Electroacupuncture preconditioning reduces cerebral ischemic injury via BDNF and SDF-1α in mice.
This study was designed to determine if electroacupuncture (EA) preconditioning improves tissue outcome and functional outcome following experimentally induced cerebral ischemia in mice. In addition, we investigated whether the expression of brain-derived neurotrophic factor (BDNF) and stromal cell derived factor-1α (SDF-1α) and infarct volume were related with improvement in neurological and motor function by interventions in this study. After treatment with EA at the acupoints 'Baihui (GV20)' and 'Dazhui (GV14)' for 20 min, BDNF was assessed in the cortical tissues based on Western blot and the SDF-1α and vascular endothelial growth factor (VEGF) levels in the plasma determined by ELISA. To assess the protective effects of EA against ischemic injury, the mice received once a day 20 min EA preconditioning for three days prior to the ischemic event. Focal cerebral ischemia was then induced by photothrombotic cortical ischemia. Infarct volumes, neurobehavioral deficit and motor deficit were evaluated 24 h after focal cerebral ischemia. The expression of BDNF protein increased significantly from 6 h, reaching a plateau at 12 h after the end of EA treatment in the cerebral cortex. Furthermore, SDF-1α, not VEGF, increased singnificantly from 12 h to 48 h after EA stimulation in the plasma. Moreover, EA preconditioning reduced the infarct volume by 43.5% when compared to control mice at 24 h after photothrombotic cortical ischemia. Consistent with a smaller infarct size, EA preconditioning showed prominent improvement of neurological function and motor function such as vestibule-motor function, sensori-motor function and asymmetric forelimb use. The expression of BDNF colocalized within neurons and SDF-1α colocalized within the cerebral vascular endothelium was observed throughout the ischemic cortex by EA. Pretreatment with EA increased the production of BDNF and SDF-1α, which elicited protective effects against focal cerebral ischemia. These results suggest a novel mechanism of EA pretreatment-induced tolerance against cerebral ischemic injury.


[Placebos in clinic and research: experimental findings and theoretical concepts].
The placebo response in medicine and psychology is incompletely understood, and is usually either mystified or down-played; a rational approach to the understanding of the placebo response, however, can be deduced from the scientific literature. We present 3 theories that may explain most of the placebo response in medical and psychological interventions: a) Regression to the mean (RTM): This refers to "errors" in measuring the outcome of therapies, that are due to small samples sizes, global assessment variables, spontaneous variability of symptoms, and other methodological reasons. b) Pavlovian Conditioning (PC): here the placebo response occurs as the consequence of successful association of diagnostic and therapeutic procedures in the past (illness history) of the individuum that resulted in symptom improvement. Neutral stimuli in the context of a treatment can thus gain therapeutic potency (become conditioned stimuli), e. g. the procedure of an injection or the colour of a drug. c) Signal detection theory (SDT): manipulation of expectencies and cognitions of the patient by suggestions and verbal instructions of the health care system will change the willingness of the patient to perceive symptoms as improved/worsened, specifically if this happens under "noisy" circumstances, e. g. with high spontaneous variability of symptoms. All three models are illustrated with examples from medical and psychological treatments or experiments, including recent findings of cortical correlates of the placebo response in functional brain imaging investigations. Potential biological mechanisms for the placebo response are discussed, including the possibility of genetic predisposition to be a placebo responder.


Common STAT3 variants are not associated with obesity or insulin resistance in female twins.
In animal models, STAT3 action in the hypothalamus and liver appears essential for normal body weight and glucose homeostasis in response to insulin. We hypothesized that variation in the STAT3 gene may be associated with body fat and/or insulin resistance in the general population. Five tagging SNPs spanning the STAT3 gene, rs8074524, rs2293152, rs2306580, rs6503695, and rs7211777 were genotyped in 2776 white female twins (mean age, 47.4+/-12.5 yrs) from the St Thomas' United Kingdom Adult Twin Registry (Twins UK). Minor allele frequencies were as follows: rs8074524 (0.19), rs2293152 (0.37), rs2306580 (0.06), rs6503695 (0.35), and rs7211777 (0.34). The minor allele of rs2293152 was associated with higher homeostasis model assessment index of insulin resistance (p=0.013) in the full cohort and confirmed in sib-transmission/disequilibrium test (TDT): (p=0.015; n=60). However, there were no associations with fasting serum insulin or glucose or with obesity variables. Although defective STAT3 action results in obesity and insulin resistance in animal models, we failed to establish any indicative associations with common SNPs in this human study.


Spontaneous epistaxis; theoretical consideration of etiologic factors and treatment.
In a study of data on 136 hemorrhages in 14 patients with posterior epistaxis it was noted that the incidence of bleeding was higher at some hours of the day than at others. The times of highest incidence were more or less inverse to hours of peak systolic blood pressure in normal subjects, suggesting that bleeding occurs as a result of dislodgement of an embolus at times of vasodilation. If that is true, then it should be possible to control bleeding by giving vasoconstrictive drugs. There are not as yet enough data on trial of that method to warrant conclusions.


Combination of vortex assisted binary solvent microextraction and QuEChERS for the determination of prothiofos, oxadiargyl, and gamma-cyhalothrin in water and pineapple samples by gas chromatography mass spectrometry.
An accurate and sensitive dispersive liquid-liquid microextraction method based on binary solvents was used to enrich prothiofos, oxadiargyl, and gamma-cyhalothrin for quantification by GC-MS. The combination of two extraction solvents (binary mixture) resulted in higher extraction efficiencies compared to the single solvent extraction systems. Parameters of the binary extraction method where optimized to enhance the extraction output of the analytes. The limits of detection calculated for the analytes ranged between 0.59 and 1.6 ng/mL. Linear calibration plots of the analytes covered wide concentration ranges with R2 values greater than 0.9996 and percent relative standard deviation lower than 10%. Spiked recovery experiments were performed well and wastewater at two different concentrations and satisfactory results (89-104%) were obtained. The binary solvent microextraction method was combined with QuEChERS to quantify the analytes in pineapple matrix, using matrix matching method to enhance the accuracy of the method to almost 100%.


