Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate.
L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.


Is bovine dentine an appropriate substitute in abrasion studies?
The study aimed to compare the wear behaviour of human and bovine dentine due to toothbrushing with different relative dentin abrasivity (RDA) toothpastes. Forty human and 40 bovine dentine samples were prepared from bovine lower incisors or human premolars roots, and baseline surface profiles were recorded. The samples were distributed to four groups (each group n = 10 human and 10 bovine samples) and brushed with fluoridated experimental toothpastes with different RDAs (group A: RDA 10, B: RDA 20, C: RDA 50, and D: RDA 100). Toothbrushing was performed in an automatic brushing machine with a brushing frequency of 60 strokes per minute and a brushing force of 2.5 N. After 2, 5, 10, and 25 min of toothbrushing, new surface profiles were recorded, and the dentine wear was calculated with a customized computer programme. The dentine wear of human and bovine dentine within the four groups was compared with unpaired t tests. No statistically significant difference was recorded for the dentine wear of human and bovine samples within the different groups.


Black carbon footprint of human presence in Antarctica.
Black carbon (BC) from fossil fuel and biomass combustion darkens the snow and makes it melt sooner. The BC footprint of research activities and tourism in Antarctica has likely increased as human presence in the continent has surged in recent decades. Here, we report on measurements of the BC concentration in snow samples from 28 sites across a transect of about 2,000 km from the northern tip of Antarctica (62°S) to the southern Ellsworth Mountains (79°S). Our surveys show that BC content in snow surrounding research facilities and popular shore tourist-landing sites is considerably above background levels measured elsewhere in the continent. The resulting radiative forcing is accelerating snow melting and shrinking the snowpack on BC-impacted areas on the Antarctic Peninsula and associated archipelagos by up to 23 mm water equivalent (w.e.) every summer.


Morphologic features of chronic hepatitis associated with primary sclerosing cholangitis and chronic ulcerative colitis.
Histologic, ultrastructural, chemical, and statistical methods were used to study liver biopsy and autopsy specimens from 43 patients who had primary sclerosing cholangitis (PSC), with or without chronic ulcerative colitis (CUC), and from 19 patients who had CUC without PSC. In all study groups, essentially the same abnormalities were found in the hepatic parenchyma outside the major bile ducts, although nondiagnostic tissue samples were observed also. Specimens from patients with extrahepatic PSC were indistinguishable from those patients with combined extra- and intrahepatic PSC. Common findings included periductal fibrosis and inflammation, portal edema and fibrosis, focal proliferation of bile ducts and ductules, focal bile duct obliteration and loss of bile ducts, copper deposition, and cholestasis. Proliferation of bile ducts in some portal tracts and obliteration or absence of bile duct in others were the most characteristic changes. In most specimens, inflammatory changes appeared mild, yet biliary cirrhosis had developed in 34% of the patients. Specimens from patients with PSC, with or without CUC, more often contained bile and strikingly increased stainable copper (Grades 2 and 3) than did specimens from patients with CUC without PSC. Hepatic copper contents, measured by atomic absorption spectrophotometry, also were higher in specimens from patients with PSC. Study of PCS specimens by transmission electron microscopy and by energy-dispersive X-ray microanalysis revealed that most copper was sequestered in lipolysosomes. The recognition of strikingly similar morphologic features in many liver specimens from patients with either PSC or CUC or both suggests that the causes of these conditions are closely related.


Changes in autonomic nervous system activity after treatment with alpha-blocker in men with lower urinary tract symptoms.
To determine changes in autonomic nervous system activity after treatment in men with lower urinary tract symptoms (LUTS), we evaluated changes in patients' symptoms, uroflowmetry, and heart rate variability (HRV) after treatment with alpha-blockers for 12 weeks. Ninety-five men who had LUTS (International Prostate Symptom Score [IPSS] ≥8) were included in this study. We divided them into two groups on the basis of a low frequency/high frequency (LF/HF) ratio of 1.6. After treatment with Xatral XL (Handok Inc., Korea) 10 mg for 3 months, we rechecked their IPSS, uroflowmetry, HRV and compared these with the baseline measurements. Fifty-four men were assigned to the low LF/HF group (group A: LF/HF ≤1.6) and 41 men to the high LF/HF group (group B: LF/HF >1.6). At baseline and 12 weeks, none of the parameters differed significantly between the groups except for HF, which is one of the parameters of HRV. IPSS, the IPSS-voiding subscore, and the IPSS-storage subscore decreased and maximal uroflow increased significantly after 12 weeks of treatment. Whereas the baseline LF/HF ratio increased from 0.89±0.407 to 1.80±1.804 after treatment in group A, it decreased from 3.93±5.471 to 1.79±1.153 in group B. The efficacies of Xatral XL were clear in both groups. We found that the LF/HF ratio in the two groups merged to a value of approximately 1.79 after treatment. We suggest that this could be a clue to the importance of balance in autonomic nervous system activity in men with LUTS.


Rationing social contact during the COVID-19 pandemic: Transmission risk and social benefits of US locations.
To prevent the spread of coronavirus disease 2019 (COVID-19), some types of public spaces have been shut down while others remain open. These decisions constitute a judgment about the relative danger and benefits of those locations. Using mobility data from a large sample of smartphones, nationally representative consumer preference surveys, and economic statistics, we measure the relative transmission reduction benefit and social cost of closing 26 categories of US locations. Our categories include types of shops, entertainments, and service providers. We rank categories by their trade-off of social benefits and transmission risk via dominance across 13 dimensions of risk and importance and through composite indexes. We find that, from February to March 2020, there were larger declines in visits to locations that our measures indicate should be closed first.


Multifunctional core-shell nanoplatforms (gold@graphene oxide) with mediated NIR thermal therapy to promote miRNA delivery.
Recent insights into the nanomedicine have revealed that nanoplatforms enhance the efficacy of carrier in therapeutic applications. Here, multifunctional nanoplatforms were utilized in miRNA-101 delivery and NIR thermal therapy to induce apoptosis in breast cancer cells. Au nanorods (NRs) or nanospheres (NSs) covered with graphene oxide (GO) were prepared and functionalized with polyethylene glycol as a stabilizer and poly-L-arginine (P-L-Arg) as a targeting agent. In nanoplatforms, coupling Au@GO prepared stable structures with higher NIR reactivity. P-L-Arg substantially enhanced the cellular uptake and gene retardation of stuffs coated by them. However, rod-shape nanoplatforms indicated better performance in cellular uptake and gene transfection than spherical ones. NIR thermal therapy was implemented to improve gene release and in synergy with miRNA-101 activated the apoptotic pathway and decreased the viability of breast cancer cell (<20%). Briefly, presented delivery systems are potentially efficient in distinguishing cancer cells, miRNA internalization and controlling apoptosis of cancer cells.


Insect symbionts in food webs.
Recent research has shown that the bacterial endosymbionts of insects are abundant and diverse, and that they have numerous different effects on their hosts' biology. Here we explore how insect endosymbionts might affect the structure and dynamics of insect communities. Using the obligate and facultative symbionts of aphids as an example, we find that there are multiple ways that symbiont presence might affect food web structure. Many symbionts are now known to help their hosts escape or resist natural enemy attack, and others can allow their hosts to withstand abiotic stress or affect host plant use. In addition to the direct effect of symbionts on aphid phenotypes there may be indirect effects mediated through trophic and non-trophic community interactions. We believe that by using data from barcoding studies to identify bacterial symbionts, this extra, microbial dimension to insect food webs can be better elucidated.This article is part of the themed issue 'From DNA barcodes to biomes'.


Effect of interposed tissue and contamination on the initial stability of a highly porous press-fit acetabular cup.
For biologic fixation, press-fit acetabular cups should have initial stability with minimal micromotion and osteoconductive surfaces in contact with bone. Inadequate exposure potentially influences initial stability by increasing the possibility of soft tissue interposition and contamination at the implant-tissue interface. A sawbone model was used to examine how interposed tissue and contamination influence initial cup stability. Seven groups (n = 4) were tested with varying levels of interposed fatty and fibrous tissue placed around the rim of the cup. 54 millimeter in diameter highly porous hemispherical acetabular cups (Stryker, Mahwah NJ) and 54 mm reamed cavities in sawbone blocks were used. Shells were seated and maximum lever out force was recorded for each sample. Cups with fibrous tissue spaced evenly along the rim had a lever out force that was 150% of the control (107 ± 6 vs. 150 ± 12N, p = 0.005), and fatty tissue contamination had a lever out force that was 140% of the control (143 ± 18 vs. 107 ± 6N, p = 0.04). Cups with fibrous tissue placed eccentrically along the rim had a lever out force that was double the control 107 ± 6 N vs. 200 ± 15 N (p = 0.001). Surprisingly, fatty tissue contamination and fibrous tissue interposition at the rim increased initial stability. The eccentrically interposed tissue forced the opposite pole of the cup into the bone, resulting in a more secure press-fit. However, soft tissue interposition decreases implant/bone apposition, and the effect on long term fixation is unknown. Statement of Clinical Significance: Soft tissue interposition between the bone and cup may provide higher initial stability, but its long-term effects are unknown. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.


Role of the C-type lectin receptors MCL and DCIR in experimental colitis.
Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract. Though its exact etiology is still unclear, it is proposed that an imbalance in the intestinal homeostasis leads to a disturbed interaction between commensal microbiota and the mucosal immune system. Previous studies have shown that both innate and adaptive immunity are involved in an overwhelming colon inflammation, and thus contribute to the pathogenesis of IBD. In innate immunity, several pattern recognition receptors such as Toll-like receptors, NOD-like receptors or C-type lectin receptors (CLRs) are involved in IBD pathogenesis. Myeloid CLRs are mainly expressed by antigen-presenting cells and bind to glycan structures present on self or foreign antigens. The Macrophage-restricted C-type lectin (MCL) and the Dendritic cell immunoreceptor (DCIR) are two poorly characterized members of the CLR family. In this study, we investigated the role of MCL and DCIR in the pathogenesis of murine colitis. Both CLRs bound to intestinal microbiota to a different extent. They modulated the production of pro-inflammatory cytokines by antigen-presenting cells upon stimulation with heat-killed microbiota and impacted subsequent T cell responses. To analyze whether MCL and DCIR contribute to the pathogenesis of IBD, the dextran sulfate sodium (DSS) murine colitis model was employed. MCL-/- as well as DCIR-/- mice exhibited only a slightly increased severity of disease compared to wild-type mice indicating a limited role for MCL and DCIR in the regulation of intestinal immunity.


