Mycorrhizal status and host genotype interact to shape plant nutrition in field grown maize (Zea mays ssp. mays).
Arbuscular mycorrhizal fungi (AMF) establish symbioses with the major cereal crops, providing plants with increased access to nutrients while enhancing their tolerance to toxic heavy metals. However, not all plant varieties benefit equally from this association. In this study, we used quantitative trait loci (QTL) mapping to evaluate the combined effect of host genotypic variation (G) and AMF across 141 genotypes on the concentration of 20 mineral elements in the leaves and grain of field grown maize (Zea mays spp. mays). Our mapping design included selective incorporation of a castor AMF-incompatibility mutation, allowing estimation of AMF, QTL and QTLxAMF effects by comparison of mycorrhizal and non-mycorrhizal plants. Overall, AMF compatibility was associated with higher concentrations of boron (B), copper (Cu), molybdenum (Mo), phosphorus (P), selenium (Se) and zinc (Zn) and lower concentrations of arsenic (As), iron (Fe), magnesium (Mg), manganese (Mn), potassium (K) and strontium (Sr). In addition to effects on individual elements, pairwise correlation matrices for element concentration differed between mycorrhizal and non-mycorrhizal plants. We mapped 22 element QTLs, including 18 associated with QTLxAMF effects that indicate plant genotype-specific differences in the impact of AMF on the host ionome. Although there is considerable interest in AMF as biofertilizers, it remains challenging to estimate the impact of AMF in the field. Our design illustrates an effective approach for field evaluation of AMF effects. Furthermore, we demonstrate the capacity of the ionome to reveal host genotype-specific variation in the impact of AMF on plant nutrition.


Alcohol increases commission error rates for a continuous performance test.
Studying the effects of alcohol on Continuous Performance Test (CPT) performance was of interest for two reasons, i.e., (1) perhaps because of the ease of the task used in previous experiments, alcohol has not been found to impair performance, and (2) CPT commission errors (described below) have been related to impulsive behavior. In this study, the CPT featured both an Immediate Memory Task (IMT) and a more difficult Delayed Memory Task (DMT). We compared the performance of 18 subjects under both alcohol and placebo conditions, using a within-subject design. Both the IMT (0.5-sec delay) and the DMT (3.5-sec delay, with distracter stimuli at 0.5-sec intervals) required the subject to respond if a briefly displayed number was identical to the one presented before it. Stimuli included target (identical match), catch (4 of 5 digits matched), and novel (random number) stimuli. On 2 separate days, subjects performed between administrations of three hourly placebo drinks or three hourly drinks containing 0.20 g/kg of alcohol (producing peak breath alcohol concentrations of approximately 0.035%). The main finding was that alcohol consumption increased responses to catch stimuli (i.e., commission errors) in the DMT. In contrast, performance in the IMT (the easier task) was unaffected by alcohol. Commission errors measured during peak breath alcohol concentrations were significantly correlated with scores on the Barratt Impulsivity Scale for both the IMT and DMT. Discriminability (A') between target and catch stimuli was reduced by alcohol for the DMT only. These data indicate that even small amounts of alcohol can produce measurable changes in CPT performance parameters if the task is of sufficient difficulty and that commission errors can be increased by alcohol consumption.


Perceptions of care coordination in cancer patient-family caregiver dyads.
To examine cancer patients and their family caregivers' perspectives of care coordination (CC) using a dyadic research design. In this pilot cross-sectional study, 54 patient-family caregiver dyads completed a validated care coordination instrument (CCI) and its parallel family caregiver instrument (CCICG) from June to September 2019. The sample available for analysis included data from 32 dyads, which included patients receiving active therapy for any cancer type and their primary family caregivers aged 18 years or older. Mixed regression models were used to examine dyadic differences. The overall family caregiver scores demonstrated a bimodal pattern; thus, we conducted analyses using aggregate data as well as by highCG and lowCG subgroups. Among dyads in the lowCG subgroup, family caregivers reported significantly lower scores than patients on the total CCI and the three CC domains: Communication, Navigation, and Operational. Caregiver gender, the absence of a patient navigator, and practice setting (hospital-based ambulatory) significantly predicted dyadic differences in the lowCG subgroup. In item-level analyses, family caregivers in the lowCG subgroup reported lower scores than patients on the items related to patient-physician communication. A subgroup of family caregivers reported poorer perception of CC than patients, suggesting that those family caregivers and providers may benefit from intervention. Further understanding of patient-family caregiver dyads' perspectives of CC can inform development of strategies to integrate family caregivers into the cancer care team, develop effective CC interventions for family caregivers, and contribute to improved quality and value of cancer care.


Oligomycin sensitivity of mitochondrial F(1)F(0)-ATPase in diabetes-prone BHE/Cdb rats.
BHE/Cdb and Sprague-Dawley rats differ in their mitochondrial DNA sequence for the ATPase 6 ("subunit a") gene. Base substitutions in this sequence result in the substitution of asparagine for aspartate at position 101 and the substitution of serine for leucine at position 129. Differences in sensitivity to oligomycin were observed. When the isolated F(1)F(0)-ATPase complex was studied and ATPase activity was assessed, that which was isolated from the BHE/Cdb rats was less sensitive to oligomycin inhibition than that which was isolated from the Sprague-Dawley rats. In contrast, when oxygen consumption was measured [oxygen phosphorylation (OXPHOS)] and a dose-response curve was generated with isolated mitochondria from these two strains, there was a shift to the left for the BHE/Cdb rat mitochondria. These mitochondria were more sensitive to oligomycin inhibition of OXPHOS than were mitochondria isolated from Sprague-Dawley rats. The OXPHOS results are consistent with those from human fibroblasts having either a normal or mutated ATPase 6 gene.


Circulating miRNA profiles provide a biomarker for severity of stroke outcomes associated with age and sex in a rat model.
Small non-coding RNA [miRNA (microRNA)] found in the circulation have been used successfully as biomarkers and mechanistic targets for chronic and acute disease. The present study investigated the impact of age and sex on miRNA expression following ischaemic stroke in an animal model. Adult (6 month) and middle-aged (11-12 months) female and male rats were subject to MCAo (middle cerebral artery occlusion) using ET-1 (endothelin-1). Circulating miRNAs were analysed in blood samples at 2 and 5 days post-stroke, and brain miRNAs were analysed at 5 days post-stroke. Although stroke-associated infarction was observed in all groups, infarct volume and sensory-motor deficits were significantly reduced in adult females compared with middle-aged females, adult males or middle-aged males. At 2 days post-stroke, 21 circulating miRNAs were differentially regulated and PCA (principal component analysis) confirmed that most of the variance was due to age. At 5 days post-stroke, 78 circulating miRNAs exhibited significantly different regulation, and most of the variance was associated with sex. A small cohort (five) of miRNAs, miR-15a, miR-19b, miR-32 miR-136 and miR-199a-3p, were found to be highly expressed exclusively in adult females compared with middle-aged females, adult males and middle-aged males. Predicted gene targets for these five miRNAs analysed for KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways revealed that the top ten KEGG pathways were related to growth factor signalling, cell structure and PI3K (phosphoinositide 3-kinase)/Akt and mTOR (mammalian target of rapamycin) signalling. Overall, the pattern of circulating miRNA expression suggests an early influence of age in stroke pathology, with a later emergence of sex as a factor for stroke severity.


The Gill Microbiota of Argopecten purpuratus Scallop Is Dominated by Symbiotic Campylobacterota and Upwelling Intensification Differentially Affects Their Abundance.
Despite the great importance of gills for bivalve mollusks (respiration, feeding, immunity), the microbiota associated with this tissue has barely been characterized in scallops. The scallop Argopecten purpuratus is an important economic resource that is cultivated in areas where coastal upwelling is intensifying by climate change, potentially affecting host-microbiota interactions. Thus, we first characterized the bacterial community present in gills from cultivated scallops (by 16S rRNA gene amplicon sequencing) and assessed their stability and functional potential in animals under farm and laboratory conditions. Results showed that under both conditions the gill bacterial community is dominated by the phylum Campylobacterota (57%), which displays a chemoautotrophic potential that could contribute to scallop nutrition. Within this phylum, two phylotypes, namely symbionts A and B, were the most abundant; being, respectively, taxonomically affiliated to symbionts with nutritional functions in mussel gills, and to uncultured bacteria present in coral mucus. Additionally, in situ hybridization and scanning electron microscopy analyses allowed us to detect these symbionts in the gills of A. purpuratus. Given that shifts in upwelling phenology can cause disturbances to ecosystems, affecting bacteria that provide beneficial functions to the host, we further assessed the changes in the abundance of the two symbionts (via qPCR) in response to a simulated upwelling intensification. The exposure to combined decreasing values in the temperature, pH, and oxygen levels (upwelling conditions) favored the dominance of symbiont B over symbiont A; suggesting that symbiont abundances are modulated by these environmental changes. Overall, results showed that changes in the main Campylobacterota phylotypes in response to upwelling intensification could affect its symbiotic function in A. purpuratus under future climate change scenarios. These results provide the first insight into understanding how scallop gill-microbial systems adapt and respond to climate change stressors, which could be critical for managing health, nutrition, and scallop aquaculture productivity.


Head and neck ultrasound: technical advances, novel applications and the role of elastography.
High-resolution ultrasound (US) provides superb anatomical detail in the superficial anatomy of the neck and has become the first-line imaging investigation for neck lumps and a crucial component of clinical pathways. In this article, a wide range of advances in neck US are described with a focus on the emerging role of ultrasound elastography. Selected examples of clinical utility are presented across a spectrum of scenarios with discussion of newer applications, service delivery, and training issues. The changing role of the neck ultrasound practitioner and the future of the technique in the head and neck are considered.


[Characterisation of major biological properties of Bordetella bacteriophages].
The main biological properties (morphology of negative colonies, parameters of adsorption and single development cycle) of B. pertussis and B. bronchiseptica phages, isolated spontaneously and by induction with mitomycin C, were studied. To compare these characteristics, one B. parapertussis indicator strain was used, and the experiments were carried out under identical conditions. Highly active sera were obtained with the use of complete Freund's adjuvant. B. pertussis phages isolated from the strains of different serovars were serologically related, but not identical, and differed in their constant characterizing their rate of neutralization with homologous antisera. The adsorption of the phages on homologous strains was more intensive than on the cells of B. parapertussis indicator strain. However, the authors failed to observe the further development of the phages in the host cells.