The ATPase activity of reptin is required for its effects on tumor cell growth and viability in hepatocellular carcinoma.
Reptin is overexpressed in most human hepatocellular carcinomas. Reptin is involved in chromatin remodeling, transcription regulation, or supramolecular complexes assembly. Its silencing leads to growth arrest and apoptosis in cultured hepatocellular carcinoma cells and stops hepatocellular carcinoma progression in xenografts. Reptin has an ATPase activity linked to Walker A and B domains. It is unclear whether every Reptin function depends on its ATPase activity. Here, we expressed Walker B ATPase-dead mutants (D299N or E300G) in hepatocellular carcinoma cells in the presence of endogenous Reptin. Then, we silenced endogenous Reptin and substituted it with siRNA-resistant wild-type (WT) or Flag-Reptin mutants. There was a significant decrease in cell growth when expressing either mutant in the presence of endogenous Reptin, revealing a dominant negative effect of the ATPase dead mutants on hepatocellular carcinoma cell growth. Substitution of endogenous Reptin by WT Flag-Reptin rescued cell growth of HuH7. On the other hand, substitution by Flag-Reptin D299N or E300G led to cell growth arrest. Similar results were seen with Hep3B cells. Reptin silencing in HuH7 cells led to an increased apoptotic cell death, which was prevented by WT Flag-Reptin but not by the D299N mutant. These data show that Reptin functions relevant for cancer are dependent on its ATPase activity, and suggest that antagonists of Reptin ATPase activity may be useful as anticancer agents.


Dynamical Collectivity and Nuclear Quantum Effects on the Intermolecular Stretching Mode of Liquid Water.
This study investigated the broadband terahertz and low-frequency Raman spectroscopy of liquid water (H2O, D2O, and H218O) over 2 decades of frequency to address long-standing challenges regarding the interpretation of the intermolecular stretching mode at around 5 THz. We experimentally demonstrated that the intermolecular stretching mode of liquid water obtained via terahertz spectroscopy is significantly redshifted and broadened compared with that via Raman. This result was rationalized by the enhanced dynamical collectivity probed by terahertz spectroscopy, although both have a common origin in the kinetic motion. Their temperature and isotope dependences emphasize the significance of oscillation mass in determining the intermolecular stretching lineshape, while quantum effects cannot be overlooked in both terahertz and low-frequency Raman spectra.


Infection of non-host model plant species with the narrow-host-range Cacao swollen shoot virus.
Cacao swollen shoot virus (CSSV) is a major pathogen of cacao (Theobroma cacao) in Africa, and long-standing efforts to limit its spread by the culling of infected trees have had very limited success. CSSV is a particularly difficult virus to study, as it has a very narrow host range, limited to several tropical tree species. Furthermore, the virus is not mechanically transmissible, and its insect vector can only be used with difficulty. Thus, the only efficient means to infect cacao plants that have been experimentally described so far are by particle bombardment or the agroinoculation of cacao plants with an infectious clone. We have genetically transformed three non-host species with an infectious form of the CSSV genome: two experimental hosts widely used in plant virology (Nicotiana tabacum and N. benthamiana) and the model species Arabidopsis thaliana. In transformed plants of all three species, the CSSV genome was able to replicate, and, in tobacco, CSSV particles could be observed by immunosorbent electron microscopy, demonstrating that the complete virus cycle could be completed in a non-host plant. These results will greatly facilitate the preliminary testing of CSSV control strategies using plants that are easy to raise and to transform genetically.


[The methodological aspects of establishing the time of death].
Methodological analysis of essence of problem concerning postmortem interval estimation is given. The authors present the most promising ways of scientific research on this problem and possibilities of practical use of results of scientific research. The general methods of postmortem interval estimation including wired-in analogy (criterial) method and methods of modelling postmortal processes (mathematical and physical) are analysed. Graph of logical structures of modes of postmortem interval estimation and means of their realization is given.


Enkephalin convertase demonstrated in the pituitary and adrenal gland by [3H]guanidinoethylmercaptosuccinic acid autoradiography: dehydration decreases neurohypophyseal levels.
[3H]Guanidinoethylmercaptosuccinic acid (GEMSA) autoradiography demonstrates the particulate form of a carboxypeptidase B-like peptide processing enzyme, enkephalin convertase (EC 22.214.171.124), in the rat pituitary and adrenal glands. The maximal number of binding sites (Bmax) for [3H]GEMSA is 20 pmol/mg protein in the intermediate lobe of the pituitary, 12.0 pmol/mg protein in the posterior pituitary lobe, 15 pmol/mg protein in the anterior pituitary lobe, 5.8 pmol/mg protein in the adrenal medulla, and less than 0.3 pmol/mg protein in the adrenal cortex. The labeling pattern is homogeneous within each of these regions. Subcellular fractionation of the bovine adrenal medulla demonstrates that [3H] GEMSA-binding sites are localized to chromaffin granules. In Brattleboro rats and dehydrated rats, the level of posterior pituitary [3H]GEMSA binding is less than 25% of that in control animals. This decrease is abolished by arginine vasopressin treatment of Brattleboro rats or rehydration of dehydrated rats. There are no changes in [3H] GEMSA binding in the supraoptic nucleus or magnocellular portion of the paraventricular nucleus of the hypothalamus under any of these conditions, suggesting that the alterations observed in the neurohypophysis result from an increased rate of loss of enkephalin convertase. The level of anterior pituitary enkephalin convertase is unchanged by dehydration, adrenalectomy, or dexamethasone or in Brattleboro rats. [3H]GEMSA labeling in the intermediate pituitary lobe is unaffected by dehydration and haloperidol treatment and in Brattleboro rats. The adrenal medullary enzyme is not altered by reserpine, hypophysectomy, or splanchnic denervation or in Brattleboro rats.