Population structure, adaptation and divergence of the meadow spittlebug, Philaenus spumarius (Hemiptera, Aphrophoridae), revealed by genomic and morphological data.
Understanding patterns of population differentiation and gene flow in insect vectors of plant diseases is crucial for the implementation of management programs of disease. We investigated morphological and genome-wide variation across the distribution range of the spittlebug Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Auchenorrhyncha, Aphrophoridae), presently the most important vector of the plant pathogenic bacterium Xylella fastidiosa Wells et al., 1987 in Europe. We found genome-wide divergence between P. spumarius and a very closely related species, P. tesselatus Melichar, 1899, at RAD sequencing markers. The two species may be identified by the morphology of male genitalia but are not differentiated at mitochondrial COI, making DNA barcoding with this gene ineffective. This highlights the importance of using integrative approaches in taxonomy. We detected admixture between P. tesselatus from Morocco and P. spumarius from the Iberian Peninsula, suggesting gene-flow between them. Within P. spumarius, we found a pattern of isolation-by-distance in European populations, likely acting alongside other factors restricting gene flow. Varying levels of co-occurrence of different lineages, showing heterogeneous levels of admixture, suggest other isolation mechanisms. The transatlantic populations of North America and Azores were genetically closer to the British population analyzed here, suggesting an origin from North-Western Europe, as already detected with mitochondrial DNA. Nevertheless, these may have been produced through different colonization events. We detected SNPs with signatures of positive selection associated with environmental variables, especially related to extremes and range variation in temperature and precipitation. The population genomics approach provided new insights into the patterns of divergence, gene flow and adaptation in these spittlebugs and led to several hypotheses that require further local investigation.


The European Association for the Study of Obesity (EASO) Endorses the Milan Charter on Urban Obesity.
The Milan Charter on Urban Obesity highlights the challenges of urban environments as a battleground for human health, as cities are often organized to subvert public health goals, and promote rather than prevent the development of obesity and consequent non-communicable diseases. The Charter articulates ten principles which detail actions and strategies through which general practitioners, diverse medical specialists, related healthcare professionals, administrators and healthcare practice managers, policy actors - within health systems and at a national level - along with experts across disciplines, and citizens, can work in cooperation to meet this challenge and improve public health. The Charter urges the adoption of decisions that deliver the following: (i) policies which enable our cities to become healthier and less obesogenic, more supportive of well-being and less health-disruptive in general, and (ii) policies that fully support primary prevention strategies, that address social stigma, and that ensure fair access to treatment for people living with obesity. The Milan Charter on Urban Obesity aims to raise awareness of our shared responsibility for the health of all citizens, and focuses on addressing the health of people living with obesity - not only as a challenge in its own right, but a gateway to other major non-communicable diseases, including cardiovascular diseases, type 2 diabetes, and some cancers.


A Novel Automated System Yields Reproducible Temporal Feeding Patterns in Laboratory Rodents.
The impact of temporal feeding patterns remains a major unanswered question in nutritional science. Progress has been hampered by the absence of a reliable method to impose temporal feeding in laboratory rodents, without the confounding influence of food-hoarding behavior. The aim of this study was to develop and validate a reliable method for supplying crushed diets to laboratory rodents in consistent, relevant feeding patterns for prolonged periods. We programmed our experimental feeding station to deliver a standard diet [StD; Atwater Fuel Energy (AFE) 13.9% fat] or high-fat diet (HFD; AFE 45% fat) during nocturnal grazing [providing 1/24th of the total daily food intake (tdF/I) of ad libitum-fed controls every 30 min] and meal-fed (3 × 1-h periods of ad libitum feeding) patterns in male rats (Sprague-Dawley: 4 wk old, 72-119 g) and mice [C57/Bl6J wild-type (WT): 6 mo old, 29-37 g], and ghrelin-null littermates (Ghr-/-; 27-34 g). Grazing yielded accurate, consistent feeding events in rats, with an approximately linear rise in nocturnal cumulative food intake [tdF/I (StD): 97.4 ± 1.5% accurate compared with manual measurement; R2 = 0.86; tdF/I (HFD): 99.0 ± 1.4% accurate; R2 = 0.86]. Meal-feeding produced 3 nocturnal meals of equal size and duration in StD-fed rats (tdF/I: 97.4 ± 0.9% accurate; R2 = 0.90), whereas the second meal size increased progressively in HFD-fed rats (44% higher on day 35 than on day 14; P < 0.01). Importantly, cumulative food intake in grazing and meal-fed rats was identical. Similar results were obtained in WT mice except that less restricted grazing induced hyperphagia (compared with meal-fed WT mice; P < 0.05 from day 1). This difference was abolished in Ghr-/- mice, with meal initiation delayed and meal duration enhanced. Neither pattern elevated corticosterone secretion in rats, but meal-feeding aligned ultradian pulses. We have established a consistent, measurable, researcher-defined, stress-free method for imposing temporal feeding patterns in rats and mice. This approach will facilitate progress in understanding the physiologic impact of feeding patterns.


Automated in situ trace element analysis of silicate materials by laser ablation inductively coupled plasma mass spectrometry.
This paper describes the automated in situ trace element analysis of solid materials by laser ablation (LA) inductively coupled plasma mass spectrometry (ICP-MS). A compact computer-controlled solid state Nd:YAG Merchantek EO UV laser ablation (LA) system has been coupled with the high sensitivity VG PQII S ICP-MS. A two-directional communication was interfaced in-house between the ICP-MS and the LA via serial RS-232 port. Each LA-ICP-MS analysis at a defined point includes a 60 s pre-ablation delay, a 60 s ablation, and a 90 s flush delay. The execution of each defined time setting by LA was corresponding to the ICP-MS data acquisition allowing samples to be run in automated cycle sequences like solution auto-sampler ICP-MS analysis. Each analytical cycle consists of four standards, one control reference material, and 15 samples, and requires about 70 min. Data produced by Time Resolved Analysis (TRA) from ICP-MS were later reduced off-line by in-house written software. Twenty-two trace elements from four reference materials (NIST SRM 613, and fused glass chips of BCR-2, SY-4, and G-2) were determined by the automated LA-ICP-MS method. NIST SRM 610 or NIST SRM 613 was used as an external calibration standard, and Ca as an internal standard to correct for drift, differences in transport efficiency and sampling yield. Except for Zr and Hf in G-2, relative standard deviations for all other elements are less than 10%. Results compare well with the data reported from literature with average limits of detection from 1 ng x g(-1) to 455 ng x g(-1) and less than 100 ng x g(-1) for most trace elements.


Phase I trial of the SPf66 malaria vaccine in a malaria-experienced population in Southeast Asia.
In preparation for a recently reported, independent field trial of SPf66 malaria vaccine efficacy in Thailand, we first established the safety and immunogenicity of two clinical lots of U.S. manufactured lots of SPf66 in a series of overlapping Phase I studies. The vaccine was produced in approved laboratories using good manufacturing practices. Two clinical lots of alum-adsorbed SPf66 were evaluated in a combined, open-label, Phase I clinical trial involving 50 healthy, malaria-experienced Karen adults and children. Volunteers were grouped by age and immunized sequentially. Group 1 had 30 adults. Group 2 had 10 children 8-15 years of age, and Group 3 had 10 children 2-6 years of age. The SPf66 vaccine was well tolerated in this malaria-experienced population. The most common side effects were erythema, induration, warmth, and tenderness at the site of injection, which typically resolved within 24-48 hr. One adult volunteer developed an acute urticarial rash following the third dose. Among adults, and to a lesser extent older children females had more local reactions than their male counterparts. Seroconversion to SPf66 by enzyme-linked immunosorbent assay occurred in 76% of volunteers receiving two or three doses. This vaccine was safe and immunogenic in malaria-experienced Karen adults and children. This study establishes the comparability of U.S.-manufactured SPf66 with that of Colombian origin, and is important for interpreting the efficacy results of U.S.-manufactured SPf66 in the same study population.


UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.
p97 is an ATP-dependent chaperone that plays an important role in endoplasmic reticulum-associated degradation but whose connections to turnover of soluble proteins remain sparse. Binding of p97 to substrates is mediated by cofactors that contain ubiquitin-binding domains. We employed "network proteomics" to show that p97 assembles with all of the 13 mammalian UBX-domain proteins. The UBX proteins that bind ubiquitin conjugates also interact with dozens of E3 ubiquitin ligases, only one of which had been previously linked to p97. In particular, UBXD7 links p97 to the ubiquitin ligase CUL2/VHL and its substrate hypoxia-inducible factor 1alpha (HIF1alpha). Depletion of p97 leads to accumulation of endogenous HIF1alpha and increased expression of a HIF1alpha target gene. The large number of ubiquitin ligases found associated with UBX proteins suggests that p97 plays a far broader role than previously anticipated in the global regulation of protein turnover.


E1-L2 activates both ubiquitin and FAT10.
Ubiquitination is catalyzed by a cascade of enzymes consisting of E1, E2, and E3. We report here the identification of an E1-like protein, termed E1-L2, that activates both ubiquitin and another ubiquitin-like protein, FAT10. Interestingly, E1-L2 can transfer ubiquitin to Ubc5 and Ubc13, but not Ubc3 and E2-25K, suggesting that E1-L2 may be specialized in a subset of ubiquitination reactions. E1-L2 forms a thioester with FAT10 in vitro, and this reaction requires the active-site cysteine of E1-L2 and the C-terminal diglycine motif of FAT10. Furthermore, endogenous FAT10 forms a thioester with E1-L2 in cells stimulated with tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma), which induce FAT10 expression. Silencing of E1-L2 expression by RNAi blocks the formation of FAT10 conjugates in cells. Deletion of E1-L2 in mice caused embryonic lethality, suggesting that E1-L2 plays an important role in embryogenesis.


