of the flaps and the best chance for flap salvage is offered by the earliest possible revision of the microanastomosis. Use of buried flaps in head and neck reconstruction makes monitoring particularly difficult and exteriorization of a segment of the flap permits a direct visualization. An original technique is presented for harvesting forearm free flaps with a secondary monitor skin paddle to externally check the status of the paddle and, when modified, can also be used for fibula and rectus abdominis flap.


[The evolution of the Th2 immune responses and its relationships with parasitic diseases and allergy].
A variety of links occur between parasites, particularly helminths, and allergic diseases--both common conditions of epidemiological importance in tropical regions. Although speculations are often made about the effects of parasitic diseases on the evolution of the immune system, the selective forces that have shaped the allergic response are unknown and probably include evolutionary mechanisms different to those traditionally reported. Helminths, infectious and antigenic sources that induce allergic-like responses, established themselves as parasites in organisms that already had cell groups related to the type 2 immunity. An essential component in the relationship between helminths and their hosts is that the former induce immunosuppression, creating a kind of balance that allows the survival of both. The development of this equilibrium undoubtedly includes adaptations in both organisms, and the survival of the parasite is the result of (a) acquiring immune suppressor mechanisms and (b) finding hosts with lower intensity of the type 2 response. This in turn suggests that although helminth infections have influenced the formation of type 2 immunity, they have not been an important selective force in the particular case of allergic response. The latter is more related to an exaggerated Th2/IgE response.


Automated ribotyping of vancomycin-resistant Enterococcus faecium isolates.
Vancomycin-resistant Enterococcus faecium (VREF) strains represent an important threat in hospital infections in the United States and are found at high frequencies in both the community and farm animals in Europe. We evaluated automated ribotyping for interlaboratory reproducibility by using the restriction enzymes EcoRI and BamHI and compared ribotyping to both amplification of fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) to assess its discriminatory power and capacity for the identification of epidemiologically important strains. Of 19 (EcoRI) and 16 (BamHI) isolates tested in duplicate in two laboratories, 18 (95%) and 16 (100%), respectively, showed reproducible ribotypes. These high reproducibility rates were obtained only after manual refinement of the automated fingerprint analysis. A group of 49 VREF strains initially selected to represent 32 distinct AFLP types were separated into 28 EcoRI ribotypes, 25 BamHI ribotypes, and 28 sequence types. Ribotyping with EcoRI and BamHI was able to discern the host-specific genogroups recently disclosed by AFLP typing and MLST and to distinguish most strains containing the esp gene, a marker specific for strains causing hospital outbreaks. An expandable ribotype identification library was created. We recommend EcoRI as the enzyme of choice for automated ribotyping of VREF strains. Given the high level of discrimination of VREF strains, the high rate of interlaboratory reproducibility, and the potential for the identification of epidemiologically important genotypes, automated ribotyping appears to be a very valuable approach for characterizing VREF strains.


Portopulmonary hypertension in liver transplant candidates.
Pulmonary vascular disorders including portopulmonary hypertension (PoPHT) are among the common complications of liver disease and are prognostically significant. Survival is very low without medical treatment and liver transplantation. With advances in medical therapy for elevated pulmonary artery pressure (PAP) and liver transplant surgery, survival of patients with PoPHT and advanced liver disease is significantly improved. Because of the prognostic significance of PoPHT and the limited donor pool, a comprehensive preoperative cardio-pulmonary assessment is of great importance in cirrhotic patients prior to transplant surgery. Therefore, a detailed transthoracic Doppler echocardiographic examination must be an essential component of this evaluation. Patients with mild PoPHT can safely undergo liver transplant surgery. In cases of moderate to severe PoPHT, right heart catheterization (RHC) should be performed. In patients with moderate to severe PoPHT on RHC (mean PAP 35-45 mmHg), vasodilator therapy should be attempted. Liver transplantation should be encouraged in cases that demonstrate a positive response. Bridging therapy with specific pulmonary arterial hypertension treatment agents should be considered until the transplant surgery and should be continued during the peri- and post-operative periods as needed.


Detergent-resistant membrane domains but not the proteasome are involved in the misfolding of a PrP mutant retained in the endoplasmic reticulum.
Inherited prion diseases are neurodegenerative pathologies related to genetic mutations in the prion protein (PrP) gene, which favour the conversion of PrP(C) into a conformationally altered pathogenic form, PrP(Sc). The molecular basis of PrP(C)/PrP(Sc) conversion, the intracellular compartment where it occurs and how this process leads to neurological dysfunction are not yet known. We have studied the intracellular synthesis, degradation and localization of a PrP mutant associated with a genetic form of Creutzfeldt-Jakob disease (CJD), PrPT182A, in transfected FRT cells. PrPT182A is retained in the endoplasmic reticulum (ER), is mainly associated with detergent-resistant microdomains (DRMs) and is partially resistant to proteinase K digestion. Although an untranslocated form of this mutant is polyubiquitylated and undergoes ER-associated degradation, the proteasome is not responsible for the degradation of its misfolded form, suggesting that it does not have a role in the pathogenesis of inherited diseases. On the contrary, impairment of PrPT182A association with DRMs by cholesterol depletion leads to its accumulation in the ER and substantially increases its misfolding. These data support the previous hypothesis that DRMs are important for the correct folding of PrP and suggest that they might have a protective role in pathological scrapie-like conversion of PrP mutants.


Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering.
Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter k D , indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion.


Optogenetic regulation of endogenous proteins.
Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.


Critical difference applied to exercise-induced salivary testosterone and cortisol using enzyme-linked immunosorbent assay (ELISA): distinguishing biological from statistical change.
Due to its noninvasive, convenient, and practical nature, salivary testosterone (sal-T) and cortisol (sal-C) are frequently used in a clinical and applied setting. However, few studies report biological and analytical error and even fewer report the 'critical difference' which is the change required before a true biological difference can be claimed. It was hypothesized that (a) exercise would result in a statistically significant change in sal-C and sal-T and (b) the exercise-induced change would be within the critical difference for both salivary hormones. In study 1, we calculated the critical difference of sal-T and sal-C of 18 healthy adult males aged 23.2 ± 3.0 years every 60 min in a seated position over a 12-h period (08:00-20:00 hours [study 1]). As proof-of-concept, sal-C and sal-T was also obtained pre and at 5 and 60 min post a maximal exercise protocols in a separate group of 17 healthy males (aged 20.1 ± 2.8 years [study 2]). The critical difference of sal-T calculated as 90 %. For sal-C, the critical difference was 148 % (study 1). Maximal exercise was associated with a statistically significant (p < 0.05) changes in sal-T and sal-C. However, these changes were all within the critical difference range. Results from this investigation indicate that a large magnitude of change for sal-C and sal-T is required before a biologically significant mean change can be claimed. Studies utilizing sal-T and sal-C should appreciate the critical difference of these measures and assess the biological significance of any statistical changes.