STAT1 gain-of-function heterozygous cell models reveal diverse interferon-signature gene transcriptional responses.
Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) is an autosomal dominant immune disorder marked by wide infectious predisposition, autoimmunity, vascular disease, and malignancy. Its molecular hallmark, elevated phospho-STAT1 (pSTAT1) following interferon (IFN) stimulation, is seen consistently in all patients and may not fully account for the broad phenotypic spectrum associated with this disorder. While over 100 mutations have been implicated in STAT1 GOF, genotype-phenotype correlation remains limited, and current overexpression models may be of limited use in gene expression studies. We generated heterozygous mutants in diploid HAP1 cells using CRISPR/Cas9 base-editing, targeting the endogenous STAT1 gene. Our models recapitulated the molecular phenotype of elevated pSTAT1, and were used to characterize the expression of five IFN-stimulated genes under a number of conditions. At baseline, transcriptional polarization was evident among mutants compared with wild type, and this was maintained following prolonged serum starvation. This suggests a possible role for unphosphorylated STAT1 in the pathogenesis of STAT1 GOF. Following stimulation with IFNα or IFNγ, differential patterns of gene expression emerged among mutants, including both gain and loss of transcriptional function. This work highlights the importance of modeling heterozygous conditions, and in particular transcription factor-related disorders, in a manner which accurately reflects patient genotype and molecular signature. Furthermore, we propose a complex and multifactorial transcriptional profile associated with various STAT1 mutations, adding to global efforts in establishing STAT1 GOF genotype-phenotype correlation and enhancing our understanding of disease pathogenesis.


Quantification of arbuscular mycorrhizal fungi root colonization in wheat, tomato, and leek using absolute qPCR.
Arbuscular mycorrhizal fungi (AMF) form symbioses with most terrestrial plants and are known to have a positive effect on plant growth and health. Different methodologies have been developed to assess the AMF-plant symbiosis. The most applied method, which involves staining of roots and microscopic observation of the AMF structures, is tedious and time-consuming and the results are highly dependent on the observer. Using quantitative polymerase chain reaction (qPCR) to quantify AMF root colonization represents a reliable, high-throughput technique that allows the assessment of numerous samples. Quantification with qPCR can be performed through two methods: relative quantification and absolute quantification. In relative quantification, the target gene is normalized with a reference gene. On the other hand, absolute quantification involves the use of a standard curve, for which template DNA is serially diluted. In a previous paper, we validated the primer pair AMG1F and AM1 for a relative quantification approach to assess AMF root colonization in Petunia. Here, we tested the same primers with an absolute quantification approach and compared the results with the traditional microscopy method. We evaluated the qPCR method with three different crops, namely, wheat (cv. Colmetta and Wiwa), tomato, and leek. We observed a strong correlation between microscopy and qPCR for Colmetta (r = 0.90, p < 0.001), Wiwa (r = 0.94, p < 0.001), and tomato (r = 0.93, p < 0.001), but no correlation for leek (r = 0.27, p = 0.268). This highlights the importance of testing the primer pair for each specific crop.


Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry.
Measurement of intracellular ionized calcium concentrations ([Ca2+]i) has been indispensable in elucidating the central role of [Ca2+]i as a trigger of cellular responses to activating stimuli. Such studies have employed the dye quin2, which has not been readily adapted to analysis of individual small cells. We show here that the calcium response of large numbers of single cells can be analyzed with the use of flow cytometry and the recently described dye, indo-1. Such analyses demonstrate for the first time the heterogeneous nature of the [Ca2+]i response to mitogenic stimuli within populations of peripheral blood lymphocytes (PBL). By simultaneous quantitation of one- or two-color surface immunofluorescence labels, some of this heterogeneity of [Ca2+]i response in PBL is shown to be related to cellular immunophenotype. Almost all T cells responded to anti-CD3 antibody; however, the response is greater among CD4+ than CD8+ cells, and within the CD4+ population the rate of response to stimulation by antibody to CD3 differed between subpopulations defined by expression of the common leukocyte marker p220. In contrast, not all T cells responded to phytohemagglutinin (PHA), even at very high doses. As with anti-CD3, after stimulation with PHA, CD4+ cells showed a larger proportion of responding cells than did CD8+ cells. In separate experiments, indo-1 was found not to impair reproductive viability of PBL, thereby providing the potential for analysis of functional activity after the separation of cells by sorting on the basis of the [Ca2+]i response to stimuli. Mixing experiments indicated that a response of a subpopulation representing as little as 0.3% of total cells could be readily detected. Thus, the flow cytometric assay with indo-1 is the first technique that allows the quantitative analysis of response differences of small subpopulations of cells and intercellular variation in [Ca2+]i.


1,4,9-Triazaspiro[5.5]undecan-2-one Derivatives as Potent and Selective METTL3 Inhibitors.
N6-methyladenosine (m6A) is the most frequent of the 160 RNA modifications reported so far. Accumulating evidence suggests that the METTL3/METTL14 protein complex, part of the m6A regulation machinery, is a key player in a variety of diseases including several types of cancer, type 2 diabetes, and viral infections. Here we report on a protein crystallography-based medicinal chemistry optimization of a METTL3 hit compound that has resulted in a 1400-fold potency improvement (IC50 of 5 nM for the lead compound 22 (UZH2) in a time-resolved Förster resonance energy transfer (TR-FRET) assay). The series has favorable ADME properties as physicochemical characteristics were taken into account during hit optimization. UZH2 shows target engagement in cells and is able to reduce the m6A/A level of polyadenylated RNA in MOLM-13 (acute myeloid leukemia) and PC-3 (prostate cancer) cell lines.