Patterns of failures after surgical resection in olfactory neuroblastoma.
Patterns of failure in patients with olfactory neuroblastoma (ONB) according to two surgical approaches, craniofacial resection (CFR) and endoscopic surgery (ENDO), have yet to be analyzed. We retrospectively reviewed 28 patients with surgically treated ONB between January 1995 and October 2017. Fourteen (50.0%) patients underwent CFR (9 CFR alone, 5 ENDO-assisted CFR) and 14 (50.0%) underwent ENDO. Nineteen (67.9%) patients underwent post-operative radiotherapy (RT). At a median follow-up of 53.8 months (range 10.4-195.3), the 5-year progression-free survival (PFS) and 10-year overall survival were 37.3% and 57.5%, respectively. Patients with adjuvant RT had a 5-year PFS of 46.7%, whereas those treated with surgery alone had a 5-year PFS of 19.4% (p = 0.01). Locoregional failure (LRF) occurred in ten patients (median 59.6 months after initial diagnosis; range 12.7-59.7). Neck node metastasis occurred in 25.0% (7 of 28). Five patients with ENDO showed LRF and underwent proper subsequent treatments with either surgery or adjuvant RT. Approximately 35.7% patients (five patients) in the CFR group experienced distant metastasis in the intracranial dura region (median 116.4 months after initial diagnosis; range 2.6-142.4). Three of four patients who developed LRF after CFR developed dura-based metastasis. Both dura-based and neck node metastasis in the delayed phase were distinct patterns of failure in ONB. Patterns of recurrence differed based on surgical approach; dura-based metastases were common after CFR. LRF was the distinct failure pattern in ENDO, but could be successfully salvaged. Treatment outcome was improved considerably with RT following surgical resection.


Public policy and programs - Missing links in growing home dialysis in the United States.
Patients with end-stage kidney disease (ESKD) require dialysis or a kidney transplant for survival and over 760 000 patients now benefit from these therapies in the United States. Dialysis in the United States in the late 1960s and early 1970s was often done in the home. After the advent of Medicare coverage for ESKD in 1972 and the subsequent easier access to center based hemodialysis, the use of home modalities dramatically declined. This stands in contrast to home dialysis uptake in other industrialized healthcare systems where both peritoneal dialysis and home hemodialysis are more frequently used. Characteristics unique to the US healthcare system as well as the cultures of providers (physicians and dialysis providers) and recipients of ESKD care are hypothesized as the main reasons for observed differences in home dialysis use. To address these issues, the Centers for Medicare and Medicaid Services have recently proposed new payment programs under an Executive Order from the President of the United States, with the explicit goal of increasing the number of patients using home dialysis. This perspective outlines policy opportunities and programs with a proven track record of home dialysis growth in other countries or hypothesized promise based on identified barriers and needs.


[Trends in mortality from drowning, Mexico, 1979-2005].
To examine the trends in mortality from drowning in Mexico and each of its federal states during the period from 1979-2005. Mortality rates and trends were estimated for the study period from Mexico's official data based on the International Classification of Diseases, 9th and 10th revisions. Results were analyzed by age group, sex, and year registered, as well as by state and size of city or town of residence. From 1979-2005, there were 107 319 deaths by drowning registered. The mortality from drowning rate decreased 65.31% (from 6.58 per 100 000 inhabitants in 1979-1981 to 2.28 per 100 000 inhabitants in 2003-2005). This decrease was evident in all the age groups (ranging from 68.10% among 5-9 year olds to 54.30% among 1-4 years) and by state (ranging from 87.19% in Colima to 36.58% in Nayarit). During the three-year period from 2003-2005, drowning mortality was relatively greater among men and boys than among women and girls (rate: 5.46 (95%CI: 5.15-5.80); among the age groups 1-4 years (4.27 per 100 000) and 60 years or older (3.37 per 100 000 inhabitants); in the Tabasco state (5.92 per 100 000 inhabitants); and among towns with fewer than 2 499 inhabitants (4.03% per 100 000 inhabitants). In Mexico, the drowning mortality drowning rate is on a downward trend, although it remains a serious public health problem. Intensified efforts are needed to harness information regarding accidentals death from this cause, both from the epidemiological surveillance angle as well as through the implementation of eradicating interventions.


Role of nonexercise activity thermogenesis in resistance to fat gain in humans.
Humans show considerable interindividual variation in susceptibility to weight gain in response to overeating. The physiological basis of this variation was investigated by measuring changes in energy storage and expenditure in 16 nonobese volunteers who were fed 1000 kilocalories per day in excess of weight-maintenance requirements for 8 weeks. Two-thirds of the increases in total daily energy expenditure was due to increased nonexercise activity thermogenesis (NEAT), which is associated with fidgeting, maintenance of posture, and other physical activities of daily life. Changes in NEAT accounted for the 10-fold differences in fat storage that occurred and directly predicted resistance to fat gain with overfeeding (correlation coefficient = 0.77, probability < 0.001). These results suggest that as humans overeat, activation of NEAT dissipates excess energy to preserve leanness and that failure to activate NEAT may result in ready fat gain.


Glycophenotyping of osteoarthritic cartilage and chondrocytes by RT-qPCR, mass spectrometry, histochemistry with plant/human lectins and lectin localization with a glycoprotein.
This study aimed to characterize the glycophenotype of osteoarthritic cartilage and human chondrocytes. Articular knee cartilage was obtained from nine osteoarthritis (OA) patients. mRNA levels for 27 glycosyltransferases were analyzed in OA chondrocytes using RT-qPCR. Additionally, N- and O-glycans were quantified using mass-spectrometry. Histologically, two cartilage areas with Mankin scores (MS) either ≤ 4 or ≥ 9 were selected from each patient representing areas of mild and severe OA, respectively. Tissue sections were stained with (1) a selected panel of plant lectins for probing into the OA glycophenotype, (2) the human lectins galectins-1 and -3, and (3) the glycoprotein asialofetuin (ASF) for visualizing β-galactoside-specific endogenous lectins. We found that OA chondrocytes expressed oligomannosidic structures as well as non-, mono- and disialylated complex-type N-glycans, and core 2 O-glycans. Reflecting B4GALNT3 mRNA presence in OA chondrocytes, LacdiNAc-terminated structures were detected. Staining profiles for plant and human lectins were dependent on the grade of cartilage degeneration, and ASF-positive cells were observed in significantly higher rates in areas of severe degeneration. In summary, distinct aspects of the glycome in OA cartilage are altered with progressing degeneration. In particular, the alterations measured by galectin-3 and the pan-galectin sensor ASF encourage detailed studies of galectin functionality in OA.


Somatostatin 2 Receptor Activation in the Rostral Ventrolateral Medulla Does Not Mediate the Decompensatory Phase of Haemorrhage.
Decompensation, a critical phase in the response to hemorrhage, is characterized by profound sympathoinhibition and the overriding of baroreflex mediated compensation. As sympathoexcitatory neurons of the rostral ventrolateral medulla (RVLM) maintain vasomotor tone and are essential for sympathetic baroreceptor reflex function, the RVLM is the likely mediator. However, how decompensation occurs is a mystery. Our previous work demonstrated that the inhibitory neuropeptide somatostatin (SST), evokes potent sympathoinhibition. Here we test the hypothesis that, in response to hypovolemia, SST in the RVLM evokes sympathoinhibition, driving decompensation and suppressing baroreflex compensation. We evaluated neuronal activation at sites that contain SST mRNA and project to the RVLM and, in SST2A expressing neurons in the RVLM. We determined the effects on cardiovascular and sympathetic responses to haemorrhage, of bilateral blockade of SST2 receptors in both the RVLM and A1 regions. Haemorrhage in conscious rats evoked c-Fos immunoreactivity in the amygdala, periaqueductal gray, and parabrachial nuclei, regions previously associated with hemorrhage, shown to contain SST and project to the RVLM. Although c-Fos labeling was found throughout the ventrolateral medulla, only a small subset of RVLM SST2A receptor expressing neurons were activated, consistent with the idea that these neurons are inhibited during hemorrhage. However, SST2 receptor antagonists bilaterally injected in the RVLM or the A1 region did not affect the decompensation response to hemorrhage. Thus somatostatin in the RVLM does not mediate decompensation. The physiological role associated with somatostatin-induced sympathoinhibition in the RVLM together with the central mechanisms responsible for decompensation remain elusive.


Impact of curricular change on the perception of the educational environment by nursing students.
This study sought to evaluate the impact of curricular change on the perception of the Educational Environment (EE) in nursing students. This was a cross-sectional study. Two consecutive cohorts were evaluated during the second year, entering 2010 (N: 58) and 2011 (N: 57) for former and new curriculum, respectively. A sociodemographic survey and perception of the EE was applied through the Dundee Ready Educational Environment Measure (DREEM) questionnaire. No differences were detected in the sociodemographic variables between the cohorts. Both groups evaluated EE more positively than negatively. The total average score of the perception of the EE by the 2010 cohort was of 132 points and by the 2011 cohort of 126 points, a statistically significant difference. Upon analyzing the survey items, it was observed that poorer perception exists of the learning atmosphere and of the social environment, as well as poorer assessment of the academic skills in the 2011 cohort compared to the 2010 cohort. The good preparation the students are receiving for the profession and the relevance of the assignments they are learning are considered strengths by the students from both groups. In spite of how positive the curricular changes could seem, perception of the EE in both cohorts does not reach the excellent category. Before any changes are made to the curriculum, it is indispensable to take into account how the academic load might affect the students.


Relationship of somatomedin-C concentration to bone age in boys with constitutional delay of growth.
Serum somatomedin-C (Sm-C) levels increase sharply during puberty, leading to difficulty in the interpretation of Sm-C values obtained from children who exhibit a discrepancy between chronological age (CA) and pubertal development. To evaluate the utility of assessing Sm-C levels on the basis of bone age (BA), we measured serum Sm-C levels in 44 boys with constitutional delay of growth (CDG). Levels of Sm-C were compared with the normative data of the Nichols Institute Reference Laboratories (NIRL), Los Angeles, by age category, substituting BA for CA. We found the mean Sm-C level in boys with CDG to be lower than that for NIRL normal subjects in each age category for both CA and BA, but the regression curve for Sm-C levels based on BA more closely approximated the NIRL regression curve than did the curve based on CA. The rise in Sm-C levels observed in NIRL normal subjects between CA 13 to 14 years is delayed in boys with CDG until CA 15 to 17 years only when a correction for BA is not made. We conclude that in boys with CDG, Sm-C levels should be interpreted on the basis of BA rather than CA, especially during the peripubertal period. The observation of blunted Sm-C levels in all age categories, even when BA was used, suggests that short children with presumed CDG may be at high risk for a "nonclassic" form of growth hormone deficiency.