Creating high reliability in health care organizations.
The objective of this paper was to present a comprehensive approach to help health care organizations reliably deliver effective interventions. Reliability in healthcare translates into using valid rate-based measures. Yet high reliability organizations have proven that the context in which care is delivered, called organizational culture, also has important influences on patient safety. MODEL FOR IMPROVEMENT: Our model to improve reliability, which also includes interventions to improve culture, focuses on valid rate-based measures. This model includes (1) identifying evidence-based interventions that improve the outcome, (2) selecting interventions with the most impact on outcomes and converting to behaviors, (3) developing measures to evaluate reliability, (4) measuring baseline performance, and (5) ensuring patients receive the evidence-based interventions. The comprehensive unit-based safety program (CUSP) is used to improve culture and guide organizations in learning from mistakes that are important, but cannot be measured as rates. We present how this model was used in over 100 intensive care units in Michigan to improve culture and eliminate catheter-related blood stream infections--both were accomplished. Our model differs from existing models in that it incorporates efforts to improve a vital component for system redesign--culture, it targets 3 important groups--senior leaders, team leaders, and front line staff, and facilitates change management-engage, educate, execute, and evaluate for planned interventions.


Screening for cardiovascular risk in asymptomatic users of the primary health care network in Lebanon, 2012-2013.
In 2012, the Ministry of Public Health in Lebanon piloted a service of multifactorial cardiovascular screening in the publicly subsidized Primary Health Care (PHC) Network. We present an epidemiological analysis of data produced during this pilot to justify the inclusion of this service in the package of essential services offered through PHC and to present a preliminary cardiovascular risk profile in an asymptomatic population. A total of 4,205 participants (two-thirds of which were women) aged at least 40 years and reportedly free from diabetes, hypertension, dyslipidemia, and cardiovascular disease (CVD) were screened. The screening protocol used a questionnaire and direct measurements to assess 5 modifiable cardiovascular risk factors; total cardiovascular risk score was calculated according to a paper-based algorithm developed by the World Health Organization and the International Society of Hypertension. Approximately 25% of the sample displayed metabolic impairments (11% for impaired blood glucose metabolism and 17% for impaired systolic blood pressure), and 6.6% were classified at total cardiovascular risk of 10% or more. Just over one-quarter of the sample was obese, almost half had a substantially elevated waist circumference, and 41% were smokers. Men were significantly more likely to screen positive for metabolic impairment than women, and women were more likely to be obese. The implementation of a multifactorial screening for CVD among asymptomatic subjects detected a substantial proportion of previously undiagnosed cases of high metabolic risk, people who could now be referred to optimal medical follow-up.


Effects of hypercholesterolaemia on physiological recruitment of coronary vascular reserve in swine.
1. The endothelium participates in the regulation of coronary vascular tone. As evidence exists from studies performed on epicardial vessels that hypercholesterolaemia impairs endothelial function, we tested the hypothesis that hypercholesterolaemia impairs coronary vascular reserve in an intact animal. 2. Domestic swine, maintained on a regular (n = 9) or a 2% high-cholesterol (n = 9) diet for 3 months were instrumented with a catheter in the left atrium for microsphere injection, a catheter in the anterior interventricular vein for venous sampling and an 82% stenosis in the left anterior descending artery. Papaverine was used to determine coronary vascular reserve. Regional coronary flow as reflected by perfusion (microsphere measurement), lactate consumption, oxygen consumption and haemodynamics were obtained at baseline, after 10 mg of papaverine and after atrial pacing at a rate of 120 beats/min and 150 beats/min. 3. Cholesterol was elevated in animals on the high cholesterol diet (350 +/- 50 mg/dl versus 99 +/- 10 mg/dl, P < 0.001). Baseline haemodynamics were similar between groups. Baseline transmural flow and its augmentation with papaverine were comparable in the two groups in the control (circumflex) and stenosed (left anterior descending artery) zones. In both groups, perfusion increased in the control zone in response to increased oxygen demand, whereas in the stenosis zone no increase was observed in either group (P not significant for normal versus high cholesterol diet). Endocardial flow reserve in the stenosis zone was exhausted in both groups. Epicardial flow in the stenosis zone increased significantly in the normal (P < 0.02) but not in the hypercholesterolaemic animals (P not significant). 4. The endocardial/epicardial ratio in the control zone at baseline revealed greater endocardial dominance in the normal compared with the hypercholesterolaemic animals (1.35 versus 1.10, P < 0.01). With papaverine, similar ratios indicated a similar reserve potential in both groups. During increased oxygen demand, normal animals continued to demonstrate endocardial dominance whereas it diminished in the hypercholesterolaemic group. In the stenosis zone, endocardial blood flow dominated at baseline in the normal animals and to a lesser extent in the hypercholesterolaemic animals (1.30 versus 1.10, P = 0.10). During increased oxygen demand, endocardial dominance decreased significantly in both groups of animals; however, it remained greater than 1.0 only in the normal animals. 5. Exposure to elevated cholesterol levels did not impair an animal's ability to augment coronary blood flow in response to an increase in oxygen demand. In contrast to this lack of effect on recruitment of coronary reserve, regional coronary blood flow was altered in the hypercholesterolaemic animals.


Ongoing unmet needs in treating estrogen receptor-positive/HER2-negative metastatic breast cancer.
Estrogen receptor-positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) advanced or metastatic breast cancer (MBC) is the most common MBC subtype and currently remains incurable, with a median overall survival of 24.8 months (95% confidence interval, 21.3-30.3). Common sites of metastases are bone, viscera, and brain, causing significant symptoms that negatively affect patient functioning, quality of life (QoL), and work productivity. Guidelines state that endocrine therapy (ET) is preferable to chemotherapy as first-line treatment for patients with ER+ MBC, regardless of limited visceral metastases, unless rapid tumor response is required or ET resistance is suspected. Although response rates up to 40% have been reported for first-line MBC treatment, the majority of initial responders eventually develop ET resistance. Notwithstanding the steep decline in efficacy between first and later lines of ET, some patients may receive chemotherapy earlier than necessary. Although new treatments have been approved for patients with ER+/HER2- advanced or MBC in the past decade, neither survival nor QoL appear to have improved significantly. Thus, there remain significant unmet needs for this patient population, including improved survival, maintaining or improving patient QoL, and emphasizing the importance of treatment selection to assist healthcare practitioners managing patient care. In this review, we identify current challenges and unmet needs in this patient population, review cutting-edge treatments, and provide clinically relevant suggestions for treatment selection that can optimize outcomes and patients' health-related QoL.


Cost and cost-effectiveness of the COMBINE study in alcohol-dependent patients.
The COMBINE (Combined Pharmacotherapies and Behavioral Intervention) clinical trial recently evaluated the efficacy of medications, behavioral therapies, and their combinations for the outpatient treatment of alcohol dependence. The costs and cost-effectiveness of these combinations are unknown and of interest to clinicians and policy makers. To evaluate the costs and cost-effectiveness of the COMBINE Study interventions after 16 weeks of treatment. A prospective cost and cost-effectiveness study of a randomized controlled clinical trial. Eleven US clinical sites. One thousand three hundred eighty-three patients having a diagnosis of primary alcohol dependence. The study included 9 treatment groups; 4 groups received medical management for 16 weeks with naltrexone, 100 mg/d, acamprosate, 3 g/d, or both, and/or placebo; 4 groups received the same therapy as mentioned earlier with combined behavioral intervention; and 1 group received combined behavioral intervention only. Incremental cost per percentage point increase in percentage of days abstinent, incremental cost per patient of avoiding heavy drinking, and incremental cost per patient of achieving a good clinical outcome. On the basis of the mean values of cost and effectiveness, 3 interventions are cost-effective options relative to the other interventions for all 3 outcomes: medical management (MM) with placebo ($409 per patient), MM plus naltrexone therapy ($671 per patient), and MM plus combined naltrexone and acamprosate therapy ($1003 per patient). To our knowledge, this is only the second prospective cost-effectiveness study with a randomized controlled clinical trial design that has been performed for the treatment of alcohol dependence. Focusing only on effectiveness, MM-naltrexone-acamprosate therapy is not significantly better than MM-naltrexone therapy. However, considering cost and cost-effectiveness, MM-naltrexone-acamprosate therapy may be a better choice, depending on whether the cost of the incremental increase in effectiveness is justified by the decision maker.