Paraneoplastic neuropathies.
This article provides an approach to the recognition and management of paraneoplastic neuropathies. Paraneoplastic neuropathies may have unique phenotypic presentations, such as sensory neuronopathy, autonomic enteric neuropathy, demyelinating neuropathy, and, rarely, motor neuropathy. Paraneoplastic sensorimotor neuropathy, on the other hand, may be indistinguishable from other common types of axonal polyneuropathy. Certain patterns of neuropathies are commonly seen with different types of cancers, but this relationship is not exclusive and not all patients whose pattern of neuropathy suggests a paraneoplastic disorder have an underlying cancer. In addition to definitive therapy for malignancy, immunomodulatory therapy, such as corticosteroids, IV immunoglobulin (IVIg), or immunosuppressants, may benefit some patients, but there are very few published treatment data for paraneoplastic neuropathies. Prompt recognition of paraneoplastic neuropathies may lead to identification and treatment of an occult cancer. Treatment can potentially arrest the progression of neuropathy.


Human chromatid ultrastructure: new observations with scanning and transmission electron microscopy.
Two new observations have been made on human chromatid/chromosome ultrastructure using both scanning and transmission electron microscopy (SEM, TEM). A bipartite, apparently half-chromatid-like structure was observed in whole human chromosomes studied with SEM and in longitudinally sectioned chromosomes analyzed with TEM. In addition, we also observed a zipper-like configuration as the parallel sister chromatids separated likely due to the supercoiled structure of the chromosome and chromatid. It is possible that either or both of these new observations resulted from our (improved) method of preparing the chromosomes for SEM and TEM.


Community-based educational intervention improved the diversity of complementary diets in western Kenya: results from a randomized controlled trial.
Lack of diversity is a major factor contributing to inadequate nutrient intakes among children during the complementary feeding period in many rural areas in developing countries. This has been attributed to inadequate feeding practices and nutrition knowledge among their caregivers. The aim of the present study was to assess the effect of an educational intervention on children's dietary diversity and nutrition knowledge of caregivers. Cluster randomization was applied and twenty matched village pairs were randomly assigned to the intervention or control group. The nutrition education intervention consisted of four sessions comprising of group trainings and cooking demonstrations that were conducted over a period of 5 months. Households in rural communities in Bondo and Teso South sub-counties, western Kenya. Caregivers with children aged 6-17 months receiving nutrition education. The children's dietary diversity scores (CDDS) and nutrition knowledge scores of the caregivers improved significantly in the intervention group at endline. The treatment effect on CDDS was positive and significant (P=0.001). The CDDS rate of the children in the intervention group was 27% larger than it would have been without the treatment effect. The intervention also had a significant effect on the caregivers' nutrition knowledge scores (incidence rate ratio=2.05; P<0.001). However, the nutrition knowledge of the caregivers did not have a significant effect on CDDS (P=0.731). The nutrition education intervention led to improvements in children's dietary diversity and nutrition knowledge of the caregivers.


The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides.
The discrete multicomponent, multienzyme cellulosome complex of anaerobic cellulolytic bacteria provides enhanced synergistic activity among the different resident enzymes to efficiently hydrolyze intractable cellulosic and hemicellulosic substrates of the plant cell wall. A pivotal noncatalytic subunit called scaffoldin secures the various enzymatic subunits into the complex via the cohesin-dockerin interaction. The specificity characteristics and tenacious binding between the scaffoldin-based cohesin modules and the enzyme-borne dockerin domains dictate the supramolecular architecture of the cellulosome. The diversity in cellulosome architecture among the known cellulosome-producing bacteria is manifest in the arrangement of their genes in either multiple-scaffoldin or enzyme-linked clusters on the genome. The recently described three-dimensional crystal structure of the cohesin-dockerin heterodimer sheds light on the critical amino acids that contribute to this high-affinity protein-protein interaction. In addition, new information regarding the regulation of cellulosome-related genes, budding genetic tools, and emerging genomics of cellulosome-producing bacteria promises new insight into the assembly and consequences of the multienzyme complex.


Epidemiological and experimental evidence for radiation-induced health effects in the progeny after exposure in utero.
It has been known for many decades that radiation exposure of the developing embryo or fetus may cause two fundamentally different types of severe health effects: on the one hand, radiation may interfere with the normal intrauterine development, on the other hand, radiation may induce leukemia and cancer which become manifest in childhood. A large amount of epidemiological and experimental data has recently been presented which might be used to improve our understanding of underlying mechanisms and setting radiation protection standards. Yet, ecological studies in the populations exposed to increased levels of radiation in regions contaminated by radioactivity released from reactor accidents (Chernobyl, Fukushima) do not provide solid evidence which would contribute to this aim. On the other hand, well designed experimental studies demonstrated the multifactorial mechanisms which lead to different health effects after radiation exposure in utero. There is no convincing evidence, neither from epidemiological nor experimental data of the existence of a dose threshold for developmental defects after radiation exposure in utero. This must be taken into account in the revision of rules and regulations of radiation protection in medicine.


Adaptive plasticity and epigenetic variation in response to warming in an Alpine plant.
Environmentally induced phenotypic plasticity may be a critical component of response to changing environments. We examined local differentiation and adaptive phenotypic plasticity in response to elevated temperature in half-sib lines collected across an elevation gradient for the alpine herb, Wahlenbergia ceracea. Using Amplified Fragment Length Polymorphism (AFLP), we found low but significant genetic differentiation between low- and high-elevation seedlings, and seedlings originating from low elevations grew faster and showed stronger temperature responses (more plasticity) than those from medium and high elevations. Furthermore, plasticity was more often adaptive for plants of low-elevation origin and maladaptive for plants of high elevation. With methylation sensitive-AFLP (MS-AFLP), we revealed an increase in epigenetic variation in response to temperature in low-elevation seedlings. Although we did not find significant direct correlations between MS-AFLP loci and phenotypes, our results demonstrate that adaptive plasticity in temperature response to warming varies over fine spatial scales and suggest the involvement of epigenetic mechanisms in this response.


[Ambulatory treatment of streptococcal bacterial endocarditis].
Bacterial endocarditis is a severe infectious disease. of which treatment is traditionally carried out in hospitalized patients through intravenous medication. The possibility of at-home or ambulatory treatment. for stringently selected cases. is attractive from the social as well as from the economic point of view. We report 6 patients with a diagnosis of bacterial endocarditis caused by Streptococcus. treated partially or completely on an outpatient basis. All of them evolved without complications and presented complete resolution of the infection.


Reproductive factors and risk of hormone receptor positive and negative breast cancer: a cohort study.
The association of reproductive factors with hormone receptor (HR)-negative breast tumors remains uncertain. Within the EPIC cohort, Cox proportional hazards models were used to describe the relationships of reproductive factors (menarcheal age, time between menarche and first pregnancy, parity, number of children, age at first and last pregnancies, time since last full-term childbirth, breastfeeding, age at menopause, ever having an abortion and use of oral contraceptives [OC]) with risk of ER-PR- (n = 998) and ER+PR+ (n = 3,567) breast tumors. A later first full-term childbirth was associated with increased risk of ER+PR+ tumors but not with risk of ER-PR- tumors (≥35 vs. ≤19 years HR: 1.47 [95% CI 1.15-1.88] p(trend) < 0.001 for ER+PR+ tumors; ≥35 vs. ≤19 years HR: 0.93 [95% CI 0.53-1.65] p(trend) = 0.96 for ER-PR- tumors; P(het) = 0.03). The risk associations of menarcheal age, and time period between menarche and first full-term childbirth with ER-PR-tumors were in the similar direction with risk of ER+PR+ tumors (p(het) = 0.50), although weaker in magnitude and statistically only borderline significant. Other parity related factors such as ever a full-term birth, number of births, age- and time since last birth were associated only with ER+PR+ malignancies, however no statistical heterogeneity between breast cancer subtypes was observed. Breastfeeding and OC use were generally not associated with breast cancer subtype risk. Our study provides possible evidence that age at menarche, and time between menarche and first full-term childbirth may be associated with the etiology of both HR-negative and HR-positive malignancies, although the associations with HR-negative breast cancer were only borderline significant.


Propeller flaps in the closure of free fibula flap donor site skin defects.
The free fibula is a versatile and commonly used free flap in microvascular reconstruction. It allows for reconstruction of both bone and soft tissue defects. In head and neck reconstruction, the skin paddle harvested along with the flap allows for the reconstruction of skin or oral mucosal defects. After skin paddle harvest, the donor site can be closed primarily or with skin grafts. Grafting the donor area is the common method used. However, this could lead to delayed healing because of the poor graft over the area of peroneal tendons. Propeller flaps have been extensively reported for closure of leg skin defects. We report a series of 10 patients in whom we used a local propeller flap for the closure of the fibula flap skin donor site. The donor defects could be satisfactorily closed without the need of a skin graft in 9 patients. This method is simple, reliable, and suitable for closing small to medium defects.


Chest CT Scan for the Diagnosis of Pediatric Pulmonary TB: Radiological Findings and Its Diagnostic Significance.
Diagnosing active TB in children remains a clinical challenge, due to difficulties in achieving a definite microbiological confirmation, aspecific clinical manifestation, low sensitivity of chest radiography (CXR). For this reason, the use of chest computed tomography (CT) scan to evaluate suspected TB pediatric cases is increasing. We retrospectively reviewed records of patients aged <16 years diagnosed with active TB at the Pediatric Infectious Disease Unit of the Catholic University of the Sacred Heart to describe CT findings and to evaluate the need for its execution for diagnosis. In 41 cases, 7 CXR were normal (17.1%) while no CT scan was evaluated as negative. In 19 cases (46.3%), CXR was considered non-probable TB pulmonary, compared with 11 of 37 cases (29.7%) of CT. In 15 cases (36.6%) CXR was described as probable for TB pulmonary, instead 26 of the 37 cases evaluated by CT (70.3%) were classified as probable TB. We describe CT findings in patients with pediatric TB. We confirmed that CT can improve the diagnostic accuracy. In particular, the comparison between the CT and CXR ability in detecting cases of pulmonary TB in accordance with the proposed radiological probability criteria, showed a superiority of CT in detecting probable TB pictures (70.3%) compared with 36.6% of the x-Ray.