The role of geography in the complex diffusion of innovations.
The urban-rural divide is increasing in modern societies calling for geographical extensions of social influence modelling. Improved understanding of innovation diffusion across locations and through social connections can provide us with new insights into the spread of information, technological progress and economic development. In this work, we analyze the spatial adoption dynamics of iWiW, an Online Social Network (OSN) in Hungary and uncover empirical features about the spatial adoption in social networks. During its entire life cycle from 2002 to 2012, iWiW reached up to 300 million friendship ties of 3 million users. We find that the number of adopters as a function of town population follows a scaling law that reveals a strongly concentrated early adoption in large towns and a less concentrated late adoption. We also discover a strengthening distance decay of spread over the life-cycle indicating high fraction of distant diffusion in early stages but the dominance of local diffusion in late stages. The spreading process is modelled within the Bass diffusion framework that enables us to compare the differential equation version with an agent-based version of the model run on the empirical network. Although both model versions can capture the macro trend of adoption, they have limited capacity to describe the observed trends of urban scaling and distance decay. We find, however that incorporating adoption thresholds, defined by the fraction of social connections that adopt a technology before the individual adopts, improves the network model fit to the urban scaling of early adopters. Controlling for the threshold distribution enables us to eliminate the bias induced by local network structure on predicting local adoption peaks. Finally, we show that geographical features such as distance from the innovation origin and town size influence prediction of adoption peak at local scales in all model specifications.


Study of morphocytochemical and immunophenotypic features of acute leukemia stem cells.
The immunophenotypic profile of hematopoietic stem cells (HSC) and hematopoietic precursor cells as well as leukemic stem cells (LSC) has been extensively studied in several laboratories worldwide. The results of our studies suggest that the standard panel for classification of acute leukemias should be supplemented with several new markers allowing us to identify more precisely the different forms of the leukemias being of the closely related origin, for example AML M6b and AML M7. The common bipotent LSC in AML M7 of low grade and AML M6b may exist analogous to precursor cell common for megakaryocytopoiesis and erythropoiesis. We have also found the similarity between blast cells in pro-B-ALL [t (4;11), 11q23] and AML M5a [t (9;11), 11q23]. Such similarity of immunophenotype and cytogenetic abnormalities in blast cells in pro-B-ALL and AML M5a may be considered as hint explaining the cases of AML M5a as a recurrence of leukemia in children with originally diagnosed pro-B-ALL.


Interdisciplinary Medication Adherence Program: The Example of a University Community Pharmacy in Switzerland.
The Community Pharmacy of the Department of Ambulatory Care and Community Medicine (Policlinique Médicale Universitaire, PMU), University of Lausanne, developed and implemented an interdisciplinary medication adherence program. The program aims to support and reinforce medication adherence through a multifactorial and interdisciplinary intervention. Motivational interviewing is combined with medication adherence electronic monitors (MEMS, Aardex MWV) and a report to patient, physician, nurse, and other pharmacists. This program has become a routine activity and was extended for use with all chronic diseases. From 2004 to 2014, there were 819 patient inclusions, and 268 patients were in follow-up in 2014. This paper aims to present the organization and program's context, statistical data, published research, and future perspectives.


Sex-Based Analysis of De Novo Variants in Neurodevelopmental Disorders.
While genes with an excess of de novo mutations (DNMs) have been identified in children with neurodevelopmental disorders (NDDs), few studies focus on DNM patterns where the sex of affected children is examined separately. We considered ∼8,825 sequenced parent-child trios (n ∼26,475 individuals) and identify 54 genes with a DNM enrichment in males (n = 18), females (n = 17), or overlapping in both the male and female subsets (n = 19). A replication cohort of 18,778 sequenced parent-child trios (n = 56,334 individuals) confirms 25 genes (n = 3 in males, n = 7 in females, n = 15 in both male and female subsets). As expected, we observe significant enrichment on the X chromosome for females but also find autosomal genes with potential sex bias (females, CDK13, ITPR1; males, CHD8, MBD5, SYNGAP1); 6.5% of females harbor a DNM in a female-enriched gene, whereas 2.7% of males have a DNM in a male-enriched gene. Sex-biased genes are enriched in transcriptional processes and chromatin binding, primarily reside in the nucleus of cells, and have brain expression. By downsampling, we find that DNM gene discovery is greatest when studying affected females. Finally, directly comparing de novo allele counts in NDD-affected males and females identifies one replicated genome-wide significant gene (DDX3X) with locus-specific enrichment in females. Our sex-based DNM enrichment analysis identifies candidate NDD genes differentially affecting males and females and indicates that the study of females with NDDs leads to greater gene discovery consistent with the female-protective effect.


Retirement investment theory explains patterns in songbird nest-site choice.
When opposing evolutionary selection pressures act on a behavioural trait, the result is often stabilizing selection for an intermediate optimal phenotype, with deviations from the predicted optimum attributed to tracking a moving target, development of behavioural syndromes or shifts in riskiness over an individual's lifetime. We investigated nest-site choice by female golden-winged warblers, and the selection pressures acting on that choice by two fitness components, nest success and fledgling survival. We observed strong and consistent opposing selection pressures on nest-site choice for maximizing these two fitness components, and an abrupt, within-season switch in the fitness component birds prioritize via nest-site choice, dependent on the time remaining for additional nesting attempts. We found that females consistently deviated from the predicted optimal behaviour when choosing nest sites because they can make multiple attempts at one fitness component, nest success, but only one attempt at the subsequent component, fledgling survival. Our results demonstrate a unique natural strategy for balancing opposing selection pressures to maximize total fitness. This time-dependent switch from high to low risk tolerance in nest-site choice maximizes songbird fitness in the same way a well-timed switch in human investor risk tolerance can maximize one's nest egg at retirement. Our results also provide strong evidence for the adaptive nature of songbird nest-site choice, which we suggest has been elusive primarily due to a lack of consideration for fledgling survival.


Caring ability of urban and rural family caregivers: association with overburden, stress and coping.
To correlate caring ability with overburden, stress and coping of urban and rural family caregivers of patients undergoing cancer treatment. Cross-sectional study, carried out in a referral hospital for cancer treatment, with urban and rural caregivers who responded the following instruments: questionnaire of sociodemographic characterization of the caregiver and the care provided, Perceived Stress scale, Burden Interview scale and Brief COPE. Pearson's correlation test was used for statistical analysis, with a significance level ≤5%. A total of 163 urban caregivers and 59 rural caregivers participated in the study. Between the caring ability and stress, a negative and moderate correlation was found in rural caregivers. In the relationship between the caring ability and the overburden, there was a statistically significant correlation in urban caregivers in the interpersonal relationship and perception of self-efficacy factor. Between coping and the caring ability, a positive and moderate correlation was identified in coping focused on the problem in the knowledge dimension in urban caregivers. Urban caregivers had greater intensity of overburden and coping focused on the problem in relation to the caring ability.


Backward masking performance in unaffected siblings of schizophrenic patients. Evidence for a vulnerability indicator.
Visual masking is a procedure that is used to assess the earliest components of visual processing. In backward masking, the identification of an initial stimulus (the target) is disrupted by a later stimulus (the mask). The masking function can be divided into an early component (e.g., up to about 60 ms) that reflects the involvement of sensory-perceptual processes, and a later component that reflects susceptibility to attentional disengagement as the mask diverts processing away from the representation of the target. Schizophrenic patients show anomalies on both masking components. It is not known whether backward masking deficits reflect enduring genetic vulnerability to schizophrenia. We assessed 32 unaffected siblings of schizophrenic patients and 52 normal control subjects on the early and late components of 4 masking conditions. The conditions differentially involved the sustained and transient visual pathways. The unaffected siblings showed poorer overall performance than control subjects on the masking procedures. More specifically, siblings showed anomalies on the early, sensory-perceptual component, but not on the later, attentional disengagement component. The backward masking performance deficits that have been observed in schizophrenic patients appear to reflect enduring vulnerability to the disorder rather than only the symptoms of the illness. This vulnerability appears to be associated with early, sensory-perceptual processes.


A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion.
The LasA protease of Pseudomonas aeruginosa can degrade elastin and is an important contributor to the pathogenesis of this organism. LasA (20 kDa) is a member of the beta-lytic endopeptidase family of extracellular bacterial proteases, and it shows high-level staphylolytic activity. We sequenced the lasA gene from strain FRD1 and overexpressed it in Escherichia coli. The lasA gene encodes a precursor, known as pre-proLasA, of 45,582 Da. Amino-terminal sequence analysis allowed the identification of the signal peptidase cleavage site and revealed that the 31-amino-acid signal peptide was removed in E. coli. The remaining proLasA (42 kDa) did not undergo autoproteolytic processing and showed little staphylolytic activity. However, it was readily processed to a 20-kDa active staphylolytic protease by incubation with trypsin or with the culture filtrate of a P. aeruginosa lasAdelta mutant. Thus, removal of the propeptide (22 kDa) was required to convert proLasA into an active protease. Although LasA protease was critical for staphylolytic activity, other proteases like elastase were found to enhance staphylolysis. Under the control of an inducible trc promoter, lasA was overexpressed in P. aeruginosa and the processing intermediates were examined. Compared with wild-type cells, the overproducing cells accumulated more 42-kDa proLasA species, and the culture supernatants of the overproducing cells showed increased levels of active 20-kDa LasA protease. Small amounts of a 25-kDa extracellular LasA-related protein, which could represent a potential processing intermediate, were also observed. To better understand the structure-function relationships in LasA protease, we tested whether His-120-X-His-122 in the mature portion of LasA plays a role in activity. This motif and surrounding sequences are conserved in the related beta-lytic protease of Achromobacter lyticus. Oligonucleotide-directed mutagenesis was used to change His-120 to Ala-120, thus forming the lasA5 allele. The product of lasA5 expressed from the chromosome of P. aeruginosa was processed to a stable, secreted 20-kDa protein (designated LasA-H120A) which was devoid of staphylolytic activity. This suggests that His-120 is essential for LasA activity and favors the possibility that proLasA processing and secretion in P. aeruginosa can proceed via mechanisms which do not involve autoproteolysis.