Continuous micro-vortex-based nanoparticle manipulation via focused surface acoustic waves.
Despite increasing demand in the manipulation of nanoscale objects for next generation biological and industrial processes, there is a lack of methods for reliable separation, concentration and purification of nanoscale objects. Acoustic methods have proven their utility in contactless manipulation of microscale objects mainly relying on the acoustic radiation effect, though the influence of acoustic streaming has typically prevented manipulation at smaller length scales. In this work, however, we explicitly take advantage of the strong acoustic streaming in the vicinity of a highly focused, high frequency surface acoustic wave (SAW) beam emanating from a series of focused 6 μm substrate wavelength interdigital transducers patterned on a piezoelectric lithium niobate substrate and actuated with a 633 MHz sinusoidal signal. This streaming field serves to focus fluid streamlines such that incoming particles interact with the acoustic field similarly regardless of their initial starting positions, and results in particle displacements that would not be possible with a travelling acoustic wave force alone. This streaming-induced manipulation of nanoscale particles is maximized with the formation of micro-vortices that extend the width of the microfluidic channel even with the imposition of a lateral flow, occurring when the streaming-induced flow velocities are an order of magnitude larger than the lateral one. We make use of this acoustic streaming to demonstrate the continuous and differential focusing of 100 nm, 300 nm and 500 nm particles.


Evidence of early ultrastructural photoreceptor abnormalities in light-induced retinal degeneration using spectral domain optical coherence tomography.
To study spatiotemporal in vivo changes in retinal morphology and quantify thickness of retinal layers in a mouse model of light-induced retinal degeneration using spectral domain optical coherence tomography (SD-OCT). BALB/c mice were exposed to 5000 lux of constant light for 3 h. SD-OCT images were taken 3 h, 24 h, 3 days, 1 week and 1 month after light exposure and were compared with histology at the same time points. SD-OCT images were also taken at 0, 1 and 2 h after light exposure in order to analyse retinal changes at the earliest time points. The thickness of retinal layers was measured using the Bioptigen software InVivoVue Diver. SD-OCT demonstrated progressive outer retinal thinning. 3 h after light exposure, the outer nuclear layer converted from hyporeflective to hyper-reflective. At 24 h, outer retinal bands and nuclear layer demonstrated similar levels of hyper-reflectivity. Significant variations in outer retinal thickness, vitreous opacities and retinal detachments occurred within days of injury. Thinning of the retina was observed at 1 month after injury. It was also determined that outer nuclear layer changes precede photoreceptor segment structure disintegration and the greatest change in segment structure occurs between 1 and 2 h after light exposure. Longitudinal SD-OCT reveals intraretinal changes that cannot be observed by histopathology at early time points in the light injury model.


Vaccination directed against the human endogenous retrovirus-K (HERV-K) gag protein slows HERV-K gag expressing cell growth in a murine model system.
Human endogenous retroviruses (HERVs) are remnants of ancestral infections and chromosomally integrated in all cells of an individual, are transmitted only vertically and are defective in viral replication. However enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed inter-alia in HIV-infected individuals and tumor patients. Therefore HERV-K might serve as a tumor-specific antigen or even as a constant target for the development of an HIV vaccine. To verify our hypothesis, we tested the immunogenicity of HERV-K Gag by using a recombinant vaccinia virus (MVA-HKcon) expressing the HERV-K Gag protein and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) and the HERV-K Gag protein (RLZ-HKGag cells). Subcutaneous application of RLZ-HKGag cells into syngenic BALB/c mice resulted in the formation of local tumors in MVA vaccinated mice. MVA-HKcon vaccination reduced the tumor growth. Furthermore, intravenous injection of RLZ-HKGag cells led to the formation of pulmonary metastases. Vaccination of tumor-bearing mice with MVA-HKcon drastically reduced the number of pulmonary RLZ-HKGag tumor nodules compared to vaccination with wild-type MVA. The data demonstrate that HERV-K Gag is a useful target for vaccine development and might offer new treatment opportunities for cancer patients.


Udock, the interactive docking entertainment system.
Protein-protein interactions play a crucial role in biological processes. Protein docking calculations' goal is to predict, given two proteins of known structures, the associate conformation of the corresponding complex. Here, we present a new interactive protein docking system, Udock, that makes use of users' cognitive capabilities added up. In Udock, the users tackle simplified representations of protein structures and explore protein-protein interfaces' conformational space using a gamified interactive docking system with on the fly scoring. We assumed that if given appropriate tools, a naïve user's cognitive capabilities could provide relevant data for (1) the prediction of correct interfaces in binary protein complexes and (2) the identification of the experimental partner in interaction among a set of decoys. To explore this approach experimentally, we conducted a preliminary two week long playtest where the registered users could perform a cross-docking on a dataset comprising 4 binary protein complexes. The users explored almost all the surface of the proteins that were available in the dataset but favored certain regions that seemed more attractive as potential docking spots. These favored regions were located inside or nearby the experimental binding interface for 5 out of the 8 proteins in the dataset. For most of them, the best scores were obtained with the experimental partner. The alpha version of Udock is freely accessible at http://udock.fr.


Cellular evidence of reverse cardiac remodeling induced by cardiac resynchronization therapy.
Left ventricular assist devices (LVADs) induce reverse cardiac remodeling by reducing myocyte size and collagen deposition. On the other hand, cardiac resynchronization therapy (CRT) induces reverse cardiac remodeling by improving electromechanical synchronization. The clinical and structural changes produced by CRT in failing myocardium are known, but whether these changes are accompanied by reverse cellular remodeling is unknown. A total of 12 patients with chronic heart failure (CHF) who underwent CRT and 15 patients who had LVAD therapy as clinically indicated and 8 healthy controls were compared. Demographics, echocardiographic data, and histologic samples from myocardial biopsies were analyzed and compared among groups. The authors found significant increases in myocyte size, myocardial fibrosis, and inflammation in both CHF groups who underwent CRT or LVAD, compared with healthy controls. After CRT or LVAD therapy, a significant decrease in myocyte size and tumor necrosis factor α (TNF-α) expression compared with healthy controls (P < .05) was found. In the CRT group, 6 of 8 patients demonstrated reduction in myocyte size and interstitial fibrosis. In addition, there was a decrease in myocyte size by 13%, total collagen by 27% and TNF-α by 49% in the CRT group vs 28%, 45%, and 45% in the LVAD group. CRT produces cellular reverse remodeling in failing human hearts that are comparable with those produced by LVAD therapy.


Pathogen-secreted proteases activate a novel plant immune pathway.
Mitogen-activated protein kinase (MAPK) cascades play central roles in innate immune signalling networks in plants and animals. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive. Here we report that pathogen-secreted proteases activate a previously unknown signalling pathway in Arabidopsis thaliana involving the Gα, Gβ, and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of an MAPK cascade. In this pathway, receptor for activated C kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G-protein signalling to downstream activation of an MAPK cascade. The protease-G-protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signalling pathways such as that elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to an MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the new protease-mediated immune signalling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.