Does uncoupling protein 2 expression qualify as marker of disease status in LRRK2-associated Parkinson's disease?
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common known genetic cause of late-onset Parkinson's disease (PD). However, the penetrance of the disease is below 50% at 60 years of age. LRRK2 is associated with the mitochondrial membrane, and mutant forms impair the function of the organelle and autophagosome clearance in human cells, including induced pluripotent stem cell-derived neurons. Elevated expression of uncoupling proteins has been identified as the cause of mitochondrial depolarization in human fibroblasts with G2019S LRRK2. To identify factors that contribute to the penetrance of LRRK2 mutations, we studied respiratory chain function, markers of mitochondrial uncoupling, oxidative stress, and autophagy in fibroblasts from affected and unaffected carriers of the G2019S mutation. Independent of disease status, all mutation carriers showed reduced mitochondrial membrane potential, increased proton leakage, and more fragmented mitochondria. However, a significant increase in the expression of uncoupling protein 2 (UCP2) was only detected in affected individuals with the G2019S mutation in LRRK2. Since oxidative stress and autophagic markers were selectively increased in some of the PD patients, we hypothesize that UCP2 expression is upregulated in response to elevated reactive oxygen species generation in affected mutation carriers and that UCP2 mRNA levels might, therefore, serve as markers of disease status in LRRK2-associated PD.


Local de novo assembly of RAD paired-end contigs using short sequencing reads.
Despite the power of massively parallel sequencing platforms, a drawback is the short length of the sequence reads produced. We demonstrate that short reads can be locally assembled into longer contigs using paired-end sequencing of restriction-site associated DNA (RAD-PE) fragments. We use this RAD-PE contig approach to identify single nucleotide polymorphisms (SNPs) and determine haplotype structure in threespine stickleback and to sequence E. coli and stickleback genomic DNA with overlapping contigs of several hundred nucleotides. We also demonstrate that adding a circularization step allows the local assembly of contigs up to 5 kilobases (kb) in length. The ease of assembly and accuracy of the individual contigs produced from each RAD site sequence suggests RAD-PE sequencing is a useful way to convert genome-wide short reads into individually-assembled sequences hundreds or thousands of nucleotides long.


Separation and assembly of deep sequencing data into discrete sub-population genomes.
Sequence heterogeneity is a common characteristic of RNA viruses that is often referred to as sub-populations or quasispecies. Traditional techniques used for assembly of short sequence reads produced by deep sequencing, such as de-novo assemblers, ignore the underlying diversity. Here, we introduce a novel algorithm that simultaneously assembles discrete sequences of multiple genomes present in populations. Using in silico data we were able to detect populations at as low as 0.1% frequency with complete global genome reconstruction and in a single sample detected 16 resolved sequences with no mismatches. We also applied the algorithm to high throughput sequencing data obtained for viruses present in sewage samples and successfully detected multiple sub-populations and recombination events in these diverse mixtures. High sensitivity of the algorithm also enables genomic analysis of heterogeneous pathogen genomes from patient samples and accurate detection of intra-host diversity, enabling not just basic research in personalized medicine but also accurate diagnostics and monitoring drug therapies, which are critical in clinical and regulatory decision-making process.


Sonic hedgehog is a critical mediator of erythropoietin-induced cardiac protection in mice.
Erythropoietin reportedly has beneficial effects on the heart after myocardial infarction, but the underlying mechanisms of these effects are unknown. We here demonstrate that sonic hedgehog is a critical mediator of erythropoietin-induced cardioprotection in mice. Treatment of mice with erythropoietin inhibited left ventricular remodeling and improved cardiac function after myocardial infarction, independent of erythropoiesis and the mobilization of bone marrow-derived cells. Erythropoietin prevented cardiomyocyte apoptosis and increased the number of capillaries and mature vessels in infarcted hearts by upregulating the expression of angiogenic cytokines such as VEGF and angiopoietin-1 in cardiomyocytes. Erythropoietin also increased the expression of sonic hedgehog in cardiomyocytes, and inhibition of sonic hedgehog signaling suppressed the erythropoietin-induced increase in angiogenic cytokine expression. Furthermore, the beneficial effects of erythropoietin on infarcted hearts were abolished by cardiomyocyte-specific deletion of sonic hedgehog. These results suggest that erythropoietin protects the heart after myocardial infarction by inducing angiogenesis through sonic hedgehog signaling.


The most personal information of all: an appraisal of genetic privacy in the shadow of the Human Genome Project.
The advent of genetics and genetic testing has given rise to unique problems for the family. The discovery of a predisposition to a genetic condition in one individual also reveals information about the genetic make-up and potential risks of family members. There is, therefore, potential for conflict over access to and control of such information. Traditionally the duty of confidentiality owed by a health care professional to a patient has provided an appropriate means by which personal health information has been kept secured. It is not clear, however, that the problems which surrounds genetic information in the familial milieu can be adequately dealt with using confidentiality. This article examines these problems and argues for the value of an appeal to the concept of privacy in seeking to resolve some of the more intractable issues.


Stabilization of DNA Structures with Poly(ethylene sodium phosphate).
The structure and stability of biomolecules under molecular crowding conditions are of interest because such information clarifies how biomolecules behave under cell-mimicking conditions. The anionic surfaces of chromatin, which is composed of DNA strands and histone complexes, are concentrated in cell nuclei and thus generate a polyanionic crowding environment. In this study, we designed and synthesized an anionic polymer, poly(ethylene sodium phosphate) (PEP·Na), which has a nucleic acid phosphate backbone and created a cell nucleus-like environment. The effects of molecular crowding with PEP·Na on the thermodynamics of DNA duplexes, triplexes, and G-quadruplexes were systematically studied. Thermodynamic analysis demonstrated that PEP·Na significantly stabilized the DNA structures; e.g., a free energy change at 25 °C for duplex formation decreased from -6.6 to -12.8 kcal/mol with 20 wt % PEP·Na. Thermodynamic parameters further indicated that the factors for the stabilization of the DNA structures were dependent on sodium ion concentration. At lower polymer concentrations, the stabilization was attributed to a shielding of the electrostatic repulsion between DNA strands by the sodium ions of PEP·Na. In contrast, at higher polymer concentrations, the DNA structures were entropically stabilized by volume exclusion, which could be enhanced by electrostatic repulsion between phosphate groups in DNA strands and in PEP·Na. Additionally, increasing PEP·Na concentration resulted in increasing enthalpy of the DNA duplex but decreasing enthalpy of DNA G-quadruplex, indicating that the polymers also promoted dehydration of the DNA strands. Thus, polyanionic crowding affects the thermodynamics of DNA structures via the sodium ions, volume exclusion, and hydration. The stabilization of DNA by the cell nucleus-like polyanionic crowding provides new information regarding DNA structures and allows for modeling reactions in cell nuclei.


A human anti-neuronal autoantibody against GABA B receptor induces experimental autoimmune agrypnia.
In the serum and cerebrospinal fluid of a patient with recurrent acute episodes of respiratory crises, autonomic symptoms and total insomnia (agrypnia), we identified a novel anti-neural complement fixing antibody directed against GABA(B) receptor (GABA(B)R). Patient purified IgG recognized a band of approximately 110 kDa on protein extracts of mouse cerebellum, cortex and brainstem and immunolabelled cultured Chinese hamster ovary (CHO) cells, transfected with human GABA(B)R1 and rat GABA(B)R2 receptors. Western blot analysis of transfected CHO homogenates showed the same band using both patient purified IgG and anti-GABA(B)R1 antibody. In order to verify the pathogenic role of these purified antibodies, we injected patient IgG intrathecally into cisterna magna of C57BL/6 mice pre-implanted with EEG electrodes and we observed severe ataxia followed by breathing depression and total suppression of slow wave sleep, as evidenced by EEG recording, in a dose-dependent manner. Immunohistochemistry on brain sections of mice injected with patient IgG showed the simultaneous presence of bound human IgG and C5b-9 deposits on Purkinje cells and cerebellar granular layer. After incubation with anti-GABA(B)R antibody, a marked reduction of receptor immunostaining was found with relative sparing of neuronal architecture. In conclusion we recognized an anti-neuronal autoantibody directed against GABA(B)R that is associated with autoimmune agrypnia and we showed that our patient purified IgG was able to induce in mice experimental autoimmune agrypnia characterized by a complex neurological syndrome affecting several CNS functions.


The punctate sites of accumulation of vaccinia virus early proteins are precursors of sites of viral DNA synthesis.
Several vaccinia virus early proteins (encoded by genes B1R, H5R and I3L) synthesized in the presence of an inhibitor of DNA synthesis localize, at least in part, to punctate inclusions that are visible by immunofluorescence in the cytoplasm of poxvirus-infected cells. It is shown that these inclusions contain DNA (visualized by DAPI staining of the infected cells) and that the number of inclusions is proportional to the amount of input virus. Their mean diameter (about 680 nm) was larger than that of purified vaccinia virus particles. When the inhibition of DNA synthesis was reversed, incorporation of BrdU into the B1R particles was demonstrated after labelling for 30 min, suggesting that these cytoplasmic focal sites correspond to viral DNA replication complexes that have initiated normally but are inhibited at the step of DNA chain elongation. These experiments suggest strongly that these inclusions are the precursors of the virosomes.


Reduction remediation of hexavalent chromium by bacterial flora in Cr(VI) aqueous solution.
Chromium(VI) is a priority pollutant in soils and wastewaters and reduction of Cr(VI) to Cr(III) is a solution to this problem. In this study a low-cost method was proposed to adapt indigenous bacteria and use them to reduce Cr(VI) in solutions. The experiment results show that Cr(VI) could be efficiently reduced by indigenous bacteria under anaerobic and pH-unadjusted conditions. After about 24 h the concentration of Cr(VI) could be reduced from 21.74 mg/L to below 0.5 mg/L. The observed Cr(VI) reduction rates were affected by temperature and pH. Cr(VI) in aqueous solutions could be reduced to Cr(III) completely and partly be incepted by the organisms. Cr(VI) reduction was enzyme-mediated. It was not an energy-conserving process but a detoxification reaction. This method could be used in an anaerobic reactor to treat low-concentration wastewater or industrial water as the last step.