Consumption of pomegranate decreases serum oxidative stress and reduces disease activity in patients with active rheumatoid arthritis: a pilot study.
Pomegranate extract (POMx) consumption has been shown to reduce the incidence and severity of collagen-induced arthritis in mice. To investigate whether pomegranate consumption affects disease activity in patients with rheumatoid arthritis (RA), in relation to their serum oxidative status. In this pilot 12 week open-labeled study eight patients with active RA consumed POMx (10 ml/day) for 12 weeks. Patients' joint status and serum oxidative status (lipid peroxidation, total thiols group, paraoxonase 1 activity) were evaluated at baseline and at week 12. Six patients completed the study. POMx consumption significantly (P < 0.02) reduced the composite Disease Activity Index (DAS28) by 17%, which could be related mostly to a significant (P < 0.005) reduction in the tender joint count (by 62%). These results were associated with a significant (P < 0.02) reduction in serum oxidative status and a moderate but significant (P < 0.02) increase in serum high density lipoprotein-associated paraoxonase 1 (PON1) activity. The addition of POMx to serum from RA patients reduced free radical-induced lipid peroxidation by up to 25%. The pomegranate consumption reduced DAS28 in RA patients, and this effect could be related to the antioxidative property of pomegranates. Dietary supplementation with pomegranates may be a useful complementary strategy to attenuate clinical symptoms in RA patients.


Informal carers and the primary care team.
The number of carers in the community is rising, and the importance of general practice in providing supportfor them has been highlighted. Caring for a disabled friend or relative has been shown to be harmful to the health of the caregiver and changes in social and family structure have led carers to become isolated and more reliant on the formal support services. However, many carersfeel that GPs do not understand their needs, and in turn many GPs and nursesfeel that they lack the relevant resources and training to take a more proactive role. The shift from hospital to community-based care and the blurring of social roles of responsibility puts additional pressure on the carers and community services. However general practice is in the best position to help and there are many things that can be done by primary care teams, such as providing information about available support, benefits and local services, which would be appreciated by informal carers.


Contact lenses: promising devices for ocular drug delivery.
In the ocular pharmacology market, there is a noteworthy unmet demand for more efficacious delivery of ocular therapeutics. Contact lenses are emerging as an alternative ophthalmic drug delivery system to resolve the drawbacks of the conventional topical application methods. Thus, contact lenses drug delivery systems have been developed to provide an increased residence time of the drug at the surface of the eye leading to enhanced bioavailability and more convenient and efficacious therapy. Several research groups have already explored the feasibility and potential of contact lenses loading conventional drugs used to treat anterior eye disorders. Drug incorporation to the lens body is achieved with techniques, like simple soaking, inclusion of drug-loaded colloidal nanoparticles, or molecular imprinting. Regardless of the technique used, key properties of the contact lens, such as transparency and oxygen permeability, should be preserved. In this article, we reviewed the different techniques used for drug delivery through contact lenses, analyzing their advantages and disadvantages, and focused on articles describing contact lens-based ophthalmic drug delivery systems with significant potential to use in ocular therapeutics.


Transoral mandibulectomy in advanced osteoradionecrosis.
Because vascularity is severely compromised following radiation therapy, aseptic necrosis with secondary infection and extensive bone destruction can result in an advanced state of osteoradionecrosis. Transoral mandibulectomy is an excellent method for dealing with this difficult problem. This article discusses technical aspects of the procedure as well as applied anatomy and a case report. Evidence in the recent literature concerning hyperbaric oxygen therapy is very encouraging.


CCR5-restricted HIV type 2 variants from long-term aviremic individuals are less sensitive to inhibition by beta-chemokines than low pathogenic HIV type 1 variants.
Many HIV-2-infected individuals maintain low, often undetectable, viral loads for prolonged periods. Virus and/or host factors that contribute to this high level of virus control are largely unknown. Previously we demonstrated that HIV-2 variants from long-term aviremic individuals have relatively low replication kinetics in vitro in comparison to HIV-1 variants. We hypothesized that the relatively low replication rates of HIV-2 in vitro as well as the high level of virus control in vivo might be explained by HIV-2 replication being more sensitive to inhibitory host factors like beta-chemokines or other CD8+ T cell-derived factors than HIV-1 replication. To test this we determined the effect of exogenously added beta-chemokines and healthy donor CD8+ T cells on the in vitro virus production of HIV-2 and HIV-1 variants from long-term nonprogressors (LTNPs). Contrary to expectations, HIV-2 replication was inhibited less efficiently by RANTES and MIP-1alpha than HIV-1 replication. CD8+ T cells from 8 of 12 healthy donors reduced HIV replication minimally 2-fold. Interestingly, cells from five of these donors inhibited HIV-1 but hardly affected HIV-2 replication, while the reverse was observed for cells from one donor. For HIV-1, but not HIV-2, the magnitude of the antiviral effect of CD8+ T cells correlated with their effect on RANTES levels in culture supernatants. Our findings indicate that RANTES is a more important factor of CD8+ T cell-associated anti-HIV-1 activity than it is of HIV-2 activity and that the benign clinical course of HIV-2 infection is not due to enhanced beta-chemokine sensitivity of HIV-2 variants.


Phytochemicals and cancer.
Epidemiological studies showing a protective effect of diets rich in fruits and vegetables against cancer have focused attention on the possibility that biologically-active plant secondary metabolites exert anti-carcinogenic activity. This huge group of compounds, now collectively termed 'phytochemicals', provides much of the flavour and colour of edible plants and the beverages derived from them. Many of these compounds also exert anti-carcinogenic effects in animal models of cancer, and much progress has been made in defining their many biological activities at the molecular level. Such mechanisms include the detoxification and enhanced excretion of carcinogens, the suppression of inflammatory processes such as cyclooxygenase-2 expression, inhibition of mitosis and the induction of apoptosis at various stages in the progression and promotion of cancer. However, much of the research on phytochemicals has been conducted in vitro, with little regard to the bioavailability and metabolism of the compounds studied. Many phytochemicals present in plant foods are poorly absorbed by human subjects, and this fraction usually undergoes metabolism and rapid excretion. Some compounds that do exert anti-carcinogenic effects at realistic doses may contribute to the putative benefits of plant foods such as berries, brassica vegetables and tea, but further research with human subjects is required to fully confirm and quantify such benefits. Chemoprevention using pharmacological doses of isolated compounds, or the development of 'customised' vegetables, may prove valuable but such strategies require a full risk-benefit analysis based on a thorough understanding of the long-term biological effects of what are often surprisingly active compounds.


Co-expression of an Erwinia chrysanthemi pectate lyase-encoding gene (pelE) and an E. carotovora polygalacturonase-encoding gene (peh1) in Saccharomyces cerevisiae.
A pectate lyase (PL)-encoding gene (pelE) from Erwinia chrysanthemi and a polygalacturonase (PG)-encoding gene (peh1) from E. carotovora were each inserted between a novel yeast expression-secretion cassette and a yeast gene terminator, and cloned separately into a yeast-centromeric shuttle vector (YCp50), generating recombinant plasmids pAMS12 and pAMS13. Transcription initiation signals present in the expression-secretion cassette were derived from the yeast alcohol dehydrogenase gene promoter (ADC1P), whereas the transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of PL and PG was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1s). A pectinase cassette comprising ADC1P-MF alpha 1s-pelE-TRP5T and ADC1P-MF alpha 1s-peh1-TRP5T was subcloned into YCp50, generating plasmid pAMS14. Subsequently, the dominant selectable Geneticin G418-resistance (GtR) marker, APH1, inserted between the yeast uridine diphosphoglucose 4-epimerase gene promoter (GAL10P) and yeast orotidine-5'-phosphate carboxylase gene terminator (URA3T), was cloned into pAMS14, resulting in plasmid pAMS15. Plasmids pAMS12, pAMS13 and pAMS14 were transformed into a laboratory strain of Saccharomyces cerevisiae, whereas pAMS15 was stably introduced into two commercial wine yeast strains. DNA-DNA and DNA-RNA hybridization analyses revealed the presence of these plasmids, and the pelE and peh1 transcripts in the yeast transformants, respectively. A polypectate agarose assay indicated the extracellular production of biologically active PL and PG by the S. cerevisiae transformants and confirmed that co-expression of the pelE and peh1 genes synergistically enhanced pectate degradation.


Plant genetics. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome.
Oilseed rape (Brassica napus L.) was formed ~7500 years ago by hybridization between B. rapa and B. oleracea, followed by chromosome doubling, a process known as allopolyploidy. Together with more ancient polyploidizations, this conferred an aggregate 72× genome multiplication since the origin of angiosperms and high gene content. We examined the B. napus genome and the consequences of its recent duplication. The constituent An and Cn subgenomes are engaged in subtle structural, functional, and epigenetic cross-talk, with abundant homeologous exchanges. Incipient gene loss and expression divergence have begun. Selection in B. napus oilseed types has accelerated the loss of glucosinolate genes, while preserving expansion of oil biosynthesis genes. These processes provide insights into allopolyploid evolution and its relationship with crop domestication and improvement.


Determinants of long-term unemployment in early adulthood: A Finnish birth cohort study.
Cumulative contributions of social and health-related determinants to long-term unemployment during early working life among young adults are poorly understood. Therefore, we used four cumulative indices of both parental and own social and health-related determinants of such unemployment among a cohort which comprised a complete census of children born in Finland in 1987. The cohort participants were registered in the Medical Birth Register, and they were followed-up through 2015 (N = 46 521). We calculated predicted probabilities for long-term unemployment (> 12 months) when participants were 25-28 years. Moreover, we examined whether the associations differed by unemployment at the municipal level. During the follow-up, 4.5% of women and 7.1% of men experienced long-term unemployment. All cumulative indices of parental and own social and health-related determinants predicted the probability of long-term unemployment. The greatest probabilities were observed for own social determinants, both in municipalities with high and low unemployment although the probabilities were higher in the high-unemployment municipalities. Of the individual determinants, poor school performance showed the strongest association with long-term unemployment among women (OR 6.65, 95% CI 5.21-8.55) and men (OR 3.70, 95% CI 2.96-4.67), after adjusting for other own social determinants. The results highlight the importance of life course social equality in the prevention of long-term unemployment in early adulthood.