Assessing the Risk of Metals and Their Mixtures in the Antarctic Nearshore Marine Environment with Diffusive Gradients in Thin-Films.
Robust environmental assessments and contaminant monitoring in Antarctic near-shore marine environments need new techniques to overcome challenges presented by a highly dynamic environment. This study outlines an approach for contaminant monitoring and risk assessment in Antarctic marine conditions using diffusive gradients in thin-films (DGT) coupled to regionally specific ecotoxicology data and environmental quality standards. This is demonstrated in a field study where DGT samplers were deployed in the near-shore marine environment of East Antarctica around the operational Casey station and the abandoned Wilkes station to measure the time-averaged biologically available fraction of metal contaminants. The incorporation of DGT-labile concentrations to reference toxicity mixture models for three Antarctic organisms predicted low toxic effects (<5% effect to the growth or development of each organism). The comparison of metal concentrations to the Australian and New Zealand default water quality guideline values (WQGVs) showed no marine site exceeding the WQGVs for 95% species protection. However, all sites exceeded the 99% WQGVs due to copper concentrations that are likely of geogenic origin (i.e., not from anthropogenic sources). This study provides evidence supporting the use of the DGT technique to monitor contaminants and assess their environmental risk in the near-shore marine environment of Antarctica.


Developing a community-centred malaria early warning system based on indigenous knowledge: Gwanda District, Zimbabwe.
Malaria continues to be a major public health problem in Sub-Saharan Africa despite efforts that have been made to prevent and control the disease for many decades. The knowledge on prediction and occurrence of the disease that communities acquired over the years has not been seriously considered in control programmes. This article reports on studies that aimed to integrate indigenous knowledge systems (IKS) on malaria into the malaria control programme in Gwanda District, Zimbabwe. The studies were conducted over a 3-year period. Data were collected using participatory rural appraisals, key informant interviews, household interviews and workshops in three wards (11, 15 and 18) with the highest malaria incidence in Gwanda District. Disease livelihoods calendars produced by the community showed their knowledge on the relationship between malaria, temperature and rainfall, and thus an understanding of malaria as a hazard. Volunteer IKS experts willing to record the indigenous environmental indicators for the occurrence of malaria in the study area were identified by the communities. Indigenous environmental indicators for the occurrence of malaria were classified as insects, plant phenology, animals, weather and cosmological indicators. Plant phenology was emphasised more than the other indicators. A community-based malaria early warning system model was developed using the identified IKS indicators in two of the wards using the ward health team as an entry point to the health system. In the model, data on indicators were collected at the village level by IKS experts, analysed at ward level by IKS experts and health workers and relayed to the district health team.


Monitoring advances including consent: learning from COVID-19 trials and other trials running in UKCRC registered clinical trials units during the pandemic.
The COVID-19 pandemic has affected how clinical trials are managed, both within existing portfolios and for the rapidly developed COVID-19 trials. Sponsors or delegated organisations responsible for monitoring trials have needed to consider and implement alternative ways of working due to the national infection risk necessitating restricted movement of staff and public, reduced clinical staff resource as research staff moved to clinical areas, and amended working arrangements for sponsor and sponsor delegates as staff moved to working from home.Organisations have often worked in isolation to fast track mitigations required for the conduct of clinical trials during the pandemic; this paper describes many of the learnings from a group of monitoring leads based in United Kingdom Clinical Research Collaboration (UKCRC) Clinical Trials Unit (CTUs) within the UK.The UKCRC Monitoring Task and Finish Group, comprising monitoring leads from 9 CTUs, met repeatedly to identify how COVID-19 had affected clinical trial monitoring. Informed consent is included as a specific issue within this paper, as review of completed consent documentation is often required within trial monitoring plans (TMPs). Monitoring is defined as involving on-site monitoring, central monitoring or/and remote monitoring.Monitoring, required to protect the safety of the patients and the integrity of the trial and ensure the protocol is followed, is often best done by a combination of central, remote and on-site monitoring. However, if on-site monitoring is not possible, workable solutions can be found using only central or central and remote monitoring. eConsent, consent by a third person, or via remote means is plausible. Minimising datasets to the critical data reduces workload for sites and CTU staff. Home working caused by COVID-19 has made electronic trial master files (TMFs) more inviting. Allowing sites to book and attend protocol training at a time convenient to them has been successful and worth pursuing for trials with many sites in the future.The arrival of COVID-19 in the UK has forced consideration of and changes to how clinical trials are conducted in relation to monitoring. Some developed practices will be useful in other pandemics and others should be incorporated into regular use.


Connecting the mechanisms of T-cell regulation: dendritic cells as the missing link.
A variety of different molecular mechanisms have been proposed to explain the suppressive action of regulatory T cells, including the production of anti-inflammatory cytokines, negative costimulatory ligands, indoleamine 2,3-dioxygenase-mediated tryptophan catabolism, CD73-mediated adenosine generation, and downregulation of antigen-presenting cells. Until now it has been unclear how important each of these different mechanisms might be and how they are coordinated. In this review, we examine the hypothesis that it is the interaction between regulatory T cells and dendritic cells that creates a local microenvironment depleted of essential amino acids and rich in adenosine that leads to the amplification of a range of different tolerogenic signals. These signals are all eventually integrated by mammalian target of rapamycin inhibition, which enables the induction of new forkhead box protein 3-expressing Tregs. If correct, this provides a molecular explanation for the in vivo phenomena of linked suppression and infectious tolerance.


Blood mercury levels in autism spectrum disorder: Is there a threshold level?
Mercury (Hg) may significantly impact the pathogenesis of autism spectrum disorders (ASDs). Lab results generated by Vitamin Diagnostics (CLIA-approved) from 2003-2007, were examined among subjects diagnosed with an ASD (n=83) in comparison to neurotypical controls (n=89). Blood Hg levels were determined by analyzing Hg content in red blood cells (RBC) using cold vapor analysis, and consistent Hg measurements were observed between Vitamin Diagnostics and the University of Rochester. Adjusted (age, gender, and date of collection) mean Hg levels were 1.9-fold significantly (P<.0001) increased among subjects diagnosed with an ASD (21.4 microg/L) in comparison to controls (11.4 microg/L). Further, an adjusted significant (P<.0005) threshold effect >15 microg/L) was observed for Hg levels on the risk of a subject being diagnosed with an ASD in comparison to controls (odds ratio=6.4). The weight of scientific evidence supports Hg as a causal factor in subjects diagnosed with an ASD.


Burden of Cardiovascular Disease among Multi-Racial and Ethnic Populations in the United States: an Update from the National Health Interview Surveys.
The study aimed to provide new evidence of health disparities in cardiovascular disease (CVD) and diabetes mellitus (DM), and to examine their associations with lifestyle-related risk factors across the U.S. multi-racial and ethnic groups. The analysis included a randomized population sample of 68,321 subjects aged ≥18 years old who participated in the U.S. 2012 and 2013 National Health Interview Surveys. Hypertension, coronary heart disease (CHD), stroke, and DM were classified according to participants' self-report of physician diagnosis. Assessments of risk factors were measured using standard survey instruments. Associations of risk factors with hypertension, CHD, stroke, and DM were analyzed using univariable and multivariable analysis methods. Non-Hispanic (NH)-Blacks had significantly higher odds of hypertension, while Hispanics had significantly lower odds of hypertension, and NH-Asians and Hispanics had significantly lower odds of stroke than NH-Whites (p < 0.001). All minority groups, NH-Blacks, NH-Asians, and Hispanics had significantly higher odds of DM, but they had significantly lower odds of CHD than NH-Whites (p < 0.001). Increased body weight, cigarette smoking, and physical inactivity were significantly associated with increased odds of hypertension, CHD, stroke, and DM (p < 0.001). However, the strengths of associations between lifestyle-related factors and the study outcomes were different across racial and ethnic groups. NH-Asians with BMI ≥30 kg/m(2) had the highest odds ratios (OR, 95% CI) for hypertension (5.37, 4.01-7.18), CHD (2.93, 1.90-4.52), and stroke (2.23, 1.08-4.61), and had the second highest odd ratios for DM (3.78, 2.68-5.35) than NH-Whites, NH-Blacks, and Hispanics. CVD and DM disproportionately affect the U.S. multi-racial and ethnic population. Although lifestyle-related risk factors are significantly associated with increased odds of CVD and DM, the magnitudes of these associations are different by race and ethnicity.