Caregiving Subgroups Differences in the Associations Between the Resilience Resources and Life Satisfaction.
Using a model of resilience, this study compared the direct and indirect associations between resilience resources (sense of mastery, openness to experience, emotion regulation, and social support) and life satisfaction among caregiving subgroups (spouses, adult-children, and parents). Participants were included from the survey of Midlife in the United States (MIDUS II). Estimates of direct and indirect relationships between the resources and life satisfaction were calculated for each subgroup, and differences in the relationships between subgroups were tested. The direct positive relationships between sense of mastery and life satisfaction were significant and stronger for spouses and parents than for adult-children. In contrast, an indirect relationship through social support between the two variables was stronger for adult-children than for spouses. Openness to experience had a direct positive link to life satisfaction among spouses, and emotion regulation was directly related to life satisfaction among parents. We suggested targeted interventions for caregiving subgroups.


Emergency nurses' knowledge of perceived barriers in pain management in Taiwan.
To explore knowledge of and perceived barriers to pain management among emergency nurses in Taiwan. Pain is the most common patient complaint in emergency departments. Quality care of these patients depends on the pain knowledge and pain management skills of emergency nurses. However, no studies have explored emergency nurses' knowledge of and perceived barriers to pain management in Taiwan. Nurse subjects (n = 249) were recruited from nine hospitals chosen by stratified sampling across Taiwan. Data were collected using the Nurses' Knowledge and Attitudes Survey-Taiwanese version, a scale to assess perceived barriers to pain management and a background information form. The overall average correct response rate for the knowledge scale was 49.2%, with a range of 4.8-89.2% for each survey question. The top barrier to managing pain was identified by these nurses as 'the responsibility of caring for other acutely ill patients in addition to a patient with pain. Knowledge of pain management had a significant, negative relationship with perceived barriers to pain management and a significant, positive relationship with extent of clinical care experience and total hours of prior pain management education. In addition, scores for knowledge and perceived barriers differed significantly by the nursing clinical ladder. Perceived barriers also differed significantly by hospital accreditation category. Our results indicate an urgent need to strengthen pain education for emergency nurses in Taiwan. The pain education should target knowledge deficits and barriers to changing pain management approaches for Taiwanese emergency nurses.


Using 3D Modeling Techniques to Enhance Teaching of Difficult Anatomical Concepts.
Anatomy is an essential component of medical education as it is critical for the accurate diagnosis in organs and human systems. The mental representation of the shape and organization of different anatomical structures is a crucial step in the learning process. The purpose of this pilot study is to demonstrate the feasibility and benefits of developing innovative teaching modules for anatomy education of first-year medical students based on three-dimensional (3D) reconstructions from actual patient data. A total of 196 models of anatomical structures from 16 anonymized computed tomography datasets were generated using the 3D Slicer open-source software platform. The models focused on three anatomical areas: the mediastinum, the upper abdomen, and the pelvis. Online optional quizzes were offered to first-year medical students to assess their comprehension in the areas of interest. Specific tasks were designed for students to complete using the 3D models. Scores of the quizzes confirmed a lack of understanding of 3D spatial relationships of anatomical structures despite standard instruction including dissection. Written task material and qualitative review by students suggested that interaction with 3D models led to a better understanding of the shape and spatial relationships among structures, and helped illustrate anatomical variations from one body to another. The study demonstrates the feasibility of one possible approach to the generation of 3D models of the anatomy from actual patient data. The educational materials developed have the potential to supplement the teaching of complex anatomical regions and help demonstrate the anatomical variation among patients.


Discordance of voluntary HIV testing with HIV sexual risk-taking and self-perceived HIV infection risk among social media-using black, Hispanic, and white young-men-who-have-sex-with-men (YMSM).
Discordance between self-perceived HIV risk and actual risk-taking may impede efforts to promote HIV testing among young adult men-who-have-sex-with-men (YMSM) in the United States (US). Understanding the extent of, and reasons for, the discordance of HIV risk self-perception, HIV risk-taking and voluntary HIV testing among black, Hispanic and white YMSM could aid in the development of interventions to increase HIV testing among this higher HIV risk population. HIV-uninfected 18-24-year-old black, Hispanic, and white YMSM were recruited from across the US through multiple social media websites. Participants were queried about their voluntary HIV testing history, perception of currently having an undiagnosed HIV infection, and condomless anal intercourse (CAI) history. We assessed the association between previous CAI and self-perceived possibility of currently having an HIV infection by HIV testing status using Cochran-Mantel-Haenszel testing. Of 2275 black, Hispanic and white social media-using 18-24 year-old YMSM, 21% had never been tested for HIV voluntarily, 87% ever had CAI with another man, 77% believed that it was perhaps possible (as opposed to not possible at all) they currently could have an undiagnosed HIV infection, and 3% who reported CAI with casual or exchange partners, but had not been tested for HIV, self-perceived having no possibility of being HIV infected. Of 471 YMSM who had not been HIV tested, 57% reported CAI with casual or exchange partners, yet self-perceived having no possibility of being HIV infected. Per the Cochran-Mantel-Haenszel test results, among those reporting HIV risk behaviors, the self-perception of possibly being HIV-infected was not greater among those who had never been tested for HIV, as compared to those who had been tested. Future interventions should emphasize promoting self-realization of HIV risk and translating that into seeking and accepting voluntary HIV testing among this higher HIV risk population.


Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study.
This study investigated the efficacy, safety, and clinical benefit of alemtuzumab (Campath-1H) for patients with relapsed or refractory B-cell chronic lymphocytic leukemia exposed to alkylating agents and having failed fludarabine therapy. Ninety-three patients received alemtuzumab in 21 centers worldwide, with the aim to obtain an overall response rate of at least 20%. Dosage was increased gradually (target 30 mg, 3 times weekly, for a maximum of 12 weeks). Infection prophylaxis was mandatory, beginning on day 8, and continuing for a minimum of 2 months after treatment. Responses were assessed at weeks 4, 8, and 12, and patients were followed for 34 months. Overall objective response in the intent-to-treat population (n = 93) was 33% (CR 2%, PR 31%). Median time to response was 1.5 months (range, 0.4-3.7 months). Median time to progression was 4.7 months overall, 9.5 months for responders. At data cut-off, 27 patients (29%) were alive; overall median survival was 16 months (95% CI: 11.8-21.9) and 32 months for responders. Nineteen responders survived more than 21 months. Clinical benefit was observed both in responders and in patients with stable disease. The most common adverse events were related to infusion, generally grade 1 or 2 in severity, occurring mainly in the first week. Grade 3 or 4 infections were reported in 25 patients (26.9%). However, only 3 (9.7%) of 31 patients who responded to alemtuzumab treatment developed grade 3 or 4 infections on the study. Alemtuzumab induced significant responses in these patients with clinical benefit in the majority and with acceptable toxicity in a high-risk group.


Nitric oxide acutely modulates hypothalamic and neurohypophyseal carbon monoxide and hydrogen sulphide production to control vasopressin, oxytocin and atrial natriuretic peptide release in rats.
Nitric oxide (NO) negatively modulates the secretion of vasopressin (AVP), oxytocin (OT) and atrial natriuretic peptide (ANP) induced by the increase in extracellular osmolality, whereas carbon monoxide (CO) and hydrogen sulphide (H2 S) act to potentiate it; however, little information is available for the osmotic challenge model about whether and how such gaseous systems modulate each other. Therefore, using an acute ex vivo model of hypothalamic and neurohypophyseal explants (obtained from male 6/7-week-old Wistar rats) under conditions of extracellular iso- and hypertonicity, we determined the effects of NO (600 μmol L-1 sodium nitroprusside), CO (100 μmol L-1 tricarbonylchloro[glycinato]ruthenium [II]) and H2 S (10 mmol L-1 sodium sulphide) donors and nitric oxide synthase (NOS) (300 μmol L-1 Nω -methyl-l-arginine [LNMMA]), haeme oxygenase (HO) (200 μmol L-1 Zn(II) deuteroporphyrin IX 2,4-bis-ethylene glycol [ZnDPBG]) and cystathionine β-synthase (CBS) (100 μmol L-1 aminooxyacetate [AOA]) inhibitors on the release of hypothalamic ANP and hypothalamic and neurohypophyseal AVP and OT, as well as on the activities of NOS, HO and CBS. LNMMA reversed hyperosmolality-induced NOS activity, and enhanced hormonal release by the hypothalamus and neurohypophysis, in addition to increasing CBS and hypothalamic HO activity. AOA decreased hypothalamic and neurohypophyseal CBS activity and hormonal release, whereas ZnDPBG inhibited HO activity and hypothalamic hormone release; however, in both cases, AOA did not modulate NOS and HO activity and ZnDPBG did not affect NOS and CBS activity. Thus, our data indicate that, although endogenous CO and H2 S positively modulate AVP, OT and ANP release, only NO plays a concomitant role of modulator of hormonal release and CBS activity in the hypothalamus and neurohypophysis and that of HO activity in the hypothalamus during an acute osmotic stimulus, which suggests that NO is a key gaseous controller of the neuroendocrine system.


Friendship as a social mechanism influencing body mass index (BMI) among emerging adults.
Social relationships have been proposed as a significant factor shaping obesity risk. The first year of college, a period of major social, behavioral, and weight changes, provides a context well-suited to tracking longitudinally the impact of shifting friendships on weight outcomes. This study sought to identify social mechanisms impacting BMI change among emerging adults. An analytic sample of 276 college students (71.0% female, 52.2% non-White) provided repeated reports of relationships and BMI was measured up to four times during 2015-2016. Stochastic actor-oriented models were used to examine change in BMI through social influence and change in friendships over time, controlling for sex and race/ethnicity. At baseline, mean BMI was 24.2±4.5 kg/m2. Overall, mean BMI increased over time; individual decreases in BMI were uncommon. There was a selection effect of BMI: participants with BMIs between 22 and 26 kg/m2 were most likely to be nominated as a friend. While participants did not select friends based on BMI similarity, participants who were reported as friends were more likely to experience convergence in BMI over time relative to the BMIs of non-friends (p = 0.015). An increase in BMI (versus stability or a decrease) was more likely for those whose friends had a higher BMI on average compared to participants whose friends had the same or lower BMI (OR = 2.85, 95% CI = 1.22, 6.71). Analyses indicated BMI affected friend selection, not through students selecting friends with similar BMI, but rather, by students avoiding friends with more extreme BMI levels.