1,25-Dihydroxyvitamin D3 inhibits the differentiation and migration of T(H)17 cells to protect against experimental autoimmune encephalomyelitis.
Vitamin D(3), the most physiologically relevant form of vitamin D, is an essential organic compound that has been shown to have a crucial effect on the immune responses. Vitamin D(3) ameliorates the onset of the experimental autoimmune encephalomyelitis (EAE); however, the direct effect of vitamin D(3) on T cells is largely unknown. In an in vitro system using cells from mice, the active form of vitamin D(3) (1,25-dihydroxyvitamin D(3)) suppresses both interleukin (IL)-17-producing T cells (T(H)17) and regulatory T cells (Treg) differentiation via a vitamin D receptor signal. The ability of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) to reduce the amount of IL-2 regulates the generation of Treg cells, but not T(H)17 cells. Under T(H)17-polarizing conditions, 1,25(OH)(2)D(3) helps to increase the numbers of IL-10-producing T cells, but 1,25(OH)(2)D(3)'s negative regulation of T(H)17 development is still defined in the IL-10(-/-) T cells. Although the STAT1 signal reciprocally affects the secretion of IL-10 and IL-17, 1,25(OH)(2)D(3) inhibits IL-17 production in STAT1(-/-) T cells. Most interestingly, 1,25(OH)(2)D(3) negatively regulates CCR6 expression which might be essential for T(H)17 cells to enter the central nervous system and initiate EAE. Our present results in an experimental murine model suggest that 1,25(OH)(2)D(3) can directly regulate T cell differentiation and could be applied in preventive and therapeutic strategies for T(H)17-mediated autoimmune diseases.


Oxidation-Induced Structure Transformation: Thin-Film Synthesis and Interface Investigations of Barium Disilicide toward Potential Photovoltaic Applications.
Barium disilicide (BaSi2) has been regarded as a promising absorber material for high-efficiency thin-film solar cells. However, it has confronted issues related to material synthesis and quality control. Here, we fabricate BaSi2 thin films via an industrially applicable sputtering process and uncovered the mechanism of structure transformation. Polycrystalline BaSi2 thin films are obtained through the sputtering process followed by a postannealing treatment. The crystalline quality and phase composition of sputtered BaSi2 are characterized by Raman spectroscopy and X-ray diffraction (XRD). A higher annealing temperature can promote crystallization of BaSi2, but also causes an intensive surface oxidation and BaSi2/SiO2 interfacial diffusion. As a consequence, an inhomogeneous and layered structure of BaSi2 is revealed by Auger electron spectroscopy (AES) and transmission electron microscopy (TEM). The thick oxide layer in such an inhomogeneous structure hinders further both optical and electrical characterizations of sputtered BaSi2. The structural transformation process of sputtered BaSi2 films then is studied by the Raman depth-profiling method, and all of the above observations come to an oxidation-induced structure transformation mechanism. It interprets interfacial phenomena including surface oxidation and BaSi2/SiO2 interdiffusion, which lead to the inhomogeneous and layered structure of sputtered BaSi2. The mechanism can also be extended to epitaxial and evaporated BaSi2 films. In addition, a glimpse toward future developments in both material and device levels is presented. Such fundamental knowledge on structural transformations and complex interfacial activities is significant for further quality control and interface engineering on BaSi2 films toward high-efficiency solar cells.


Association of vascular function and estimated cardiovascular risk in patients with rheumatoid arthritis.
Rheumatoid arthritis (RA) patients should receive cardiovascular (CV) risk assessment. For this purpose CV risk calculators are available. In addition, parameters of vascular function can be measured and used for risk prediction. Aim of the present study was to assess the association of these two concepts. 287 RA patients (58.4±12.6 years) and 232 controls (49.9±13.4 years) were included in this cross-sectional study. We calculated 10 year CV risk with SCORE and QRISK2. For SCORE we used the recommended multiplier of 1.5 in eligible RA patients and estimated the risk also in patients younger than 40 years (mSCORE (0-65)). Augmentation index (AIx) and central pulse pressure (PP), markers of vascular integrity and CV risk, were assessed by pulse wave analysis (PWA). Primary endpoint was the correlation of AIx and the estimated CV risk using mSCORE (0-65). In RA patients AIx showed a statistically significant correlation with mSCORE (0-65) (rho=0.3374; p<0.0001) and QRISK2 (rho=0.3307; p<0.0001). The correlations of central PP with mSCORE (0-65) (rho=0.4692; p<0.0001) and QRISK2 (rho=0.5828; p<0.0001) were also statistically significant. Increasing quartiles of central PP were associated with an increased odds of being in the "high risk" category according to SCORE (OR 2.18; 95% CI 1.58-3.01) or QRISK2 (OR 2.18; 95% CI 1.75-2.72). In control patients we also found a correlation of AIx and central PP with SCORE (0-65) and QRISK2. Parameters of central haemodynamics correlate with calculated CV risk. However, both do not give exactly the same information. The question arises whether a combination of both concepts would result in an improved CV risk prediction.


DIBI, a 3-hydroxypyridin-4-one chelator iron-binding polymer with enhanced antimicrobial activity.
Depriving microorganisms of bioavailable iron is a promising strategy for new anti-infective agents. The new, highly water-soluble, low molecular weight co-polymer DIBI was developed to selectively bind iron(iii) ions as a tris chelate and acts as a standalone anti-infective. Minimum inhibitory concentration (MIC) studies show DIBI is effective against representative reference strains for Gram-positive and Gram-negative bacteria S. aureus and A. baumannii, and the fungus C. albicans. Compared to the small molecule iron chelators, deferiprone and deferoxamine, DIBI outclassed these by factors of 100 to 1000 for inhibition of initial growth. DIBI and a series of related co-polymers (Mw of 2-9 kDa) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization of a chelating 3-hydroxypyridin-4-one (HPO) methacrylamide monomer and N-vinylpyrrolidone (NVP). Full incorporation of the HPO monomer into the co-polymers from the reaction solution was determined by 1H NMR spectroscopy and ranged from 4.6 to 25.6 mol%. UV-vis spectroscopy showed that all the HPO in DIBI binds readily to iron(iii) in a tris chelate mode to the maximum theoretical iron(iii) binding capacity of the co-polymer. Chemical characterization including single crystal X-ray diffraction analyses of the O-benzyl protected and the functional HPO monomer are discussed. By design, DIBI is highly water soluble; the highest mass fraction in water tested was 70% w/w, without the need of organic co-solvents.


In vivo DNA-binding properties of a yeast transcription activator protein.
UV light can serve as a molecular probe to identify DNA-protein interactions at nucleotide level resolution from intact yeast cells. We have used the photofootprinting technique to determine during which of three regulated states (uninduced, induced, and catabolite repressed) the transcriptional activator protein encoded by GAL4 binds to its recognition sites within the GAL1-GAL10 upstream activating sequence (UASG). GAL4 protein is bound to at least four, and probably five, related sequence blocks within UASG under both induced and uninduced states. GAL4-dependent photofootprints are lost under conditions of catabolite repression. We observed no footprint patterns unique to catabolite-repressed cells, which suggests that binding of a repressor to the UASG is not involved in this process. Photofootprints of the GAL10 TATA element are strictly correlated with transcription: uninduced, catabolite-repressed, and delta gal4 cells exhibit footprints characteristic of the inactive promoter; induced and delta gal80 cells, which express GAL10 constitutively, display footprints unique to the actively transcribed gene.


Solar Fenton and solar TiO2 catalytic treatment of ofloxacin in secondary treated effluents: evaluation of operational and kinetic parameters.
Two different technical approaches based on advanced oxidation processes (AOPs), solar Fenton homogeneous photocatalysis (hv/Fe(2+)/H(2)O(2)) and heterogeneous photocatalysis with titanium dioxide (TiO(2)) suspensions were studied for the chemical degradation of the fluoroquinolone ofloxacin in secondary treated effluents. A bench-scale solar simulator in combination with an appropriate photochemical batch reactor was used to evaluate and select the optimal oxidation conditions of ofloxacin spiked in secondary treated domestic effluents. The concentration profile of the examined substrate during degradation was determined by UV/Vis spectrophotometry. Mineralization was monitored by measuring the dissolved organic carbon (DOC). The concentrations of Fe(2+) and H(2)O(2) were the key factors for the solar Fenton process, while the most important parameter of the heterogeneous photocatalysis was proved to be the catalyst loading. Kinetic analyses indicated that the photodegradation of ofloxacin can be described by a pseudo-first-order reaction. The rate constant (k) for the solar Fenton process was determined at different Fe(2+) and H(2)O(2) concentrations whereas the Langmuir-Hinshelwood (LH) kinetic expression was used to assess the kinetics of the heterogeneous photocatalytic process. The conversion of ofloxacin depends on several parameters based on the various experimental conditions, which were investigated. A Daphnia magna bioassay was used to evaluate the potential toxicity of the parent compound and its photo-oxidation by-products in different stages of oxidation. In the present study solar Fenton has been demonstrated to be more effective than the solar TiO(2) process, yielding complete degradation of the examined substrate and DOC reduction of about 50% in 30 min of the photocatalytic treatment.