The molecular significance of amyloid beta-peptide for Alzheimer's disease.
Alzheimer's disease is the most common form of dementia. Although the majority of the cases occur sporadically, in some rare cases Alzheimer's disease is genetically inherited. Pathologically, Alzheimer's disease is characterized by the accumulation of senile plaques with-in the extracellular space of brain regions known to be important for intellectual functions. In addition to senile plaques, deposits of identical biochemical composition are found in the walls of meningeal and cerebral blood vessels. Senile plaques are surrounded by degenerating neurons indicating a toxic interference of amyloid plaques with neurons. The major component of senile plaques is the 4kDa amyloid beta-peptide. This peptide has been shown to exhibit neurotoxic properties when added to cultured neurons, or injected into rat brains. Amyloid beta-peptide is derived from a high molecular weight precursor, the beta-amyloid precursor protein, by proteolytic processing. Mutations responsible for the early onset of Alzheimer's disease in some families are found within the gene coding for the beta-amyloid precursor protein. These mutations strongly influence the generation of amyloid beta-peptide resulting in a significant overproduction of the peptide or the generation of elongated forms which are known to aggregate and precipitate much faster. Moreover, mutations found in other genes known to cause early onset of Alzheimer's disease have been shown to interfere directly with the production or precipitation of amyloid beta-peptide.


The administration of intranasal live attenuated influenza vaccine induces changes in the nasal microbiota and nasal epithelium gene expression profiles.
Viral infections such as influenza have been shown to predispose hosts to increased colonization of the respiratory tract by pathogenic bacteria and secondary bacterial pneumonia. To examine how viral infections and host antiviral immune responses alter the upper respiratory microbiota, we analyzed nasal bacterial composition by 16S ribosomal RNA (rRNA) gene sequencing in healthy adults at baseline and at 1 to 2 weeks and 4 to 6 weeks following instillation of live attenuated influenza vaccine or intranasal sterile saline. A subset of these samples was submitted for microarray host gene expression profiling. We found that live attenuated influenza vaccination led to significant changes in microbial community structure, diversity, and core taxonomic membership as well as increases in the relative abundances of Staphylococcus and Bacteroides genera (both p < 0.05). Hypergeometric testing for the enrichment of gene ontology terms in the vaccinated group reflected a robust up-regulation of type I and type II interferon-stimulated genes in the vaccinated group relative to controls. Translational murine studies showed that poly I:C administration did in fact permit greater nasal Staphylococcus aureus persistence, a response absent in interferon alpha/beta receptor deficient mice. Collectively, our findings demonstrate that although the human nasal bacterial community is heterogeneous and typically individually robust, activation of a type I interferon (IFN)-mediated antiviral response may foster the disproportionate emergence of potentially pathogenic species such as S. aureus. This study was registered with Clinicaltrials.gov on 11/3/15, NCT02597647 .


Gastric ulcer diagnosed on a plain abdominal radiograph.
On occasion gastric ulcers may be recognized on plain abdominal radiographs. A case in which a gastric ulcer was diagnosed on the plain abdominal radiograph, illustrating the importance of careful scrutiny of the plain film in all cases of acute abdomen, is presented. The existence of the lesser curvature ulcer was confirmed on barium contrast studies.


Physician privacy concerns when disclosing patient data for public health purposes during a pandemic influenza outbreak.
Privacy concerns by providers have been a barrier to disclosing patient information for public health purposes. This is the case even for mandated notifiable disease reporting. In the context of a pandemic it has been argued that the public good should supersede an individual's right to privacy. The precise nature of these provider privacy concerns, and whether they are diluted in the context of a pandemic are not known. Our objective was to understand the privacy barriers which could potentially influence family physicians' reporting of patient-level surveillance data to public health agencies during the Fall 2009 pandemic H1N1 influenza outbreak. Thirty seven family doctors participated in a series of five focus groups between October 29-31 2009. They also completed a survey about the data they were willing to disclose to public health units. Descriptive statistics were used to summarize the amount of patient detail the participants were willing to disclose, factors that would facilitate data disclosure, and the consensus on those factors. The analysis of the qualitative data was based on grounded theory. The family doctors were reluctant to disclose patient data to public health units. This was due to concerns about the extent to which public health agencies are dependable to protect health information (trusting beliefs), and the possibility of loss due to disclosing health information (risk beliefs). We identified six specific actions that public health units can take which would affect these beliefs, and potentially increase the willingness to disclose patient information for public health purposes. The uncertainty surrounding a pandemic of a new strain of influenza has not changed the privacy concerns of physicians about disclosing patient data. It is important to address these concerns to ensure reliable reporting during future outbreaks.


Tracking immunodynamics by identification of S-G2/M-phase T cells in human peripheral blood.
The ready availability of human blood makes it the first choice for immuno-monitoring. However, this has been largely confined to static metrics, particularly resting T cell phenotypes. Conversely, dynamic assessments have mostly relied on cell stimulation in vitro which is subject to multiple variables. Here, immunodynamic insights from the peripheral blood are shown to be obtainable by applying a revised approach to cell-cycle analysis. Specifically, refined flow cytometric protocols were employed, assuring the reliable quantification of T cells in the S-G2/M phases of the cell-cycle (collectively termed "T Double S" for T cells in S-phase in Sanguine: in short "TDS" cells). Without protocol refinement, TDS could be either missed, as most of them layed out of the conventional lymphocyte gates, or confused with cell doublets artefactually displaying high DNA-content. To illustrate the nature of TDS cells, and their relationship to different immunodynamic scenarios, we examined them in healthy donors (HD); infectious mononucleosis (IM) patients versus asymptomatic EBV+ carriers; and recently-diagnosed T1D patients. TDS were reproducibly more abundant among CD8+ T cells and a defined subset of T-regulatory CD4+ T cells, and were substantially increased in IM and a subset of T1D patients. Of note, islet antigen-reactive TDS cell frequencies were associated with an aggressive T cell effector phenotype, suggesting that peripheral blood can reflect immune events within tissues in T1D, and possibly in other organ-specific autoimmune diseases. Our results suggest that tracking TDS cells may provide a widely applicable means of gaining insight into ongoing immune response dynamics in a variety of settings, including tissue immunopathologies where the peripheral blood has often not been considered insightful.