DECTIN-1: A modifier protein in CTLA-4 haploinsufficiency.
Autosomal dominant loss-of-function (LoF) variants in cytotoxic T-lymphocyte associated protein 4 (CTLA4) cause immune dysregulation with autoimmunity, immunodeficiency and lymphoproliferation (IDAIL). Incomplete penetrance and variable expressivity are characteristic of IDAIL caused by CTLA-4 haploinsufficiency (CTLA-4h), pointing to a role for genetic modifiers. Here, we describe an IDAIL proband carrying a maternally inherited pathogenic CTLA4 variant and a paternally inherited rare LoF missense variant in CLEC7A, which encodes for the β-glucan pattern recognition receptor DECTIN-1. The CLEC7A variant led to a loss of DECTIN-1 dimerization and surface expression. Notably, DECTIN-1 stimulation promoted human and mouse regulatory T cell (Treg) differentiation from naïve αβ and γδ T cells, even in the absence of transforming growth factor-β. Consistent with DECTIN-1's Treg-boosting ability, partial DECTIN-1 deficiency exacerbated the Treg defect conferred by CTL4-4h. DECTIN-1/CLEC7A emerges as a modifier gene in CTLA-4h, increasing expressivity of CTLA4 variants and acting in functional epistasis with CTLA-4 to maintain immune homeostasis and tolerance.


The impact of a monthly rest day on avian influenza virus isolation rates in retail live poultry markets in Hong Kong.
Retail live poultry markets (LPMs) may act as a reservoir of avian influenza viruses (AIV). In this study we test the hypothesis that a rest day in the LPMs where the stalls are completely emptied of poultry, cleansed, and restocked will reduce the isolation rates of avian influenza viruses. The isolation rate of H9N2 subtype viruses from chicken was significantly lower after the rest day than prior to it, indicating its impact in reducing transmission. In contrast, Newcastle disease virus (NDV) isolation rates appear unaffected by this intervention, possibly reflecting differences in herd immunity or virus transmission dynamics.


The Challenges of Providing Culturally Competent Care Within a Disability Focused Team: A Phenomenological Exploration of Staff Experiences.
The study explored how service providers address the challenges of providing culturally competent care within disability services in the United Kingdom. Focus groups and interpretative phenomenological analysis were used to explore the experiences of 20 service providers from a range of professions, working with South Asian families. Two superordinate themes emerged. The first concentrated on difficulties with language and communication and the challenges posed by the use of interpreters. The second included five subthemes that broadly described a five-stage process of dealing with barriers and engagement to service delivery, followed by reflections on the effort made to provide culturally competent care in the face of limited resources and training. The study makes recommendations for cultural competency within health and social care services.


Augmentation of endothelial function by endothelin antagonism in human saphenous vein conduits.
Cerebral revascularization with saphenous vein (SV) conduits is used in the management of hard-to-treat lesions that require deliberate arterial occlusion and in selected patients with occlusive vascular disease. Endothelial dysfunction is thought to contribute to acute perioperative vasospasm and chronic graft atherosclerosis. In the present study the authors examined the contribution of the potent vasoconstrictor endothelin-1 (ET-1) to endothelial dysfunction in human SVs. The effects of an ET(A/B) receptor antagonist (bosentan), an ET(A) receptor antagonist (BQ-123), and an ET(B) receptor antagonist (BQ-788) on in vitro endothelium-dependent and -independent responses were studied in human SVs. Vascular segments were obtained in 34 patients who had undergone revascularization procedures, and isometric dose-response curves (DRCs) were constructed using the isolated tissue bath procedure as follows: 1) cumulative DRCs to norepinephrine; and 2) DRCs to acetylcholine (ACh) and sodium nitroprusside in the absence and presence of bosentan, BQ-123, or BQ-788. Maximal vasodilatory responses and sensitivity were compared between groups. In the presence of bosentan (Experiment 1) and BQ-123 or BQ-788 (Experiment 2), ACh responses were significantly augmented (percent maximum relaxation values: 7+/-2 [control] compared with 17+/-3 [bosentan], p < 0.002 [Experiment 1]; and 12+/-2 [control] compared with 29+/-2 [BQ-123] and 25+/-2 [BQ-788], p < 0.003 and p < 0.002, respectively [Experiment 2]). The sensitivity of SVs to ACh was unaffected by treatment. These beneficial effects were specific for the endothelium. Blockade of ET receptors significantly improves endothelial function in SVs. Furthermore, these effects appear to be independently and maximally mediated by antagonism of either ET(A) or ET(B) receptors. Interventions aimed at improving endothelial function may serve to counter perioperative vasospasm and impede atherosclerosis in SVs used for revascularization procedures.


Eight-year follow-up of school-based intervention on childhood overweight--the Kiel Obesity Prevention Study.
The aim of this study was to evaluate the 8-year outcome of school-based intervention on weight status, lifestyle and blood pressure (BP) as part of the Kiel Obesity Prevention Study (KOPS). Within a quasi-randomized controlled trial, 240 intervention (I) and 952 non-intervention (NI) students at age 6 and 14 years were assessed in schools. Six nutrition units followed by 20-min running games were performed within the first year at school. Primary outcome was the 8-year change in body mass index standard deviation score (BMI-SDS) according to German references. Effective intervention was tested using multilevel linear regression analysis. Eight-year changes in BMISDS were +0.18 and +0.22 with increases in prevalence of overweight from 8.3 to 10.4% and 7.0 to 11.2% in I and NI students, respectively. Cumulative 8-year incidence of overweight was 5.9% and 7.1% in I and NI students, respectively. There was no overall effect of intervention, but a significant interaction was shown between the intervention and the socio-economic status (SES), which demonstrated that in high SES, the 8-year change in BMI-SDS was in favour of I (-0.17 in I and +0.17 in NI; p < 0.01). Intervention had no measurable effects on lifestyle and BP. School-based health promotion has some favourable and sustained effects on 8-year changes in BMI-SDS, which are most pronounced in students of high SES families. The data argue in favour of further preventive measures.