Pharmacogenetics-based population pharmacokinetic analysis of efavirenz in HIV-1-infected individuals.
Besides CYP2B6, other polymorphic enzymes contribute to efavirenz (EFV) interindividual variability. This study was aimed at quantifying the impact of multiple alleles on EFV disposition. Plasma samples from 169 human immunodeficiency virus (HIV) patients characterized for CYP2B6, CYP2A6, and CYP3A4/5 allelic diversity were used to build up a population pharmacokinetic model using NONMEM (non-linear mixed effects modeling), the aim being to seek a general approach combining genetic and demographic covariates. Average clearance (CL) was 11.3 l/h with a 65% interindividual variability that was explained largely by CYP2B6 genetic variation (31%). CYP2A6 and CYP3A4 had a prominent influence on CL, mostly when CYP2B6 was impaired. Pharmacogenetics fully accounted for ethnicity, leaving body weight as the only significant demographic factor influencing CL. Square roots of the numbers of functional alleles best described the influence of each gene, without interaction. Functional genetic variations in both principal and accessory metabolic pathways demonstrate a joint impact on EFV disposition. Therefore, dosage adjustment in accordance with the type of polymorphism (CYP2B6, CYP2A6, or CYP3A4) is required in order to maintain EFV within the therapeutic target levels.


Effects of Cage Position and Light Transmission on Home Cage Activity and Circadian Entrainment in Mice.
Light is known to exert powerful effects on behavior and physiology, including upon the amount and distribution of activity across the day/night cycle. Here we use home cage activity monitoring to measure the effect of differences in home cage light spectrum and intensity on key circadian activity parameters in mice. Due to the relative positioning of any individually ventilated cage (IVC) with regard to the animal facility lighting, notable differences in light intensity occur across the IVC rack. Although all mice were found to be entrained, significant differences in the timing of activity onset and differences in activity levels were found between mice housed in standard versus red filtering cages. Furthermore, by calculating the effective irradiance based upon the known mouse photopigments, a significant relationship between light intensity and key circadian parameters are shown. Perhaps unsurprisingly given the important role of the circadian photopigment melanopsin in circadian entrainment, melanopic illuminance is shown to correlate more strongly with key circadian activity parameters than photopic lux. Collectively, our results suggest that differences in light intensity may reflect an uncharacterized source of variation in laboratory rodent research, with potential consequences for reproducibility. Room design and layout vary within and between facilities, and caging design and lighting location relative to cage position can be highly variable. We suggest that cage position should be factored into experimental design, and wherever possible, experimental lighting conditions should be characterized as a way of accounting for this source of variation.


Phosphorylation of myosin light chain and phosphorylase in tracheal smooth muscle in response to KCl and carbachol.
It has been proposed that Ca2+-dependent myosin light chain (P-light chain) phosphorylation in smooth muscle permits cycling of myosin cross-bridges within myofibrillar elements for muscle shortening, but a second Ca2+-dependent regulatory mechanism is responsible for force generation. Accordingly, we examined P-light chain phosphorylation and another Ca2+-dependent protein phosphorylation reaction, phosphorylase a formation, in bovine tracheal smooth muscle during isometric force generation elicited by the cholinergic agonist carbachol or KCl depolarization, two stimuli thought to increase the concentration of sarcoplasmic free Ca2+ by mobilizing different pools of Ca2+. Increases in P-light chain phosphorylation reached maximal values of 0.79 and 0.59 mole of phosphate per mole of P-light chain at 1 min and then declined during maintained isometric force developed in response to 1 microM carbachol and 60 mM KCl, respectively. Carbachol elicited approximately twice the amount of force as found in the presence of KCl, and yet a more rapid rate of decline in the phosphate content of P-light chain was apparent. Decreases in maximal levels of phosphorylase a also occurred during carbachol-mediated isometric force maintenance, yet did not occur with KCl stimulation. Concentration-dependent responses with carbachol and KCl showed a positive relationship between the extent of P-light chain phosphorylation and extent of developed isometric force after 1 min of contraction with both stimuli. Under no conditions was force generated without P-light chain phosphorylation. The concentration dependence of phosphorylase a formation with KCl was similar to isometric force and P-light chain phosphorylation. However, concentrations of carbachol necessary to stimulate phosphorylase a formation were much higher than those required for stimulation of isometric force and P-light chain phosphorylation. Furthermore, carbachol attenuated the stimulation of phosphorylase a formation by isoproterenol. Thus, carbachol appears to have both an inhibitory and stimulatory effect on phosphorylase a formation in bovine tracheal smooth muscle. These results also indicate that maintained isometric force in smooth muscle may be dependent upon the maximal extent of P-light chain phosphorylation obtained during an early temporal transient in phosphorylation.


A pilot study of user satisfaction and perceived helpfulness of the Swedish version of the mobile app PTSD Coach.
Background: There is a need for accessible interventions in the aftermath of traumatic events with documented efficacy for preventing or reducing negative mental health consequences. The PTSD Coach is a mobile app that has shown to be effective in reducing symptoms of posttraumatic stress (PTSS). Objective: The purpose of the current study was to evaluate the user satisfaction, perceived helpfulness and potential reductions of PTSS and symptoms of depression among participants using the Swedish version of the PTSD Coach. Method: This was an uncontrolled pre-test post-test open trial including participants recruited from the community via advertisement and from an ongoing observational study who had experienced a potentially traumatic event in the last five years. Participants had access to the Swedish PTSD Coach app for four weeks. Results: Eleven participants (mean age = 38.6, female = 8) completed the study. Nine of the participants met criteria for full or partial PTSD. Results from the PTSD Coach Survey indicated that participants found the app slightly to moderately helpful and were slightly to moderately satisfied with the app. Nominal but not statistically significant reductions of medium effect sizes in PTSS (PCL-5) and depression (PHQ-9) from pre- to post-assessment were found. In interviews, participants indicated that they found elements such as learning about PTSD, breathing exercises and monitoring symptoms helpful in managing symptoms. However, several participants indicated that they had not used the app as much as they had intended to. Participants also had suggestions for improvements such as enhanced app structure and better guidance regarding how to use the app. Conclusions: The perceived helpfulness and user satisfaction were slightly lower compared to research on the original version of the app. Experiences from the study are discussed and a future controlled study of the Swedish version of the PTSD Coach is suggested.


Mapping-linked quantitative trait loci using Bayesian analysis and Markov chain Monte Carlo algorithms.
A Bayesian method for mapping linked quantitative trait loci (QTL) using multiple linked genetic markers is presented. Parameter estimation and hypothesis testing was implemented via Markov chain Monte Carlo (MCMC) algorithms. Parameters included were allele frequencies and substitution effects for two biallelic QTL, map positions of the QTL, and markers, allele frequencies of the markers, and polygenic and residual variances. Missing data were polygenic effects and multi-locus marker-QTL genotypes. Three different MCMC schemes for testing the presence of a single or two linked QTL on the chromosome were compared. The first approach includes a model indicator variable representing two unlinked QTL, affecting the trait, one linked and one unlinked QTL, or both QTL linked with the markers. The second approach incorporates an indicator variable for each QTL into the model for phenotype, allowing or not allowing for a substitution effect of a QTL, on phenotype, and the third approach is based on model determination by reversible jump MCMC. Methods were evaluated empirically by analyzing simulated granddaughter designs. All methods identified correctly a second, linked QTL and did not reject the one-QTL model when there was only a single QTL, and no additional or an unlinked QTL.


Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing.
The glial cells missing (gcm) gene in Drosophila encodes a transcription factor that determines the choice between glial and neuronal fates. We report here the isolation of two mammalian gcm homologs, Gcm1 and Gcm2, and the characterization of their expression patterns during embryonic development. Although Gcm2 is expressed in neural tissues at a low level, the major sites of expression for both of the mammalian genes are nonneural, suggesting that the functions of the mammalian homologs have diverged and diversified. However, when expressed ectopically, Gcm1 can substitute functionally for Drosophila gcm by transforming presumptive neurons into glia. Thus, certain biochemical properties, although not the specificity of the tissue in which the gene is expressed, have been conserved through the evolution of the Gcm gene family.


The population history of endogenous retroviruses in mule deer (Odocoileus hemionus).
Mobile elements are powerful agents of genomic evolution and can be exceptionally informative markers for investigating species and population-level evolutionary history. While several studies have utilized retrotransposon-based insertional polymorphisms to resolve phylogenies, few population studies exist outside of humans. Endogenous retroviruses are LTR-retrotransposons derived from retroviruses that have become stably integrated in the host genome during past infections and transmitted vertically to subsequent generations. They offer valuable insight into host-virus co-evolution and a unique perspective on host evolutionary history because they integrate into the genome at a discrete point in time. We examined the evolutionary history of a cervid endogenous gammaretrovirus (CrERVγ) in mule deer (Odocoileus hemionus). We sequenced 14 CrERV proviruses (CrERV-in1 to -in14), and examined the prevalence and distribution of 13 proviruses in 262 deer among 15 populations from Montana, Wyoming, and Utah. CrERV absence in white-tailed deer (O. virginianus), identical 5' and 3' long terminal repeat (LTR) sequences, insertional polymorphism, and CrERV divergence time estimates indicated that most endogenization events occurred within the last 200000 years. Population structure inferred from CrERVs (F ST = 0.008) and microsatellites (θ = 0.01) was low, but significant, with Utah, northwestern Montana, and a Helena herd being particularly differentiated. Clustering analyses indicated regional structuring, and non-contiguous clustering could often be explained by known translocations. Cluster ensemble results indicated spatial localization of viruses, specifically in deer from northeastern and western Montana. This study demonstrates the utility of endogenous retroviruses to elucidate and provide novel insight into both ERV evolutionary history and the history of contemporary host populations.