Regulation of endothelial progenitor cell function by micrornas.
After cardiac injury, endothelial progenitor cells (EPC) are mobilized from the bone marrow and participate in cardiac repair by increasing revascularization of injured tissues. These cells have been studied actively in the past few years, but their exact phenotype and function are still controversial. In clinical trials, injection of progenitor cells has shown modest benefits. A better understanding of the biology of EPC will allow improving their therapeutic potential. MicroRNAs (miRNAs) are tiny single-stranded non-coding RNAs that negatively regulate gene expression post transcriptionally. They are involved in multiple biological pathways regulating cardiac pathophysiology. Modern technologies using miRNA inhibitors and miRNA mimics have been developed and allow modifying the expression and hence the biological effects of miRNAs. The role of miRNAs in cardiac cells has been extensively investigated. Recent studies suggest that miRNAs play significant roles in EPC, and therefore might be used to improve the regenerative capacities of EPC. In this review, we will first provide a brief overview of the role of EPC in cardiovascular disease. Then, we will summarize current knowledge on the role of miRNAs in EPC and we will discuss how miRNAs may be used to enhance the capacity of EPC to repair the injured heart.


Working from home, quality of life, and perceived productivity during the first 50-day COVID-19 mitigation measures in Austria: a cross-sectional study.
To explore changes in quality of life and perceived productivity, focusing on the effects of working from home during the first COVID-19 50-day mitigation period in Austria. We conducted an Austrian-representative online survey (N = 1010) of self-reported life- and work-related changes during the first COVID-19 50-day mitigation period (March 16 through May 1 2020) compared to the situation before. We used multinominal logistic regression models to identify correlates of improved/decreased quality of life in the entire sample, and of improved/decreased productivity in a subsample of the working population (N = 686). We also calculated age- and multivariable-adjusted ORs and 95% CIs of an improved/decreased quality of life and an improved/decreased productivity by work from home status. During the COVID-19 mitigation period, quality of life improved in 17.5%, but decreased in 20.7% of the general Austrian population; perceived productivity at work increased in 12.7%, but decreased in 30.2% of the working population. Working from home during the mitigation period was associated with an increased quality of life (vs. none, partially: OR 2.07, 95% CI 1.09-3.91; all the time: 3.69, 1.86-7.29). In contrast, perceived productivity seemed to decrease when people worked from home (vs. none, partially: 1.42, 0.86-2.35; all the time: 1.48, 0.85-2.58). Working from home and related benefits were not equally distributed among gender, age, and educational attainment. A transition to more flexibility of workplace and working hours for employees could have important positive consequences for family and professional life, for stakeholders, for public health, and ultimately for the environment.


Effects of development of wind energy and associated changes in land use on bird densities in upland areas.
Wind energy development is the most recent of many pressures on upland bird communities and their habitats. Studies of birds in relation to wind energy development have focused on effects of direct mortality, but the importance of indirect effects (e.g., displacement, habitat loss) on avian community diversity and stability is increasingly being recognized. We used a control-impact study in combination with a gradient design to assess the effects of wind farms on upland bird densities and on bird species grouped by habitat association (forest and open-habitat species). We conducted 506 point count surveys at 12 wind-farm and 12 control sites in Ireland during 2 breeding seasons (2012 and 2013). Total bird densities were lower at wind farms than at control sites, and the greatest differences occurred close to turbines. Densities of forest species were significantly lower within 100 m of turbines than at greater distances, and this difference was mediated by habitat modifications associated with wind-farm development. In particular, reductions in forest cover adjacent to turbines was linked to the observed decrease in densities of forest species. Open-habitat species' densities were lower at wind farms but were not related to distance from turbines and were negatively related to size of the wind farm. This suggests that, for these species, wind-farm effects may occur at a landscape scale. Our findings indicate that the scale and intensity of the displacement effects of wind farms on upland birds depends on bird species' habitat associations and that the observed effects are mediated by changes in land use associated with wind-farm construction. This highlights the importance of construction effects and siting of turbines, tracks, and other infrastructure in understanding the impacts of wind farms on biodiversity.


The minimally important difference in clinical practice for patient-centered outcomes including health assessment questionnaire, fatigue, pain, sleep, global visual analog scale, and SF-36 in scleroderma.
We studied a large clinical practice and multicenter database to estimate the minimally important difference (MID) in systemic sclerosis (SSc) using global rating of change anchors for the Health Assessment Questionnaire-Disability Index (HAQ-DI) and visual analog scale (VAS) in pain, fatigue, sleep, global status, and the Medical Outcomes Study Short-Form 36 (SF-36) in clinical practice. Longitudinal data were collected from a scleroderma clinic on patients with scleroderma (n = 109) who had completed the HAQ-DI and pain/fatigue/sleep/global status VAS (0 to 100 mm) questionnaires at 2 consecutive visits, and rated their change in overall status since the last visit as much better, better, same, worse, or much worse. Data were extracted from the Canadian Scleroderma Research Group (CSRG) database (n = 341) for 2 consecutive annual visits where the patients had completed HAQ-DI and SF-36, and the SF-36 "change in health" item. For the single site, the mean baseline HAQ-DI was 0.895 and 0.911 at followup, with a mean change of 0.016. The MID estimates for improvement and worsening respectively were -0.0125 (0.125, 75th percentile)/0.042 (0.217, 75th percentile) for HAQ-DI, -8.00/3.61 for pain, -10.00/3.79 (25.32) for fatigue, -18.50/5.92 for sleep, and -6.70/4.05 for global VAS. In the CSRG, baseline scores were 0.787 for HAQ-DI, 37.20 for the Physical Component Summary (PCS) of SF-36, and 48.57 for the Mental Component Summary (MCS). The MID estimates for improvement and worsening were -0.037 (0.250, 75th percentile)/0.140 (0.375, 75th percentile) for HAQ-DI, 2.18/-1.74 for PCS, and 1.33/-2.61 for MCS. This study provides MID estimates in SSc from 2 large databases for commonly used patient-reported outcomes in a clinical practice setting, which could differ from MID in trials.


Detection and genotyping of torque teno virus (TTV) in healthy blood donors and patients infected with HBV or HCV in Qatar.
Torque Teno virus (TTV) has been associated with non A-G hepatitis. The goal of this study was to estimate the infection rates and genotypic characteristics of TTV in the State of Qatar. A total of 644 blood samples representing different nationalities: (i) Qatari (118) and (ii) non-Qatari (526) nationals (mostly from Arab and South Eeast Asia countries) were tested for the presence of TTV DNA by nested PCR. The majority (573) of the blood samples belonged to healthy blood donors, whereas 54 and 53 of the blood samples belonged to patients infected with hepatitis B virus (HBV) and hepatitis C virus (HCV), respectively. The results obtained showed that the TTV infection rates in the healthy blood donors, and those infected with HBV or HCV patients were 81.4, 90.75 and 84.9%, respectively. Significant association between TTV viremia and age, or nationality was observed. Sequence analysis of PCR fragments amplified from the 5'-untranslated region (5'-UTR) of all (531) TTV positive samples showed that 65.5% (348/531) of the PCR fragment sequences were classified into main genogroup 3, followed by main genogroups 5 (24%), 2 (5.8%), and 1 (4.7%). Genogroup 4 was not detected among the our studied subjects. Phylogenetic and pairwise analyses using sequences from TTV viremic samples also showed an overall close similarity to the main genogroup 3. In conclusion, there was no significant difference in the rates of TTV detection among Qataris and non-Qataris and several genotypes, mainly genotype 3, were isolated.