TBX5 mutations in non-Holt-Oram syndrome (HOS) malformed hearts.
The T-box transcription factor Tbx5 is important in mammalian cardiac development. Mutations in the human TBX5 gene cause Holt-Oram syndrome (HOS), a disorder characterized by heart and upper limb deformities. To determine the role of TBX5 in non-HOS patients with complex cardiac malformations, we analyzed 68 explanted hearts from unrelated patients with various cardiac abnormalities including atrial (ASD), ventricular (VSD) and atrioventricular septal defects (AVSD). Direct sequencing detected nine mutations in diseased cardiac tissues of patients, eight of which are novel. Six mutations would affect amino acids in the T-domain, and one (c.236C>T, p.Ala79Val) is within the recently identified nuclear localization signal (NLS1) region. Further, mutations were found in patients with ASD and AVSD, but not with VSD; and mutations were absent in normal heart tissue of same patients, thus indicating somatic origin. Our results suggest a possible role of somatically occurring TBX5 mutations in congenital heart disease. We show for the first time TBX5 mutations in non-HOS associated cardiac malformations and we identified a novel missense mutation that would impact nuclear localization of TBX5.


Jaagsiekte sheep retrovirus and enzootic nasal tumor virus promoters drive gene expression in all airway epithelial cells of mice but only induce tumors in the alveolar region of the lungs.
Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats, while the closely related enzootic nasal tumor virus type 1 (ENTV-1) and ENTV-2 induce tumors in the nasal epithelium of sheep and goats, respectively. When expressed using a strong Rous sarcoma virus promoter, the envelope proteins of these viruses induce tumors in the respiratory tract of mice, but only in the distal airway. To examine the role of the retroviral long terminal repeat (LTR) promoters in determining tissue tropism, adeno-associated virus (AAV) vectors expressing alkaline phosphatase under the control of the JSRV, ENTV-1, or ENTV-2 LTRs were generated and administered to mice. The JSRV LTR was active in all airway epithelial cells, while the ENTV LTRs were active in the nasal epithelium and alveolar type II cells but poorly active in tracheal and bronchial epithelial cells. When vectors were administered systemically, the ENTV-1 and -2 LTRs were inactive in major organs examined, whereas the JSRV showed high-level activity in the liver. When a putative transcriptional enhancer from the 3' end of the env gene was inserted upstream of the JSRV and ENTV-1 LTRs in the AAV vectors, a dramatic increase in transgene expression was observed. However, intranasal administration of AAV vectors containing any combination of ENTV or JSRV LTRs and Env proteins induced tumors only in the lower airway. Our results indicate that mice do not provide an adequate model for nasal tumor induction by ENTV despite our ability to express genes in the nasal epithelium.


[Dung beetles (Coleoptera: Scarabaeinae) of the northwestern slope of the Sierra Nevada of Santa Marta, Colombia].
The community structure of dung beetles in the middle and lower river basin of the Gaira river, Sierra Nevada de Santa Marta, Colombia, is described. Four sites were selected along an altitudinal gradient of 50-940 m for sampling from June to October, 2004. Dung beetles were captured using modified pitfall traps and manual recollections. We captured 7,872 individuals belonging to 29 species, distributed in 15 genera and five tribes of Scarabaeinae. Canthon and Onthophagus were the most diverse genera, each represented by six species. The sampled sites shared the following species: Onthophagus acuminatus Harold, O. clypeatus Blanchard, O. marginicollis Harold. Bocatoma was the most diverse site with 23 species; whereas Port Mosquito presented the highest abundance, with 3,262 individuals. Seven species represented 89% of all captures: Canthidium sp., Dichotomius sp., Uroxys sp. 1, Uroxys sp. 2, O. marginicollis, O. clypeatus and O. acuminatus. Of the 29 captured species, 17 belonged to the functional group of diggers and 10 were ball-rollers. We did not observe significant among-site differences in community structure. Abiotic factors such as altitude, temperature and humidity cannot explain observed variation in community structure across sites, indicating other variables such as vegetation cover, density of the vegetation and soil type may play a role in the community structure of these insects.


Inferring Perspective Versus Getting Perspective: Underestimating the Value of Being in Another Person's Shoes.
People use at least two strategies to solve the challenge of understanding another person's mind: inferring that person's perspective by reading his or her behavior (theorization) and getting that person's perspective by experiencing his or her situation (simulation). The five experiments reported here demonstrate a strong tendency for people to underestimate the value of simulation. Predictors estimated a stranger's emotional reactions toward 50 pictures. They could either infer the stranger's perspective by reading his or her facial expressions or simulate the stranger's perspective by watching the pictures he or she viewed. Predictors were substantially more accurate when they got perspective through simulation, but overestimated the accuracy they had achieved by inferring perspective. Predictors' miscalibrated confidence stemmed from overestimating the information revealed through facial expressions and underestimating the similarity in people's reactions to a given situation. People seem to underappreciate a useful strategy for understanding the minds of others, even after they gain firsthand experience with both strategies.


Relapse rates with long-term antidepressant drug therapy: a meta-analysis.
Several long-term double-blind placebo controlled trials have shown prophylactic antidepressant therapy in unipolar depression. The goal of this work was to conduct a meta-analysis that would incorporate the most recent trials and evaluate their overall level of efficacy and relapse prevention over time. We performed a comprehensive literature search. The extracted data from selected studies were used to construct a regression model and evaluate the effect of treatment, time on medication, severity of illness, age, gender, and number of previous episodes. Across 11 maintenance treatment studies, the relapse rate was significantly different at 1 year for active drug (23%) versus placebo (51%). In addition, time on medication significantly affected the relapse rate. Prophylactic antidepressant drug therapy appears efficacious in preventing future relapses across a range of illness severity as well as age. More studies are needed to explore the effects of various acute antidepressant strategies and the direct influence of treatment resistance on relapse outcomes.