Traumatic Spinal Cord Injury-Repair and Regeneration.
Traumatic spinal cord injuries (SCI) have devastating consequences for the physical, financial, and psychosocial well-being of patients and their caregivers. Expediently delivering interventions during the early postinjury period can have a tremendous impact on long-term functional recovery. This is largely due to the unique pathophysiology of SCI where the initial traumatic insult (primary injury) is followed by a progressive secondary injury cascade characterized by ischemia, proapoptotic signaling, and peripheral inflammatory cell infiltration. Over the subsequent hours, release of proinflammatory cytokines and cytotoxic debris (DNA, ATP, reactive oxygen species) cyclically adds to the harsh postinjury microenvironment. As the lesions mature into the chronic phase, regeneration is severely impeded by the development of an astroglial-fibrous scar surrounding coalesced cystic cavities. Addressing these challenges forms the basis of current and upcoming treatments for SCI. This paper discusses the evidence-based management of a patient with SCI while emphasizing the importance of early definitive care. Key neuroprotective therapies are summarized including surgical decompression, methylprednisolone, and blood pressure augmentation. We then review exciting neuroprotective interventions on the cusp of translation such as Riluzole, Minocycline, magnesium, therapeutic hypothermia, and CSF drainage. We also explore the most promising neuroregenerative strategies in trial today including Cethrin™, anti-NOGO antibody, cell-based approaches, and bioengineered biomaterials. Each section provides a working knowledge of the key preclinical and patient trials relevant to clinicians while highlighting the pathophysiologic rationale for the therapies. We conclude with our perspectives on the future of treatment and research in this rapidly evolving field.


A Donor Utilization Index to Assess the Utilization and Discard of Deceased Donor Kidneys Perceived as High Risk.
An increasing number of patients on the waitlist for a kidney transplant indicates a need to effectively utilize as many deceased donor kidneys as possible while ensuring acceptable outcomes. Assessing regional and center-level organ utilization with regards to discard can reveal regional variation in suboptimal deceased donor kidney acceptance patterns stemming from perceptions of risk. We created a weighted donor utilization index from a logistic regression model using high-risk donor characteristics and discard rates from 113,640 deceased donor kidneys procured for transplant from 2010 to 2016, and used it to examine deceased donor kidney utilization in 182 adult transplant centers with >15 annual deceased donor kidney transplants. Linear regression and correlation were used to analyze differences in donor utilization indexes. The donor utilization index was found to significantly vary by Organ Procurement and Transplantation Network region (P<0.001), revealing geographic trends in kidney utilization. When investigating reasons for this disparity, there was no significant correlation between center volume and donor utilization index, but the percentage of deceased donor kidneys imported from other regions was significantly associated with donor utilization for all centers (rho=0.39; P<0.001). This correlation was found to be particularly strong for region 4 (rho=0.83; P=0.001) and region 9 (rho=0.82; P=0.001). Additionally, 25th percentile time to transplant was weakly associated with the donor utilization index (R2=0.15; P=0.03). There is marked center-level variation in the use of deceased donor kidneys with less desirable characteristics both within and between regions. Broader utilization was significantly associated with shorter time to transplantation.


Pilot trial of low-dose naltrexone and quality of life in multiple sclerosis.
To evaluate the efficacy of 4.5mg nightly naltrexone on the quality of life of multiple sclerosis (MS) patients. This single-center, double-masked, placebo-controlled, crossover study evaluated the efficacy of 8 weeks of treatment with 4.5mg nightly naltrexone (low-dose naltrexone, LDN) on self-reported quality of life of MS patients. Eighty subjects with clinically definite MS were enrolled, and 60 subjects completed the trial. Ten withdrew before completing the first trial period: 8 for personal reasons, 1 for a non-MS-related adverse event, and 1 for perceived benefit. Database management errors occurred in 4 other subjects, and quality of life surveys were incomplete in 6 subjects for unknown reasons. The high rate of subject dropout and data management errors substantially reduced the trial's statistical power. LDN was well tolerated, and serious adverse events did not occur. LDN was associated with significant improvement on the following mental health quality of life measures: a 3.3-point improvement on the Mental Component Summary score of the Short Form-36 General Health Survey (p = 0.04), a 6-point improvement on the Mental Health Inventory (p < 0.01), a 1.6-point improvement on the Pain Effects Scale (p =.04), and a 2.4-point improvement on the Perceived Deficits Questionnaire (p = 0.05). LDN significantly improved mental health quality of life indices. Further studies with LDN in MS are warranted.


Pivotal Advance: Phospholipids determine net membrane surface charge resulting in differential localization of active Rac1 and Rac2.
In this investigation, we used primary murine neutrophils to demonstrate that local changes in membrane phospholipid composition alter the net cytoplasmic membrane surface charge, which results in selective recruitment of Rac1 or Rac2 based on the net charge of their respective C-terminal domains. Murine neutrophils undergoing chemotaxis or carrying out phagocytosis were transfected with K-ras4B-derived membrane charge biosensors and lipid markers, which allowed us to simultaneously monitor the levels of PIP(2), PIP(3), and PS and net membrane charge of the newly developing phagosome membrane and plasma membrane. Our results indicate that the combination of PIP(2), PIP(3), and PS generates a high negative charge (-8) at the plasma membrane of actin-rich pseudopods, where active Rac1 preferentially localizes during phagosome formation. The lipid metabolism that occurs during phagosome maturation results in the localized depletion of PIP(2), PIP(3), and partial decrease in PS. This creates a moderately negative net charge that correlates with the localization of active Rac2. Conversely, the accumulation of PIP(3) at the leading-edge membrane during chemotaxis generates a polarized accumulation of negative charges that recruits Rac1. These results provide evidence that alterations in membrane lipid composition and inner-membrane surface charge are important elements for the recruitment of differentially charged proteins and localization of signaling pathways during phagocytosis and chemotaxis in neutrophils.


Level of agreement between questionnaire measures of alcohol dependence, alcoholism and problem drinking in a sample presenting at a specialist alcohol treatment service.
Two questionnaire measures of the alcohol dependence syndrome, one of alcoholism, and one of alcohol-related problems were compared in a sample of 101 subjects attending a specialist alcohol treatment unit. There was substantial agreement between the two measures of alcohol dependence, and lesser agreement between any other pairing. The measures of alcohol dependence also showed the strongest correlation with reported weekly alcohol consumption. The findings are compared with previous research, and the suitability of the various questionnaires as case-defining instruments in empirical investigations is discussed.


Phosphoramidites of base-modified 2'-deoxyinosine isosteres and solid-phase synthesis of d(GCI*CGC) oligomers containing an ambiguous base.
Novel phosphoramidites (1,2) of appropriately protected 2'-deoxyinosine isosteres (I*) such as allopurinol 2'-deoxyribofuranoside (4a) and 7-deaza-2'-deoxyinosine (4b) have been synthesized. They were employed together with the phosphoramidite of 2'-deoxyinosine in solid-phase synthesis of d(GCI*CGC) hexamers (12a-d). From thermodynamic data of these alternating hexamers it was shown that allopurinol 2'-deoxyribofuranoside destabilizes such duplexes less strongly than 2'-deoxyinosine. Additionally, the phosphoramidite of 7-deaza-2'-deoxyinosine (2) exhibits an extraordinary stability of the N-glycosylic bond. Since the new phosphoramidites are structurally related to 2'-deoxyinosine, they can be used in the construction of hybridization probes containing an ambiguous base.


Emotional fluidity in chronic care.
In this exploratory opinion piece, we define emotional fluidity as a state of changing emotions which influence choice of, attitudes and behaviour towards, and response to, chronic health care and therapy. This manifests clinically as changing opinions regarding suitability of modern health care, and as or varying, responses to suggestions and interventions from the health care team. Diabetes distress is an extreme example of emotional fluidity. Emotional fluidity also encompasses the variability of the health care professional's response to emotional and medical needs of persons seeking care. This can be inter-individual or intra-individual, and can be influenced by compassion fatigue. Health care professionals should be aware of emotional fluidity, and should be able to address its negative impact, as well as amplify its positive influence.


Assessment of Long-term Follow-up of Randomized Trial Participants by Linkage to Routinely Collected Data: A Scoping Review and Analysis.
Follow-up of participants in randomized trials may be limited by logistic and financial factors. Some important randomized trials have been extended well beyond their original follow-up period by linkage of individual participant information to routinely collected data held in administrative records and registries. To perform a scoping review of randomized clinical trials extended by record linkage to characterize this literature and explore any additional insights into treatment effectiveness provided by long-term follow-up using record linkage. A literature search in Embase, CINAHL, MEDLINE, and the Cochrane Register of Controlled Trials was performed for the period January 1, 1945, through November 25, 2016. Various combinations of search terms were used, as there is no accepted terminology. Determination of study eligibility and extraction of information about trial characteristics and outcomes, for both original and extended trial reports, were performed in duplicate. Assessment of study eligibility and data extraction were performed independently by 2 reviewers. All analyses were descriptive. Outcomes in the pairs of original and extended trials were categorized according to whether any benefits or harms from interventions were sustained, were lost, or emerged during long-term follow-up. A total of 113 extended trials were included in the study. Linkage to administrative and registry data extended follow-up by between 1 and 55 years. The most common interventions were pharmaceuticals (47 [41.6%]), surgery (19 [16.8%]), and disease screening (19 [16.8%]). End points most frequently studied through record linkage included mortality (88 [77.9%]), cancer (41 [36.3%]), and cardiovascular events (37 [32.7%]). One hundred four trial extensions (92.0%) were analyzed according to the original trial randomization. The reports provided details of 155 analyses of study outcomes. Seventy-four analyses (47.7%) identified statistically significant benefits in the trial extension phase. In 21 of these (28.4%), benefits were significant only in this period. Null results in both the original and extended trials were seen in 34 of the analyses (21.9%). Loss of significant benefits of an intervention were seen in 12 analyses (7.7%). Statistically significant harms were seen in 16 trial extension analyses (10.3%), and in 14 of these (87.5%), the harms were significant only in the trial extension phase. Trial extension by linkage to routinely collected data is a versatile underused approach that may add critical insights beyond those of the original trial. Some beneficial and harmful outcomes of interventions are captured only in the extension phase of randomized trials.