The respiratory microbiome in idiopathic pulmonary fibrosis.
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease of unknown cause. Current evidence suggests that it arises in genetically susceptible individuals as a consequence of an aberrant wound-healing response following repetitive alveolar injury. Overt respiratory infection and immunosuppression carry a high mortality, while polymorphisms in genes related to epithelial integrity and host defence predispose to IPF. Recent advances in sequencing technologies have allowed the use of molecular microbial technologies to characterise the respiratory microbiota in patients with IPF. Studies have suggested that changes in the overall bacterial burden are related to disease progression and highlighted significant differences between the microbiota in IPF subjects and healthy controls. Indeed differences in the microbiota between IPF patients may differentiate those with stable compared to progressive disease. As our understanding of the IPF microbiome evolves, along with refinement and advances in sampling and sequencing methodologies we may be able to use microbial signatures as a biomarker to guide prognostication and even treatment stratification in this devastating disease.


Three-dimensional structure of the argininosuccinate lyase frequently complementing allele Q286R.
Argininosuccinate lyase (ASL) catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate, a reaction involved in the biosynthesis of arginine in all species and in the production of urea in ureotelic species. In humans, mutations in the enzyme result in the autosomal recessive disorder argininosuccinic aciduria. Intragenic complementation has been demonstrated to occur at the ASL locus, with two distinct classes of ASL-deficient strains having been identified, the frequent and high-activity complementers. The frequent complementers participate in the majority of the complementation events observed and were found to be either homozygous or heterozygous for a glutamine to arginine mutation at residue 286. The three-dimensional structure of the frequently complementing allele Q286R has been determined at 2.65 A resolution. This is the first high-resolution structure of human ASL. Comparison of this structure with the structures of wild-type and mutant duck delta1 and delta2 crystallins suggests that the Q286R mutation may sterically and/or electrostatically hinder a conformational change in the 280's loop (residues 270-290) and domain 3 that is thought to be necessary for catalysis to occur. The comparison also suggests that residues other than R33, F333, and D337 play a role in maintaining the structural integrity of domain 1 and reinforces the suggestion that residues 74-89 require a particular conformation for catalysis. The electron density has enabled the structure of residues 6-18 to be modeled for the first time. Residues 7-9 and 15-18 are in type IV beta-turns and are connected by a loop. The conformation observed is stabilized, in part, by a salt bridge between the side chains of R12 and D18. Although the disease causing mutation R12Q would disrupt this salt bridge, it is unclear why this mutation has such a significant effect on the catalytic activity as residues 1-18 are disordered in all other delta-crystallin structures determined to date.


Monitoring individual and joint action outcomes in duet music performance.
We investigated whether people monitor the outcomes of their own and their partners' individual actions as well as the outcome of their combined actions when performing joint actions together. Pairs of pianists memorized both parts of a piano duet. Each pianist then performed one part while their partner performed the other; EEG was recorded from both. Auditory outcomes (pitches) associated with keystrokes produced by the pianists were occasionally altered in a way that either did or did not affect the joint auditory outcome (i.e., the harmony of a chord produced by the two pianists' combined pitches). Altered auditory outcomes elicited a feedback-related negativity whether they occurred in the pianist's own part or the partner's part, and whether they affected individual or joint action outcomes. Altered auditory outcomes also elicited a P300 whose amplitude was larger when the alteration affected the joint outcome compared with individual outcomes and when the alteration affected the pianist's own part compared with the partner's part. Thus, musicians engaged in joint actions monitor their own and their partner's actions as well as their combined action outcomes, while at the same time maintaining a distinction between their own and others' actions and between individual and joint outcomes.


Campylobacter jejuni CsrA complements an Escherichia coli csrA mutation for the regulation of biofilm formation, motility and cellular morphology but not glycogen accumulation.
Although Campylobacter jejuni is consistently ranked as one of the leading causes of bacterial diarrhea worldwide, the mechanisms by which C. jejuni causes disease and how they are regulated have yet to be clearly defined. The global regulator, CsrA, has been well characterized in several bacterial genera and is known to regulate a number of independent pathways via a post transcriptional mechanism, but remains relatively uncharacterized in the genus Campylobacter. Previously, we reported data illustrating the requirement for CsrA in several virulence related phenotypes of C. jejuni strain 81-176, indicating that the Csr pathway is important for Campylobacter pathogenesis. We compared the Escherichia coli and C. jejuni orthologs of CsrA and characterized the ability of the C. jejuni CsrA protein to functionally complement an E. coli csrA mutant. Phylogenetic comparison of E. coli CsrA to orthologs from several pathogenic bacteria demonstrated variability in C. jejuni CsrA relative to the known RNA binding domains of E. coli CsrA and in several amino acids reported to be involved in E. coli CsrA-mediated gene regulation. When expressed in an E. coli csrA mutant, C. jejuni CsrA succeeded in recovering defects in motility, biofilm formation, and cellular morphology; however, it failed to return excess glycogen accumulation to wild type levels. These findings suggest that C. jejuni CsrA is capable of efficiently binding some E. coli CsrA binding sites, but not others, and provide insight into the biochemistry of C. jejuni CsrA.


Sézary Syndrome: Clinical and Biological Aspects.
Sézary syndrome (SS) is a rare and aggressive type of cutaneous T cell lymphoma (CTCL) characterized by an intensely pruritic, exfoliative rash, known as erythroderma, with cutaneous and systemic dissemination of clonal CD4+ T cells into the blood and lymph nodes. This review aims to present recent advancements in the biological and clinical aspects of SS. We begin by providing an overview of the diagnostic criteria for SS and reviewing some of its epidemiological and clinical aspects. We then discuss updates in the etiology of this elusive disease and the genetic and molecular landscapes that define it. Finally, we provide a short overview of the current therapeutic strategies for SS as well as recent advances in the prognosis of this disease. A brief set of recommendations is provided regarding future directions in research and therapy.


Rapid HIV testing at gay pride events to reach previously untested MSM: U.S., 2009-2010.
We offered rapid HIV testing at social events frequented by young men who have sex with men (MSM), a group disproportionately affected by the HIV epidemic. We tested 1,312 MSM; of those MSM, 1,072 (81.7%) reported HIV testing history. Of those reporting HIV testing history, 550 (51.3%) were non-Hispanic black and 404 (37.7%) were aged <25 years. One hundred twenty-eight (11.9%) had never tested for HIV; 77 (7.2%) were preliminarily positive, with 15 (19.5%) being first-time testers. Factors associated with no previous HIV test included young age (13-24 years) (adjusted odds ratio [AOR] = 3.5, 95% confidence interval [CI] 1.9, 6.5) and non-Hispanic black (AOR=3.2, 95% CI 1.6, 6.4) or Hispanic (AOR=2.8, 95% CI 1.2, 6.3) race/ethnicity. HIV testing at Gay Pride events reaches young, previously untested MSM. This venue-based HIV testing approach at nonclinical sociocultural events is an additional strategy for HIV prevention goals to increase the number of people aware of their HIV infection with subsequent linkage to HIV care.