Rate limiting step of the allosteric activation of the bacterial adhesin FimH investigated by molecular dynamics simulations.
The bacterial adhesin FimH is a model for the study of protein allostery because its structure has been resolved in multiple configurations, including the active and the inactive state. FimH consists of a pilin domain (PD) that anchors it to the rest of the fimbria and an allosterically regulated lectin domain (LD) that binds mannose on the surface of infected cells. Under normal conditions, the two domains are docked to each other and LD binds mannose weakly. However, in the presence of tensile force generated by shear the domains separate and conformational changes propagate across LD resulting in a stronger bond to mannose. Recently, the crystallographic structure of a variant of FimH has been resolved, called FimH FocH $$ {\mathrm{FimH}}^{\mathrm{FocH}} $$ , where PD contains 10 mutations near the inter-domain interface. Although the X-ray structures of FimH and FimH FocH $$ {\mathrm{FimH}}^{\mathrm{FocH}} $$ are almost identical, experimental evidence shows that FimH FocH $$ {\mathrm{FimH}}^{\mathrm{FocH}} $$ is activated even in the absence of shear. Here, molecular dynamics simulations combined with the Jarzynski equality were used to investigate the discrepancy between the crystallographic structures and the functional assays. The results indicate that the free energy barrier of the unbinding process between LD and PD is drastically reduced in FimH FocH $$ {\mathrm{FimH}}^{\mathrm{FocH}} $$ . Rupture of inter-domain hydrogen bonds involving R166 constitutes a rate limiting step of the domain separation process and occurs more readily in FimH FocH $$ {\mathrm{FimH}}^{\mathrm{FocH}} $$ than FimH. In conclusion, the mutations in FimH FocH $$ {\mathrm{FimH}}^{\mathrm{FocH}} $$ shift the equilibrium toward an equal occupancy of bound and unbound states for LD and PD by reducing a rate limiting step.


Establishing need and population priorities to improve the health of homeless and vulnerably housed women, youth, and men: A Delphi consensus study.
Homelessness is one of the most disabling and precarious living conditions. The objective of this Delphi consensus study was to identify priority needs and at-risk population subgroups among homeless and vulnerably housed people to guide the development of a more responsive and person-centred clinical practice guideline. We used a literature review and expert working group to produce an initial list of needs and at-risk subgroups of homeless and vulnerably housed populations. We then followed a modified Delphi consensus method, asking expert health professionals, using electronic surveys, and persons with lived experience of homelessness, using oral surveys, to prioritize needs and at-risk sub-populations across Canada. Criteria for ranking included potential for impact, extent of inequities and burden of illness. We set ratings of ≥ 60% to determine consensus over three rounds of surveys. Eighty four health professionals and 76 persons with lived experience of homelessness participated from across Canada, achieving an overall 73% response rate. The participants identified priority needs including mental health and addiction care, facilitating access to permanent housing, facilitating access to income support and case management/care coordination. Participants also ranked specific homeless sub-populations in need of additional research including: Indigenous Peoples (First Nations, Métis, and Inuit); youth, women and families; people with acquired brain injury, intellectual or physical disabilities; and refugees and other migrants. The inclusion of the perspectives of both expert health professionals and people with lived experience of homelessness provided validity in identifying real-world needs to guide systematic reviews in four key areas according to priority needs, as well as launch a number of working groups to explore how to adapt interventions for specific at-risk populations, to create evidence-based guidelines.


Thrombospondins: A Role in Cardiovascular Disease.
Thrombospondins (TSPs) represent extracellular matrix (ECM) proteins belonging to the TSP family that comprises five members. All TSPs have a complex multidomain structure that permits the interaction with various partners including other ECM proteins, cytokines, receptors, growth factors, etc. Among TSPs, TSP1, TSP2, and TSP4 are the most studied and functionally tested. TSP1 possesses anti-angiogenic activity and is able to activate transforming growth factor (TGF)-β, a potent profibrotic and anti-inflammatory factor. Both TSP2 and TSP4 are implicated in the control of ECM composition in hypertrophic hearts. TSP1, TSP2, and TSP4 also influence cardiac remodeling by affecting collagen production, activity of matrix metalloproteinases and TGF-β signaling, myofibroblast differentiation, cardiomyocyte apoptosis, and stretch-mediated enhancement of myocardial contraction. The development and evaluation of TSP-deficient animal models provided an option to assess the contribution of TSPs to cardiovascular pathology such as (myocardial infarction) MI, cardiac hypertrophy, heart failure, atherosclerosis, and aortic valve stenosis. Targeting of TSPs has a significant therapeutic value for treatment of cardiovascular disease. The activation of cardiac TSP signaling in stress and pressure overload may be therefore beneficial.


Effects of suspended sediment on the biodegradation and mineralization of phenanthrene in river water.
High suspended sediment (SPS) concentration commonly exists in many Asian rivers. Furthermore, climate change can cause high floods and lead to the resuspension of sediments and soil erosion, resulting in high SPS concentration in many natural waters. This research studied the impact of the presence of SPS and organic C composition of SPS on the biodegradation and mineralization of phenanthrene (PHE). Three sediments, including original sediment (OS), 375 degrees C (S375), and 600 degrees C (S600) combusted sediment, were studied. A flask-based 14C-respirometer system was applied to study the mineralization of [14C]PHE by Agrobacterium sp. The mineralization rate of PHE in the absence of SPS was significantly lower than that with the presence of OS and S600 but higher than that with S375, suggesting that the effect of the presence of sediment on PHE mineralization depended on its organic C composition. The residual levels of PHE in the S375 and OS systems were about 1.5 times that of the S600 system after incubation for 2 d. After 26-d incubation, the mineralization rates of PHE were 34.64, 29.40, and 14.00% in the OS, S600, and S375 systems, respectively. The first-order rate constants of the OS and S600 systems were about three times that of the S375 system. The net influence of SPS on the biodegradation and mineralization rates of PHE was dependent on its effects on compound bioavailability and bacteria population. This study suggested that black C played a key role in reducing the mineralization rates of PHE in sediments-even without aging.