Implications of ventricular arrhythmia "bursts" with normal epicardial flow, myocardial blush, and ST-segment recovery in anterior ST-elevation myocardial infarction reperfusion: a biosignature of direct myocellular injury "downstream of downstream".
Establishing epicardial flow with percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction (STEMI) is necessary but not sufficient to ensure nutritive myocardial reperfusion. We evaluated whether adding myocardial blush grade (MBG) and quantitative reperfusion ventricular arrhythmia "bursts" (VABs) surrogates provide a more informative biosignature of optimal reperfusion in patients with Thrombolysis in Myocardial Infarction (TIMI) 3 flow and ST-segment recovery (STR). Anterior STEMI patients with final TIMI 3 flow had protocol-blinded analyses of simultaneous MBG, continuous 12-lead electrocardiogram (ECG) STR, Holter VABs, and day 5-14 SPECT imaging infarct size (IS) assessments. Over 20 million cardiac cycles from >4500 h of continuous ECG monitoring in subjects with STR were obtained. IS and clinical outcomes were examined in patients stratified by MBG and VABs. VABs occurred in 51% (79/154) of subjects. Microcirculation (MBG 2/3) was restored in 75% (115/154) of subjects, of whom 53% (61/115) had VABs. No VABs were observed in subjects without microvascular flow (MBG of 0). Of 115 patients with TIMI 3 flow, STR, and MBG 2/3, those with VABs had significantly larger IS (median: 23.0% vs 6.0%, p=0.001). Multivariable analysis identified reperfusion VABs as a factor significantly associated with larger IS (p=0.015). Despite restoration of normal epicardial flow, open microcirculation, and STR, concomitant VABs are associated with larger myocardial IS, possibly reflecting myocellular injury in reperfusion settings. Combining angiographic and ECG parameters of epicardial, microvascular, and cellular response to STEMI intervention provides a more predictive "biosignature" of optimal reperfusion than do single surrogate markers.


Chloride transport inhibition by piretanide and MK-196 in bullfrog corneal epithelium.
Two new diuretic agents, piretanide and MK-196, inhibited short-circuit current (SCC) across the isolated frog corneal epithelium. The effect is explained as an inhibition of active Cl transport. A definite decrease in SCC and an increase in electrical resistance was observed with both diuretics in concentrations as low as 10(-6) M. Piretanide, at 10(-4) M, reduced the SCC by 90%, reduced th unidirectional forward Cl flux from 0.60 to 0.28 mueq x h-1 x cm-2, and increased the resistance by 60%. There was no effect on the Cl backflux. At 10(-4) M, MK-196 reduced the SCC by 83% and increased the resistance by 72%, from 1.68 to 2.91 k omega x cm2. Replacement of Cl by SO4 in the bathing solutions resulted in a larger increase in resistance, from 1.68 to 3.80 k omega x cm2. The diuretics had no effect on active Na transport across the corneal epithelium. After the permeability of the apical side was increased by amphotericin B, the drugs could not inhibit the Cl-originated SCC. These results suggest that piretanide and MK-196 selectively inhibit active Cl transport in the cornea by blocking Cl permeability of the apical side of the epithelial cells.


Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-independent inhibitors.
Thrombolytic therapy induces a procoagulant state characterized by elevated plasma levels of fibrinopeptide A (FPA), but the responsible mechanism is uncertain. Washed plasma clots were incubated in citrated plasma in the presence or absence of tissue plasminogen activator (t-PA), and FPA generation was monitored as an index of unopposed thrombin activity. FPA levels are almost twofold higher in the presence of t-PA than in its absence. This primarily reflects the action of thrombin bound to soluble fibrin degradation products because (a) there is progressive FPA generation even after clots are removed from t-PA-containing plasma, and (b) clot lysates produce concentration-dependent FPA generation when incubated in citrated plasma. Using thrombin-agarose affinity chromatography, (DD)E and fragment E but not D-dimer were identified as the thrombin-binding fibrin fragments, indicating that the thrombin-binding site is located within the E domain. Heparin inhibits thrombin bound to fibrin degradation products less effectively than free thrombin. In contrast, D-Phe-Pro-ArgCH2Cl, hirudin and hirugen inhibit free thrombin and thrombin bound to fibrin degradation products equally well. Thrombin bound to soluble fibrin degradation products is primarily responsible for the increase in FPA levels that occurs when a clot undergoes t-PA-induced lysis. Like clot-bound thrombin, thrombin bound to fibrin derivatives is protected from inhibition by heparin but susceptible to inactivation by direct thrombin inhibitors. These findings help to explain the superiority of direct thrombin inhibitors over heparin as adjuncts to thrombolytic therapy.


The youngest children in each school cohort are overrepresented in referrals to mental health services.
To investigate whether the youngest children in each school cohort are overrepresented as users of specialist mental health services. Dates of birth were obtained for all 9,157 children and adolescents referred to specialist mental health services in 3 London boroughs from 2008 to 2011. The actual frequency of referrals by month of birth is compared to the expected frequency of referrals as determined by birth statistics for the relevant age group. August-born children, who are the youngest in their cohorts in England, represent 9.38% of referrals but only 8.59% of the population in the relevant age segment. Hence, August-born children are overrepresented in referrals to specialist mental health services (P value = .007). September- and October-born children, who are the oldest in their cohorts, are underrepresented: September-born children represent 8.62% of the population but 7.99% of referrals to mental health services (P value = .032), and October-born children are 8.56% of the population but 7.86% of referrals (P value = .016). Being among the youngest in a school cohort is associated with a higher risk of referral to mental health services, while being among the oldest is a protective factor.


Rectal Eversion Technique: A Method to Achieve Very Low Rectal Transection and Anastomosis With Particular Value in Laparoscopic Cases.
Transection of the rectum at the anorectal junction is required for proper resection in ulcerative colitis and restorative proctocolectomy. Achieving stapled transection at the pelvic floor is often challenging, particularly during laparoscopic proctectomy. Transanal mucosectomy and handsewn anastomosis are frequently used to achieve adequate resection. Rectal eversion provides an alternative for low anorectal transection and maintains the ability to perform stapled anastomosis. The purpose of this article is to describe a technique for low anorectal transection. The work was conducted at tertiary care center by 2 colon and rectal surgeons on patients undergoing total proctocolectomy with creation of ileal pouch rectal anastomosis for ulcerative colitis. We measured the ability to achieve low stapled anastomosis. Very low transection was achieved, allowing for creation of IPAA without leaving significant rectal cuff. This study was limited because it is an early experience that was not performed in the setting of a scientific investigation. No sphincter or bowel functional data were obtained or evaluated. Rectal eversion technique provides an alternative to mucosectomy when low pelvic transection is difficult to achieve. See Video at http://links.lww.com/DCR/A441.


Identification of a novel cis-acting element conferring sulfur deficiency response in Arabidopsis roots.
SULTR1;1 high-affinity sulfate transporter is highly regulated in the epidermis and cortex of Arabidopsis roots responding to sulfur deficiency (-S). We identified a novel cis-acting element involved in the -S-inducible expression of sulfur-responsive genes in Arabidopsis. The promoter region of SULTR1;1 was dissected for deletion and gain-of-function analysis using luciferase (LUC) reporter gene in transgenic Arabidopsis. The 16-bp sulfur-responsive element (SURE) from -2777 to -2762 of SULTR1;1 promoter was sufficient and necessary for the -S-responsive expression, which was reversed when supplied with cysteine and glutathione (GSH). The SURE sequence contained an auxin response factor (ARF) binding sequence (GAGACA). However, SURE was not responsive to naphthalene acetic acid, indicating its specific function in the sulfur response. The base substitution analysis indicated the significance of a 5-bp sequence (GAGAC) within the conserved ARF binding site as a core element for the -S response. Microarray analysis of early -S response in Arabidopsis roots indicated the presence of SURE core sequences in the promoter regions of -S-inducible genes on a full genome GeneChip array. It is suggested that SURE core sequences may commonly regulate the expression of a gene set required for adaptation to the -S environment.


Bridging the gap: western rock skinks (Trachylepis sulcata) have a short history in South Africa.
Phylogeographic patterns in wide-ranging species in southern Africa remain largely unexplored, especially in areas north of South Africa. Here, we investigate population structuring, demographic history, and the colonization pattern of the western rock skink (Trachylepis sulcata), a rock-dwelling species with a range extending from southwestern South Africa into Angola. Using 1056 bp from the mitochondrial marker ND2 and > 2.5 kb from three nuclear genes (EXPH5, KIF24, RAG-1), we constructed allele networks, generated extended Bayesian skyline plots and performed population clustering analyses. Analyses of historical demographic patterns show an overall southward range expansion from Northern Namibia into Southern Namibia and South Africa, although we find contrasting genetic breaks across these geographic regions using nuclear and mitochondrial data. We suggest that mtDNA has introgressed across a nuclear break corresponding to the Knersvlakte region of South Africa, a previously proposed biogeographic barrier for rupicolous species. This pattern of mitochondrial variation contrasts sharply to that of other South African taxa previously investigated, which all show significant mtDNA differentiation across the Knersvlakte region. Additionally, while other taxa show divergences dating to the Pliocene, T. sulcata appears to be a recent arrival in southern Africa, having crossed this barrier and colonized South Africa in the mid-Pleistocene. The complex phylogeographic history of T. sulcata corroborates the intricate patterns of genetic variation found in South African taxa and provides novel insight into historical processes affecting species distributed across Namibia.


Ventricular pacing site separation by cardiac computed tomography: validation for the prediction of clinical response to cardiac resynchronization therapy.
Cardiac Resynchronization Therapy (CRT) fails to provide benefit in up to one-third of patients. Maximizing the geographic separation of right and left ventricular pacing lead sites has been suggested as one way to improve response. Cardiac CT provides an opportunity to explore 3-dimensional inter-lead distance (ILD) measures for the prediction of CRT response. The objective of this study was to investigate associations between standardized measures of ILD by cardiac CT and echocardiographic response to CRT. Forty-two consecutive patients undergoing CRT had serial clinical and echocardiographic evaluations performed in addition to a post-procedural cardiac-gated CT with blinded measurement of direct and circumferential (via the myocardium) ILD measures. Clinical response to CRT, the primary clinical outcome, was defined as a ≥15% reduction in LVESV using echocardiography at 6-months. The mean age and ejection fraction was 63.6 ± 8.9 years and 25.2 ± 7.8%, respectively. The primary outcome occurred in 35 of 42 patients (83%). Both direct and circumferential CT-based ILD measures were associated with the primary outcome by univariate analysis. Receiver Operator Characteristic analysis identified Circumferential ILD to have the strongest predictive accuracy (AUC 0.78). Inter- and intra-observer reproducibility of CT-derived ILD measures was excellent. Circumferential ILD measures on cardiac CT are predictive of clinical response to CRT. Incorporation of these measures into the selection of optimal pacing targets, particularly from pre-procedural CT coronary vein imaging may be of therapeutic benefit and warrants further investigation.