Synchrotron Imaging Shows Effect of Ventilator Settings on Intrabreath Cyclic Changes in Pulmonary Blood Volume.
Despite the importance of dynamic changes in the regional distributions of gas and blood during the breathing cycle for lung function in the mechanically ventilated patient, no quantitative data on such cyclic changes are currently available. We used a novel gated synchrotron computed tomography imaging to quantitatively image regional lung gas volume (Vg), tissue density, and blood volume (Vb) in six anesthetized, paralyzed, and mechanically ventilated rabbits with normal lungs. Images were repeatedly collected during ventilation and steady-state inhalation of 50% xenon, or iodine infusion. Data were acquired in a dependent and nondependent image level, at zero end-expiratory pressure (ZEEP) and 9 cm H2O (positive end-expiratory pressure), and a tidal volume (Vt) of 6 ml/kg (Vt1) or 9 ml/kg (Vt2) at an Inspiratory:Expiratory ratio of 0.5 or 1.7 by applying an end-inspiratory pause. A video showing dynamic decreases in Vb during inspiration is presented. Vb decreased with positive end-expiratory pressure (P = 0.006; P = 0.036 versus Vt1-ZEEP and Vt2-ZEEP, respectively), and showed larger oscillations at the dependent image level, whereas a 45% increase in Vt did not have a significant effect. End-inspiratory Vb minima were reduced by an end-inspiratory pause (P = 0.042, P = 0.006 at nondependent and dependent levels, respectively). Normalized regional Vg:Vb ratio increased upon inspiration. Our data demonstrate, for the first time, within-tidal cyclic variations in regional pulmonary Vb. The quantitative matching of regional Vg and Vb improved upon inspiration under ZEEP. Further study is underway to determine whether these phenomena affect intratidal gas exchange.


Evidence for a limit to human lifespan.
Driven by technological progress, human life expectancy has increased greatly since the nineteenth century. Demographic evidence has revealed an ongoing reduction in old-age mortality and a rise of the maximum age at death, which may gradually extend human longevity. Together with observations that lifespan in various animal species is flexible and can be increased by genetic or pharmaceutical intervention, these results have led to suggestions that longevity may not be subject to strict, species-specific genetic constraints. Here, by analysing global demographic data, we show that improvements in survival with age tend to decline after age 100, and that the age at death of the world's oldest person has not increased since the 1990s. Our results strongly suggest that the maximum lifespan of humans is fixed and subject to natural constraints.


Differential regulation of aldosterone synthase and 11beta-hydroxylase transcription by steroidogenic factor-1.
11beta-Hydroxylase (hCYP11B1) and aldosterone synthase (hCYP11B2) are closely related isozymes with distinct roles in cortisol and aldosterone production respectively. Aldosterone synthase catalyzes the final step in aldosterone biosynthesis and is expressed only in the zona glomerulosa of the normal adrenal. 11beta-Hydroxylase catalyzes the final reaction in the production of cortisol and is expressed at higher levels in the zona fasciculata. The mechanisms causing differential expression of these genes are not well defined. Herein, we demonstrate contrasting roles for the orphan receptor steroidogenic factor-1 (SF-1) in the regulation of human (h) CYP11B1 and hCYP11B2. Human NCI-H295R (H295R) or mouse Y-1 cells were transiently transfected with luciferase reporter constructs containing 5'-flanking regions of hCYP11B1, hCYP11B2, human 17alpha-hydroxylase (hCYP17), human cholesterol side-chain cleavage (hCYP11A1) or mouse (m) cyp11b2 (mcyp11b2). Co-transfection of vectors encoding SF-1 increased expression of hCYP11B1, hCYP11A1 and hCYP17 constructs, but inhibited hCYP11B2 reporter activity. Murine, bovine and human SF-1 were unable to increase transcription of hCYP11B2 in H295R cells. Both hCYP11B2 and mcyp11b2 promoter constructs were inhibited similarly by human SF-1. In mouse Y-1 cells, reporter expression of hCYP11B2 and mcyp11b2 was very low compared with hCYP11B1 constructs, suggesting that this adrenal cell model may not be appropriate for studies of CYP11B2. Electrophoretic mobility shift assay demonstrated that SF-1 interacted with an element from both hCYP11B1 and hCYP11B2. However, mutation of this element, termed Ad4, did not prevent agonist stimulation of hCYP11B2 by angiotensin II or forskolin but blocked activity of hCYP11B1. In some, but not all, reports of genetic linkage analysis, a naturally occurring single nucleotide polymorphism within the Ad4 element of hCYP11B2 (-344C/T) has been associated with cardiovascular disease. Herein, we have demonstrated that this polymorphism influenced binding of SF-1 in electrophoretic mobility shift assays, with the C allele binding SF-1 more strongly than the T allele. However, when hCYP11B2 constructs containing these alleles were transfected into H295R cells, there was no difference in agonist-stimulated expression or the response of either reporter construct to co-expression with human SF-1. Taken together, these data suggest that SF-1 and the Ad4 element are not major regulators of adrenal hCYP11B2 gene expression. Thus far, hCYP11B2 is the first steroid hydroxylase gene which is not positively regulated by SF-1.


Neurobehavioral development in children with potential exposure to pesticides.
Children may be at higher risk than adults from pesticide exposure, due to their rapidly developing physiology, unique behavioral patterns, and interactions with the physical environment. This preliminary study conducted in Ecuador examines the association between household and environmental risk factors for pesticide exposure and neurobehavioral development. We collected data over 6 months in the rural highland region of Cayambe, Ecuador (2003-2004). Children age 24-61 months residing in 3 communities were assessed with the Ages and Stages Questionnaire and the Visual Motor Integration Test. We gathered information on maternal health and work characteristics, the home and community environment, and child characteristics. Growth measurements and a hemoglobin finger-prick blood test were obtained. Multiple linear regression analyses were conducted. Current maternal employment in the flower industry was associated with better developmental scores. Longer hours playing outdoors were associated with lower gross and fine motor and problem solving skills. Children who played with irrigation water scored lower on fine motor skills (8% decrease; 95% confidence interval = -9.31 to -0.53), problem-solving skills (7% decrease; -8.40 to -0.39), and Visual Motor Integration test scores (3% decrease; -12.00 to 1.08). These results suggest that certain environmental risk factors for exposure to pesticides may affect child development, with contact with irrigation water of particular concern. However, the relationships between these risk factors and social characteristics are complex, as corporate agriculture may increase risk through pesticide exposure and environmental contamination, while indirectly promoting healthy development by providing health care, relatively higher salaries, and daycare options.


Silk-based biomaterials functionalized with fibronectin type II promotes cell adhesion.
The objective of this work was to exploit the fibronectin type II (FNII) module from human matrix metalloproteinase-2 as a functional domain for the development of silk-based biopolymer blends that display enhanced cell adhesion properties. The DNA sequence of spider dragline silk protein (6mer) was genetically fused with the FNII coding sequence and expressed in Escherichia coli. The chimeric protein 6mer+FNII was purified by non-chromatographic methods. Films prepared from 6mer+FNII by solvent casting promoted only limited cell adhesion of human skin fibroblasts. However, the performance of the material in terms of cell adhesion was significantly improved when 6mer+FNII was combined with a silk-elastin-like protein in a concentration-dependent behavior. With this work we describe a novel class of biopolymer that promote cell adhesion and potentially useful as biomaterials for tissue engineering and regenerative medicine. This work reports the development of biocompatible silk-based composites with enhanced cell adhesion properties suitable for biomedical applications in regenerative medicine. The biocomposites were produced by combining a genetically engineered silk-elastin-like protein with a genetically engineered spider-silk-based polypeptide carrying the three domains of the fibronectin type II module from human metalloproteinase-2. These composites were processed into free-standing films by solvent casting and characterized for their biological behavior. To our knowledge this is the first report of the exploitation of all three FNII domains as a functional domain for the development of bioinspired materials with improved biological performance. The present study highlights the potential of using genetically engineered protein-based composites as a platform for the development of new bioinspired biomaterials.