Genetic diversity and differentiation of 12 eastern Adriatic and western Dinaric native sheep breeds using microsatellites.
Nuclear genetic diversity and differentiation of 341 sheep belonging to 12 sheep breeds from Croatia and Bosnia and Herzegovina were examined. The aim of the study was to provide the understanding of the genetic structure and variability of the analysed pramenka sheep populations, and to give indications for conservation strategies based on the population diversity and structure information. The genetic variation of the sheep populations, examined at the nuclear level using 27 microsatellite loci, revealed considerable levels of genetic diversity, similar to the diversity found in other European indigenous low-production sheep breeds. Population-specific alleles were detected at most loci and in breeds analysed. The observed heterozygosity ranged from 0.643 (in Lika pramenka) to 0.743 (in Vlasic pramenka), and the expected heterozygosity ranged from 0.646 (in Lika pramenka) to 0.756 (in Dalmatian pramenka). Significant inbreeding coefficients were found for half of the populations studied and ranged from 0.040 (Pag island sheep) to 0.091 (Kupres pramenka). Moderate genetic differentiation was found between the studied sheep populations. The total genetic variability observed between different populations was 5.29%, whereas 94.71% of the variation was found within populations. Cres island sheep, Lika pramenka and Istrian sheep were identified as the most distinct populations, which was confirmed by the factorial analysis of correspondence and supported through a bootstrapping adjustment to correct for the difference in the sample sizes. The population structure analysis distinguished 12 clusters for the 12 sheep breeds analysed. However, the cluster differentiation was low for Dalmatian, Vlasic, Stolac and Krk pramenka. This systematic study identified Lika pramenka and Rab island sheep as those with the lowest diversity, whereas Istrian sheep and Pag island sheep had the highest. Conservation actions are proposed for Istrian, Rab and Cres island sheep, Lika and Kupres pramenka because of high estimated coefficients of inbreeding.


Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting.
A fundamental feature of immune systems is the ability to distinguish pathogenic from self and commensal elements, and to attack the former but tolerate the latter. Prokaryotic CRISPR-Cas immune systems defend against phage infection by using Cas nucleases and small RNA guides that specify one or more target sites for cleavage of the viral genome. Temperate phages include viruses that can integrate into the bacterial chromosome, and they can carry genes that provide a fitness advantage to the lysogenic host. However, CRISPR-Cas targeting that relies strictly on DNA sequence recognition provides indiscriminate immunity both to lytic and lysogenic infection by temperate phages-compromising the genetic stability of these potentially beneficial elements altogether. Here we show that the Staphylococcus epidermidis CRISPR-Cas system can prevent lytic infection but tolerate lysogenization by temperate phages. Conditional tolerance is achieved through transcription-dependent DNA targeting, and ensures that targeting is resumed upon induction of the prophage lytic cycle. Our results provide evidence for the functional divergence of CRISPR-Cas systems and highlight the importance of targeting mechanism diversity. In addition, they extend the concept of 'tolerance to non-self' to the prokaryotic branch of adaptive immunity.


Change detection in newborns using a multiple deviant paradigm: a study using magnetoencephalography.
Mismatch responses are elicited to changes in sound streams in healthy newborns. In the ideal case, these responses can predict cognitive problems later in life. We employed a multiple deviant paradigm for a fast assessment of the ability of the newborn brain to respond to various types of acoustic changes. In 12 healthy newborns, we recorded an electroencephalogram (EEG) and magnetoencephalogram while presenting auditory stimuli. Between repeated stimuli, four types of acoustic changes (frequency, intensity, duration, and a gap) were presented, varying in deviance magnitude. One major response was present in the neonatal evoked potentials and fields at 250-260 ms. Magnetic mismatch responses were elicited to all change types except for the duration deviant and they were positive in polarity. The frequency deviant elicited more positive EEG amplitudes than the standard, whereas the response to the duration deviant was more negative. These results show that newborns can detect changes to at least four types of deviances within a sound stream. Furthermore, the use of magneto- and electroencephalography is complementary in newborns, since the methods may reveal different outcomes. Further studies are warranted to determine whether the present study design can play a role in testing auditory function in clinical infant populations.


Adolescent neurological development and its implications for adolescent substance use prevention.
Recent technological advancements have facilitated the study of adolescent neurological development and its implications for adolescent decision-making and behavior. This article reviews findings from the adolescent neurodevelopment and substance use prevention literatures. It also discusses how findings from these two distinct areas of adolescent development can complement each other and be used to build more developmentally appropriate interventions for preventing adolescent substance use. Specifically, a combination of child-centered and family-based strategies is advocated based on extant neurological and prevention literature. EDITORS' STRATEGIC IMPLICATIONS: Researchers are encouraged to take up the authors' challenge and study the links between adolescent neurological development/decision making ability and the long term efficacy of comprehensive interventions for preventing adolescent substance use.