Potential anti-influenza effective plants used in Turkish folk medicine: A review.
Due to the outbreaks such as SARS, bird flu and swine flu, which we frequently encounter in our century, we need fast solutions with no side effects today more than ever. Due to having vast ethnomedical experience and the richest flora (34% endemic) of Europe and the Middle East, Turkey has a high potential for research on this topic. Plants that locals have been using for centuries for the prevention and treatment of influenza can offer effective alternatives to combat this problem. In this context, 224 herbal taxa belonging to 45 families were identified among the selected 81 studies conducted in the seven regions of Turkey. However, only 35 (15.6%) of them were found to be subjected to worldwide in vitro and in vivo research conducted on anti-influenza activity. Quercetin and chlorogenic acid, the effectiveness of which has been proven many times in this context, have been recorded as the most common (7.1%) active ingredients among the other 56 active substances identified. This study has been carried out to reveal the inventory of plant species that have been used in flu treatment for centuries in Turkish folk medicine, which could be used in the treatment of flu or flu-like pandemics, such as COVID 19, that humanity has been suffering with, and also compare them with experimental studies in the literature. The investigation was conducted in two stages on the subject above by using electronic databases, such as Web of Science, Scopus, ScienceDirect, ProQuest, Medline, Cochrane Library, EBSCO, HighWire Press, PubMed and Google Scholar. The results of both scans are presented in separate tables, together with their regional comparative analysis. Data obtained on taxa are presented in a table, including anti-influenza mechanism of actions and the active substances. Rosa canina (58.7%) and Mentha x piperita (22.2%) were identified as the most common plants used in Turkey. Also, Sambucus nigra (11.6%), Olea europaea (9.3%), Eucalyptus spp., Melissa officinalis, and Origanum vulgare (7.0%) emerged as the most investigated taxa. This is the first nationwide ethnomedical screening work conducted on flu treatment with plants in Turkey. Thirty-nine plants have been confirmed in the recent experimental anti-influenza research, which strongly shows that these plants are a rich pharmacological source. Also, with 189 (84.4%) taxa, detections that have not been investigated yet, they are an essential resource for both national and international pharmacological researchers in terms of new natural medicine searches. Considering that the production of antimalarial drugs and their successful use against COVID-19 has begun, this correlation was actually a positive and remarkable piece of data, since there are 15 plants, including Centaurea drabifolia subsp. Phlocosa (an endemic taxon), that were found to be used in the treatment of both flu and malaria.


HIF-2 Complex Dissociation, Target Inhibition, and Acquired Resistance with PT2385, a First-in-Class HIF-2 Inhibitor, in Patients with Clear Cell Renal Cell Carcinoma.
The heterodimeric transcription factor HIF-2 is arguably the most important driver of clear cell renal cell carcinoma (ccRCC). Although considered undruggable, structural analyses at the University of Texas Southwestern Medical Center (UTSW, Dallas, TX) identified a vulnerability in the α subunit, which heterodimerizes with HIF1β, ultimately leading to the development of PT2385, a first-in-class inhibitor. PT2385 was safe and active in a first-in-human phase I clinical trial of patients with extensively pretreated ccRCC at UTSW and elsewhere. There were no dose-limiting toxicities, and disease control ≥4 months was achieved in 42% of patients. We conducted a prospective companion substudy involving a subset of patients enrolled in the phase I clinical trial at UTSW (n = 10), who were treated at the phase II dose or above, involving multiparametric MRI, blood draws, and serial biopsies for biochemical, whole exome, and RNA-sequencing studies. PT2385 inhibited HIF-2 in nontumor tissues, as determined by a reduction in erythropoietin levels (a pharmacodynamic marker), in all but one patient, who had the lowest drug concentrations. PT2385 dissociated HIF-2 complexes in ccRCC metastases, and inhibited HIF-2 target gene expression. In contrast, HIF-1 complexes were unaffected. Prolonged PT2385 treatment resulted in the acquisition of resistance, and we identified a gatekeeper mutation (G323E) in HIF2α, which interferes with drug binding and precluded HIF-2 complex dissociation. In addition, we identified an acquired TP53 mutation elsewhere, suggesting a possible alternate mechanism of resistance. These findings demonstrate a core dependency on HIF-2 in metastatic ccRCC and establish PT2385 as a highly specific HIF-2 inhibitor in humans. New approaches will be required to target mutant HIF-2 beyond PT2385 or the closely related PT2977 (MK-6482).


Nonsteroidal anti-inflammatory drugs activate carbonic anhydrase by a direct mechanism of action.
Previous studies by this research team proved that vasodilating prostaglandins (PGs) E1, E2 and I2 inhibit carbonic anhydrase (CA) in vitro and in vivo, which suggested involvement of CA in gastric acid secretion inhibition and the increase of gastric mucosa blood flow produced by this group of PGs. Relying on these findings, as well as on our clinical observations, we studied in vitro and in vivo the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on CA I and CA II. We also followed in vitro the effects on these isozymes of NSAIDs associated to histamine, Ca, PGE2 and acetazolamide. The results show that the NSAIDs used here, which reduce the activity of cyclooxygenase and PG production, activated CA I and CA II in a dose-dependent manner by a mechanism of the noncompetitive type. Histamine and Ca added to NSAIDs amplified the activating effect of the latter on CA II. Association of PGE2 or acetazolamide to NSAIDs reduced NSAID-induced activation of CA I and CA II. Indomethacin abolished the inhibitory effect of acetazolamide on CA I and CA II. Our data imply that between CA and cyclooxygenase there is an inverse relationship, CA activation being accompanied by reduction of cyclooxygenase activity, a reduction achieved by the pH modifications induced by CA activation. In this way, cyclooxygenase, inhibition occurs "via CA," with the pH variations it brings about.


Changes in the topography of the sea urchin egg after fertilization.
Changes in the topography of the sea urchin egg after fertilization were studied by scanning and transmission electron microscopy. Strongylocentrotus purpuratus eggs were treated with dithiothreitol to modify the vitelline layer and to prevent formation of a fertilization membrane. Dithiothreitol treatment caused the microvilli to become more irregular in shape, length, and diameter than those of untreated eggs. The microvilli were similarly modified by trypsin treatment. This effect did not appear to be due to disruption of cytoskeletal elements beneath the plasma membrane, for neither colchicine nor cytochalasin B altered microvillar morphology. Thus, it appears that the vitelline layer may act in the maintenance of surface form of unfertilized eggs. Since dithiothreitol-treated eggs did not elevate a fertilization membrane, scanning electron microscopy could be used to directly observe modifications in the egg plasma membrane after fertilization. The wave of cortical granule exocytosis initiated at the point of attachment of the fertilizing sperm was characterized by the appearance of pits that subsequently opened, releasing the cortical granule contents and leaving depressions upon the egg surface. The perigranular membranes inserted during exocytosis were seen as smooth patches between the microvillous patches remaining from the original egg surface. This produced a mosaic surface with more than double the amount of membrane of unfertilized eggs. The mosaic surface subsequently reorganized to accommodate the inserted membrane material by elongation of microvilli. Blebs and membranous whorls present before reorganization suggested the existence of an unstable intermediate state of plasma membrane reorganization. Exocytosis and mosaic membrane formation were not blocked by colchicine or cytochalasin B, but microvillar elongation was blocked by cytochalasin B treatment.


The relationship between mathematics and language: academic implications for children with specific language impairment and English language learners.
The present study examined the relationship between mathematics and language to better understand the nature of the deficit and the academic implications associated with specific language impairment (SLI) and academic implications for English language learners (ELLs). School-age children (N = 61; 20 SLI, 20 ELL, 21 native monolingual English [NE]) were assessed using a norm-referenced mathematics instrument and 3 experimental computer-based mathematics games that varied in language demands. Group means were compared with analyses of variance. The ELL group was less accurate than the NE group only when tasks were language heavy. In contrast, the group with SLI was less accurate than the groups with NE and ELLs on language-heavy tasks and some language-light tasks. Specifically, the group with SLI was less accurate on tasks that involved comparing numerical symbols and using visual working memory for patterns. However, there were no group differences between children with SLI and peers without SLI on language-light mathematics tasks that involved visual working memory for numerical symbols. Mathematical difficulties of children who are ELLs appear to be related to the language demands of mathematics tasks. In contrast, children with SLI appear to have difficulty with mathematics tasks because of linguistic as well as nonlinguistic processing constraints.


Control of antimicrobial resistance in Iran: the role of international factors.
Antimicrobial resistance (AMR) is currently causing various challenges for all countries around the world. Accordingly, the WHO is placing a great emphasis on the global partnership and allinaces to drive countries towards developing policy guidances and a strategic framework for AMR contatiment. This study thus seeks to elaborate on the international factors underlying AMR management in Iran. Semi-structured interviews were conducted with managers from the Ministry of health (n = 14), Iran veterinary organization (n = 4), the national professional associations (n = 3) and researchers (n = 3), between November 2018 and July 2019. Participants were selected using purposeful and snowball sampling. Interviews were recorded and transcribed verbatim and were subsequently coded and analyzed thematically using MAXQDA software (V.18) and reported. International enabling and predisposing factors were identified in relation to the AMR control in the country. Enabling factors included knowledge transfer, facilitation in policy agenda setting, formulation and implementaion process, and AMR monitoring. Predisposing factors, alternatively, encompassed the migration of infectious patients, trafficking of medicine and livestock from neighboring countries, and the imposed sanctions. Nowadays, AMR is taken cognizance of as a global challenge, thus to be addressed effectively, needs an international consensus more than ever. This harmony would not certainly underrate national efforts, but instead, is needed to reinforce such efforts through e.g. technical and financial assistance. It is suggested for policymakers to use all available political and legal means such as health diplomacy to establish humanitarian channels in order to enhance global convention and remove possible barriers as the sanctions and reduce their adverse consequences for AMR control.


Estrogen receptor/Sp1 complexes are required for induction of cad gene expression by 17beta-estradiol in breast cancer cells.
The cad gene is trifunctional and expresses carbamoylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for pyrimidine biosynthesis. Cad gene activities are induced in MCF-7 human breast cancer cells, and treatment of MCF-7 or ZR-75 cells with 10 nM 17beta-estradiol (E2) resulted in a 3- to 5-fold increase in cad mRNA levels in both cell lines. The mechanism of hormone-induced cad gene expression was further investigated using constructs containing the growth-responsive -90 to +115 (pCAD1) region of the cad gene promoter. E2 induced reporter gene (luciferase) activity in MCF-7 and ZR-75 cells transfected with pCAD1, which contains three upstream GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the cad gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required for E2 responsiveness. Results of electrophoretic mobility shift and chromatin immunoprecipitation assays show that both Sp1 and estrogen receptor alpha interact with the GC-rich region of the cad gene promoter. Moreover, in transactivation assays with pCAD1, hormone-induced transactivation was inhibited by cotransfection with dominant-negative Sp1 expression plasmid and small inhibitory RNA for Sp1, which silences Sp1 expression in the cells. These results demonstrate that, in common with many other genes involved in E2-induced cell proliferation, the cad gene is also regulated by a nonclassical ERalpha/Sp1-mediated pathway.