[Epidemiology of human immunodeficiency virus infection and AIDS in Chile].
In Chile, the first cases of AIDS were reported 23 years ago, and since then, through December 2006, 17.235 persons have been notified with HIV infection or AIDS1. To the year 2005, there have been 5.288 fatal cases of AIDS. The last available data indicates that notification rates for AIDS and HIV infection in 2006 were 2.5 and 4.5 per 100.000 inhabitants, respectively, and mortality rate for AIDS in 2005 was 2.4 per 100.000 inhabitants. Trend analysis shows a decline in the notification rate among men, both for HIV infection and AIDS, which could be a real decrease or a sub notification bias. In Chile, like in other countries of the region, variations in the epidemiologic pattern were observed considering age group, gender, educational level and geographic distribution of the population. Currently, the Chilean Ministry of Health has implemented both a surveillance and monitoring system on line, in order to improve the quality and opportunity of the information, therefore providing an important tool to HIV infection/AIDS prevention and control strategies.


Layer-by-layer self-assembly of glucose oxidase and Os(Bpy)2CIPyCH2NH-poly(allylamine) bioelectrode.
The uptake of glucose oxidase (GOx) onto a polycationic redox polymer (PAA-Os)-modified surface, by adsorption from dilute aqueous GOx solutions, was followed by the quartz crystal microbalance (QCM) and shows double exponential kinetics. The electrochemistry of the layer-by-layer-deposited redox-active polymer was followed by cyclic voltammetry in glucose-free solutions, and the enzyme catalysis mediated by the redox polymer was studied in beta-D-glucose-containing solutions. AFM studies of the different layers showed the existence of large two dimension enzyme aggregates on the osmium polymer for 1 microM GOx and less aggregation for 50 nM GOx solutions. When the short alkanethiol, 2,2'-diaminoethyldisulfide was preadsorbed onto gold, a monoexponential adsorption law was observed, and single GOx enzyme molecules could be seen on the surface where the enzyme was adsorbed from 50 nM GOx in water.


Human CD3+ T lymphocytes that express neither CD4 nor CD8 antigens.
CD3+ T lymphocytes expressing neither CD4 nor CD8 antigens exist in normal human peripheral blood in low frequency (approximately 3% of lymphocytes). The CD3+,4-,8- phenotype was stably maintained after in vitro culture in IL-2. Culture of CD3+,4-,8- cells in only rIL-2 generated cytotoxic T cells that lysed NK-sensitive and NK-insensitive tumor cell targets without MHC restriction. These experiments clearly show that phenotypically and functionally competent T cells expressing neither CD4 nor CD8 are present in normal peripheral blood.


Surgical approaches to the treatment of early breast cancer.
Various methods have been explored for treating early breast cancer with conservation of the breast. Although local recurrence increased, early trials indicated that in patients with clinical stage I disease (T1 or T2 NO or N1a), wide excision and radiotherapy of the breast and gland fields resulted in the same distant recurrence and survival rates as the radical operation. It was suggested that local control of the disease was not important in the ultimate prognosis, that minimal surgery incurred no penalty and that breast cancer would soon be treated by radiotherapy alone. Four recent developments affected this forecast. 1. Assays of hormone receptors in primary tumours give important information on prognosis in early cancer. 2. In clinical stage II disease (T1 or T2 N1b), limited surgery exacts a penalty in terms of distant recurrence and survival. 3. The number in involved axillary lymph nodes must be known to evaluate prognosis 4. Adjuvant chemo- or endocrine therapy is effective in patients with extensive involvement of axillary nodes. Currently, only modified radical mastectomy effectively fulfils the requirements resulting from these developments.


Gout and the risk of Alzheimer's disease: a population-based, BMI-matched cohort study.
While gout is associated with cardiovascular (CV)-metabolic comorbidities and their sequelae, the antioxidant effects of uric acid may have neuroprotective benefits. We evaluated the potential impact of incident gout on the risk of developing Alzheimer's disease (AD) in a general population context. We conducted an age-matched, sex-matched, entry-time-matched and body mass index (BMI)-matched cohort study using data from The Health Improvement Network, an electronic medical record database representative of the UK general population, from 1 January 1995 to 31 December 2013. Up to five non-gout individuals were matched to each case of incident gout by age, sex, year of enrolment and BMI. We compared incidence rates of AD between the gout and comparison cohorts, excluding individuals with prevalent gout or dementia at baseline. Multivariate hazard ratios (HRs) were calculated, while adjusting for smoking, alcohol use, physician visits, social deprivation index, comorbidities and medication use. We repeated the same analysis among patients with incident osteoarthritis (OA) as a negative control exposure. We identified 309 new cases of AD among 59 224 patients with gout (29% female, mean age 65 years) and 1942 cases among 238 805 in the comparison cohort over a 5-year median follow up (1.0 vs 1.5 per 1000 person-years, respectively). Univariate (age-matched, sex-matched, entry-time-matched and BMI-matched) and multivariate HRs for AD among patients with gout were 0.71 (95% CI 0.62 to 0.80) and 0.76 (95% CI 0.66 to 0.87), respectively. The inverse association persisted among subgroups stratified by sex, age group (<75 and ≥75 years), social deprivation index and history of CV disease. The association between incident OA and the risk of incident AD was null. These findings provide the first general population-based evidence that gout is inversely associated with the risk of developing AD, supporting the purported potential neuroprotective role of uric acid.


Comparative effectiveness between mosunetuzumab monotherapy clinical trial and real-world data in relapsed/refractory follicular lymphoma in third or subsequent lines of systemic therapy.
A comparison of clinical outcomes in the third or subsequent line (3 L+) of systemic therapy between a real-world data (RWD) external control cohort and a mosunetuzumab single-arm clinical trial cohort is presented. Data for 3 L + patients with relapsed/refractory follicular lymphoma (FL) were obtained from the mosunetuzumab single-arm trial (n = 90) and a US electronic health records database (n = 158), with patients meeting key eligibility criteria from the trial, balanced on pre-specified prognostic factors. Overall response and complete response rates were 80% and 60% in the mosunetuzumab cohort and 75% and 33% in the RWD cohort, odds ratios of 1.23 (95% CI, 0.52-2.93) and 3.18 (95% CI, 1.41-7.17), respectively. Hazard ratios for progression-free survival and overall survival were 0.82 (95% CI, 0.53-1.27) and 0.43 (95% CI, 0.19-0.94). These findings support a clinically meaningful benefit of mosunetuzumab monotherapy as a chemotherapy-free option for the 3 L + FL population.


The Social Context of Partnered Older Adults' Insomnia Symptoms.
The current study included an examination of social factors that mitigate or exacerbate insomnia symptoms among older adults who are married or living with a partner. We first examined the unique effects of spousal support and strain on insomnia symptoms and then evaluated the degree to which extramarital social factors (e.g., friend support) moderated spousal influences. Data came from Waves 2 and 3 of the National Social Life, Health, and Aging Project. Our sample consisted of 495 participants who were either married or cohabitating with a partner (M age in years = 69.84, SD = 8.08). Spousal strain-but not support-predicted higher insomnia symptoms 5 years later. Spousal influences on sleep, however, were moderated by extramarital factors in nuanced ways. Findings highlight the importance of taking into account older adults' wider social context when examining the ways in which sleep is sensitive to positive and negative aspects of marital quality.


Measuring Optic Nerve Sheath Diameter with Bedside Ocular Ultrasonography in Patients with Carbon Monoxide Poisoning.
To investigate the relationship between optic nerve sheath diameter (ONSD) and clinical and carboxyhemoglobin levels in patients with carbon monoxide poisoning. This prospective cross-sectional study enrolled 55 consecutive adult patients with carbon monoxide poisoning. The demographic and clinical characteristics of the patients and the diameters of the optic nerve sheaths of both eyes of those patients at the time of admission and at the 6th hour after receiving 100% oxygen therapy and/or hyperbaric oxygen therapy were determined. The ages, genders, arterial blood pressure values, symptoms, carboxyhemoglobin levels, and bilateral ONSD values (measured sonographically before and after the oxygen therapy) of the patients were recorded in a data collection form. Measurements of ONSD before and after treatment were observed significantly (P = .01). Significant decreases were observed in ONSDs after treatment (P < .05). However, no significant difference was observed between ONSDs at the time of admission and after the treatment of patients receiving 2-hour single-session hyperbaric oxygen therapy and 100% oxygen therapy with a reservoir mask (P > .05). The changing of ONSD, which is an indirect indicator of increased intracranial pressure, is a promising method to use at emergency service for patients with carbon monoxide poisoning.


Electrospray ionization with higher-energy collision dissociation tandem mass spectrometry toward characterization of ceramides as [M + Li]+ ions: Mechanisms of fragmentation and structural identification.
Ceramide is a huge lipid family consisting of diversified structures in which various modifications are seen in the fatty acyl chain and the long chain base (LCB). In this contribution, a higher collision energy (HCD) linear ion-trap mass spectrometric method (LIT MSn) was applied to study the mechanisms underlying the fragmentation processes of ceramide molecules in 12 subclasses, which were desorbed by ESI as the [M + Li]+ ions. Multiple sets of fragment ions reflecting the fatty acyl chain and LCB were observed in the HCD MS2 spectra for all the ceramide classes, resulting in unambiguous definition of the ceramide structures, including the chain length and the modification (α-hydroxy-, β-hydroxy-, ω-hydroxy-FA) of the fatty acyl moiety, and the types of LCB (sphingosine, phytosphigosine, 6-hydroxy-sphingosine). Thereby, this approach permits differentiation of isomeric structures and ceramide species in the biological specimen can be unveiled in detail. By application of sequential MS3, the double bond position along the fatty acyl chain of the molecule can be located, and a complete structural characterization of ceramides can be achieved.


The impact of proton-pump inhibitors administered with tyrosine kinase inhibitors in patients with metastatic renal cell carcinoma.
Tyrosine kinase inhibitors (TKIs) are the backbone of the systemic treatment for patients with metastatic renal cell carcinoma (mRCC). TKIs such as pazopanib and cabozantinib can interact with other drugs concomitantly administered, particularly with proton-pump inhibitors (PPIs), possibly impacting the effectiveness of the anticancer treatment and patients outcome. Few data are available about this interaction. We conducted a multicenter retrospective observational data collection of patients with mRCC treated with pazopanib or cabozantinib between January 2012 and December 2020 in nine Italian centers. Univariate and multivariate analyses were performed. The aim was to describe the impact of baseline concomitant PPIs on the outcome of patients to pazopanib and cabozantinib in terms of response, progression-free survival (PFS) and overall survival (OS), toxicity, and treatment compliance. The use of PPI in our study population (301 patients) significantly influenced the effectiveness of TKIs with worse PFS (16.3 vs. 9.9 months; P < 0.001) and OS (30.6 vs. 18.4 months; P = 0.013) in patients taking PPI at TKI initiation. This detrimental effect was maintained both in the pazopanib and cabozantinib groups. The use of PPI influenced the toxicity and TKI treatment compliance with a reduction of dose or schedule modifications, and treatment interruptions in the population taking PPIs. Our study demonstrates that the use of PPIs can significantly influence the outcome and compliance of patients with mRCC to TKI treatment, suggesting the importance of a more careful selection of patients who need a gastroprotective therapy, avoiding indiscriminate use of PPIs.


Verapamil preserves adenine nucleotide pool in cardiomyopathic Syrian hamster.
There is a decrease in total adenine nucleotides, cyclic AMP (cAMP), ATP/total ADP, and phosphocreatine (PCr)/creatine (Cr) both in situ and in the perfused heart in the heart failure stage of the cardiomyopathic Syrian hamster. There were decreases in developed pressure, dP/dt, and O2 consumption associated with the decrease in total adenine nucleotides and cAMP. Cardiomyopathic Syrian hamsters (180-240 days old) with congestive heart failure were given water with the calcium entry blocker, verapamil, as an additive 2 mo before death. In the cardiomyopathic group given verapamil the adenine nucleotides, cAMP, and high-energy phosphates were preserved and cardiac performance was not significantly different from that of the verapamil-treated healthy hamsters at the time of death. Pretreatment of cardiomyopathic animals with verapamil (6.6 mg verapamil/ml water consumed by drinking) resulted in significantly higher ATP/total ADP and PCr/Cr compared with nontreated cardiomyopathic hamsters. This is the first report demonstrating that a calcium entry blocker may improve cardiac performance and preserve total adenine nucleotides during the heart failure stage of the cardiomyopathic hamster.


Elevating the levels of Sox2 in embryonal carcinoma cells and embryonic stem cells inhibits the expression of Sox2:Oct-3/4 target genes.
Recent studies have identified large sets of genes in embryonic stem and embryonal carcinoma cells that are associated with the transcription factors Sox2 and Oct-3/4. Other studies have shown that Sox2 and Oct-3/4 work together cooperatively to stimulate the transcription of their own genes as well as a network of genes required for embryogenesis. Moreover, small changes in the levels of Sox2:Oct-3/4 target genes alter the fate of stem cells. Although positive feedforward and feedback loops have been proposed to explain the activation of these genes, little is known about the mechanisms that prevent their overexpression. Here, we demonstrate that elevating Sox2 levels inhibits the endogenous expression of five Sox2:Oct-3/4 target genes. In addition, we show that Sox2 repression is dependent on the binding sites for Sox2 and Oct-3/4. We also demonstrate that inhibition is dependent on the C-terminus of Sox2, which contains its transactivation domain. Finally, our studies argue that overexpression of neither Oct-3/4 nor Nanog broadly inhibits Sox2:Oct-3/4 target genes. Collectively, these studies provide new insights into the diversity of mechanisms that control Sox2:Oct-3/4 target genes and argue that Sox2 functions as a molecular rheostat for the control of a key transcriptional regulatory network.


Proteomics and transcriptomics of the BABA-induced resistance response in potato using a novel functional annotation approach.
Induced resistance (IR) can be part of a sustainable plant protection strategy against important plant diseases. β-aminobutyric acid (BABA) can induce resistance in a wide range of plants against several types of pathogens, including potato infected with Phytophthora infestans. However, the molecular mechanisms behind this are unclear and seem to be dependent on the system studied. To elucidate the defence responses activated by BABA in potato, a genome-wide transcript microarray analysis in combination with label-free quantitative proteomics analysis of the apoplast secretome were performed two days after treatment of the leaf canopy with BABA at two concentrations, 1 and 10 mM. Over 5000 transcripts were differentially expressed and over 90 secretome proteins changed in abundance indicating a massive activation of defence mechanisms with 10 mM BABA, the concentration effective against late blight disease. To aid analysis, we present a more comprehensive functional annotation of the microarray probes and gene models by retrieving information from orthologous gene families across 26 sequenced plant genomes. The new annotation provided GO terms to 8616 previously un-annotated probes. BABA at 10 mM affected several processes related to plant hormones and amino acid metabolism. A major accumulation of PR proteins was also evident, and in the mevalonate pathway, genes involved in sterol biosynthesis were down-regulated, whereas several enzymes involved in the sesquiterpene phytoalexin biosynthesis were up-regulated. Interestingly, abscisic acid (ABA) responsive genes were not as clearly regulated by BABA in potato as previously reported in Arabidopsis. Together these findings provide candidates and markers for improved resistance in potato, one of the most important crops in the world.


Crosstalk of four kinds of cell deaths defines subtypes of cutaneous melanoma for precise immunotherapy and chemotherapy.
Cell death patterns can give therapeutic and biological clues that facilitate the development of individualized treatments for this lethal form of skin cancer. We employed unsupervised clustering to establish robust classifications based on the four kinds of cell death-associated gene expression of 462 melanoma patients in the Cancer Genome Atlas (TCGA) and tested their reproducibility in two independent melanoma cohorts of 558 patients. We then used dimensionality reduction of graph learning to display the different characteristics of cell death patterns and immune microenvironments. We examined 570 cell death-associated gene expression data of melanoma patients for exploration, independent verification, and comprehensive classification of five reproducible melanoma subtypes (CS1 to CS5) with different genomic and clinical features. Patients in death-inactive subtypes (CS1, CS2, and CS5) had the least immune and stromal cell infiltration, and their prognosis was the poorest. A death-active subtype (CS4), on the other hand, had the highest infiltrated immune and stromal cells and elevated immune-checkpoints. As a result, these patients had the highest response to immunotherapy and the best prognosis. An additional subtype (CS3) had more diversified cell death and immune characteristics with moderate prognoses. Based on graph learning, we successfully divided the CS3 subtype into two subgroups (group A and group B) with distinct survival outcomes and immune features. Finally, we identified eight potential chemical drugs that were specifically targeted for the therapy of melanoma subtypes. This research defines the intrinsic subtypes of melanoma based on the crosstalk of four kinds of cell deaths, which affords a blueprint for clinical strategies and guiding precise immunotherapy and chemotherapy for melanoma patients.


Targeted lipid nanoparticles for RNA therapeutics and immunomodulation in leukocytes.
Abnormalities in leukocytes' function are associated with many immune related disorders, such as cancer, autoimmunity and susceptibility to infectious diseases. Recent developments in Genome-wide-association-studies give rise to new opportunities for novel therapeutics. RNA-based modalities, that allow a selective genetic manipulation in vivo, are powerful tools for personalized medicine, enabling downregulation or expression of relevant proteins. Yet, RNA-based therapeutics requires a delivery modality to facilitate the stability, uptake and intracellular release of the RNA molecules. The use of lipid nanoparticles as a drug delivery approach improves the payloads' stability, pharmacokinetics, bio-distribution and therapeutic benefit while reducing side effects. Moreover, a wide variety of targeting moieties allow a precise and modular manipulation of gene expression, together with the ability to identify and selectively affect disease-relevant leukocytes-subsets. Altogether, RNA-based therapeutics, targeting leukocytes subsets, is believed to be one of the most promising therapeutic concepts of the near future, addressing pressing issues in cancer and inflammation heterogeneity.


Evidence that Extreme Dilutions of Paclitaxel and Docetaxel Alter Gene Expression of In Vitro Breast Cancer Cells.
Gene expression analysis of cells treated with extreme dilutions or micro amounts of drugs has been used to provide useful suggestions about biological responses. However, most of the previous studies were performed on medicines being prepared from a variety of herbal and metal sources. This study investigated the effects of ultramolecular dilution of the taxane anti-cancer drugs, which are not commonly used in homeopathic medicines, on mRNA expression profiles of five key genes (p53, p21, COX-2, TUBB2A and TUBB3) in the breast cancer cell line MCF-7. MCF-7 cells were exposed to paclitaxel (Taxol) or docetaxel (Taxotere) preparations (6X, 5C and 15C dilutions prepared from pharmacological concentration of 25 nmol/L) for 72 hours. The cell culture groups were evaluated with the trypan blue dye exclusion method for the proliferation/cytotoxicity rates, immuno-staining β-tubulin for microtubule organization, and reverse transcription polymerase chain reaction for gene expression levels.Fold-change in gene expression was determined by the ΔΔCt method. The administration of diluted preparations had little or no cytotoxic effect on MCF-7 cells, but altered the expression of genes analyzed with a complex effect. According to the ΔΔCt method with a five-fold expression difference (p < 0.05) as a cut-off level, ultra-high dilutions of paclitaxel and docetaxel showed differential effects on the studied genes with a concentration-independent activity. Furthermore, the dilutions disrupted the microtubule structure of MCF-7 cells, suggesting that they retain their biological activity. Despite some limitations, our findings demonstrate that gene expression alterations also occur with ultra-high dilutions of taxane drugs.


Comparative genomic hybridization: a comparison with molecular and cytogenetic analysis.
Comparative genomic hybridization (CGH) is a powerful technique for detecting copy number changes throughout the genome. We describe the development of a versatile image analysis program for CGH studies. Several methods for the production of metaphases which give optimum hybridization signals have also been assessed. CGH analysis was performed on DNA samples from several different and clinically relevant specimens: amniotic fluid cells trisomic for a single chromosome, lymphoblastoid cell lines with abnormalities involving single chromosome bands, malignant cell lines and biopsy material from primary ovarian carcinomas. The results were compared with those derived from G-banding, chromosome painting, and molecular genetic techniques. Our data demonstrate that CGH was able to detect a wide range of quantitative genetic alterations including duplication or deletion of single chromosome bands. CGH analysis also indicated the presence of genetic abnormalities that were not detected by other cytogenetic or molecular approaches. Moreover, our CGH methodology allowed the ready comparison of CGH results from different tumors, a process which greatly facilitated identification of shared genetic changes.