Model Uncertainty and Bayesian Model Averaged Benchmark Dose Estimation for Continuous Data.
The benchmark dose (BMD) approach has gained acceptance as a valuable risk assessment tool, but risk assessors still face significant challenges associated with selecting an appropriate BMD/BMDL estimate from the results of a set of acceptable dose-response models. Current approaches do not explicitly address model uncertainty, and there is an existing need to more fully inform health risk assessors in this regard. In this study, a Bayesian model averaging (BMA) BMD estimation method taking model uncertainty into account is proposed as an alternative to current BMD estimation approaches for continuous data. Using the "hybrid" method proposed by Crump, two strategies of BMA, including both "maximum likelihood estimation based" and "Markov Chain Monte Carlo based" methods, are first applied as a demonstration to calculate model averaged BMD estimates from real continuous dose-response data. The outcomes from the example data sets examined suggest that the BMA BMD estimates have higher reliability than the estimates from the individual models with highest posterior weight in terms of higher BMDL and smaller 90th percentile intervals. In addition, a simulation study is performed to evaluate the accuracy of the BMA BMD estimator. The results from the simulation study recommend that the BMA BMD estimates have smaller bias than the BMDs selected using other criteria. To further validate the BMA method, some technical issues, including the selection of models and the use of bootstrap methods for BMDL derivation, need further investigation over a more extensive, representative set of dose-response data.


Individual classification of children with epilepsy using support vector machine with multiple indices of diffusion tensor imaging.
Support vector machines (SVM) have recently been demonstrated to be useful for voxel-based MR image classification. In the present study we sought to evaluate whether this method is feasible in the classification of childhood epilepsy intractability based on diffusion tensor imaging (DTI), with adequate accuracy. We applied SVM in conjunction DTI indices of fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (RD) and axial diffusivity (AD). DTI studies have reported white matter abnormalities in childhood-onset epilepsy, but the mechanisms underlying these abnormalities are not well understood. The aim of this study was to examine the relationship between epileptic seizures and cerebral white matter abnormalities identified by DTI in children with active compared to remitted epilepsy utilizing an automated and unsupervised classification method. The DTI data were tensor-derived indices including FA, MD, AD and RD in 49 participants including 20 children with epilepsy 5-6 years after seizure onset as compared to healthy controls. To determine whether there was normalization of white matter diffusion behavior following cessation of seizures and treatment, the epilepsy subjects were grouped into those with active versus remitted epilepsy. Group comparisons were previously made examining FA, MD and RD via whole-brain tract-based spatial statistics (TBSS). The SVM analysis was undertaken with the WEKA software package with 10-fold cross validation. Weighted sensitivity, specificity and accuracy were measured for all the DTI indices for two classifications: (1) controls vs. all children with epilepsy and (2) controls vs. children with remitted epilepsy vs. children with active epilepsy. Using TBSS, significant differences were identified between controls and all children with epilepsy, between controls and children with active epilepsy, and also between the active and remitted epilepsy groups. There were no significant differences between the remitted epilepsy and controls on any DTI measure. In the SVM analysis, the best predictor between controls and all children with epilepsy was MD, with a sensitivity of 90-100% and a specificity between 96.6 and 100%. For the three-way classification, the best results were for FA with 100% sensitivity and specificity. DTI-based SVM classification appears promising for distinguishing children with active epilepsy from either those with remitted epilepsy or controls, and the question that arises is whether it will prove useful as a prognostic index of seizure remission. While SVM can correctly identify children with active epilepsy from other groups' diagnosis, further research is needed to determine the efficacy of SVM as a prognostic tool in longitudinal clinical studies.


Multimerization of the heptad repeat regions of the SARS-CoV 2 spike protein.
The heptad repeat 1 and 2 (HR1, HR2) regions in the spike protein of SARS-CoV 2 play a key role in the fusogenic mechanism of the virus with the host cell. During the fusion process they are thought to rearrange into an interdomain multimer. Functional fragments of the heptad repeat 1 and 2 regions in the spike protein of SARS-CoV 2 were chemically synthesized, labeled with nitrofurazone (NBD) and their interactions investigated by fluorescence spectroscopy. Steady state emission, fluorescence quenching, anisotropy and lifetime measurements in combination with a fluorophore dilution scheme were used to dissect multimer formation of HR1 and HR2 in quantitative detail. In addition, the investigation of the multimers by homo-FRET (via anisotropy) and lifetime measurements reveals new insights into the mechanism of fluorophore-fluorophore interactions in biological samples.


Lack of RNase L attenuates macrophage functions.
Macrophages are one of the major cell types in innate immunity against microbial infection. It is believed that the expression of proinflammatory genes such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and cyclooxygenase-2 (Cox-2) by macrophages is also crucial for activation of both innate and adaptive immunities. RNase L is an interferon (IFN) inducible enzyme which is highly expressed in macrophages. It has been demonstrated that RNase L regulates the expression of certain inflammatory genes. However, its role in macrophage function is largely unknown. Bone marrow-derived macrophages (BMMs) were generated from RNase L(+/+)and (-/-) mice. The migration of BMMs was analyzed by using Transwell migration assays. Endocytosis and phagocytosis of macrophages were assessed by using fluorescein isothiocyanate (FITC)-Dextran 40,000 and FITC-E. coli bacteria, respectively. The expression of inflammatory genes was determined by Western Blot and ELISA. The promoter activity of Cox-2 was measured by luciferase reporter assays. Lack of RNase L significantly decreased the migration of BMMs induced by M-CSF, but at a less extent by GM-CSF and chemokine C-C motif ligand-2 (CCL2). Interestingly, RNase L deficient BMMs showed a significant reduction of endocytic activity to FITC-Dextran 40,000, but no any obvious effect on their phagocytic activity to FITC-bacteria under the same condition. RNase L impacts the expression of certain genes related to cell migration and inflammation such as transforming growth factor (TGF)-β, IL-1β, IL-10, CCL2 and Cox-2. Furthermore, the functional analysis of the Cox-2 promoter revealed that RNase L regulated the expression of Cox-2 in macrophages at its transcriptional level. Taken together, our findings provide direct evidence showing that RNase L contributes to innate immunity through regulating macrophage functions.


The role of SPP/SPPL intramembrane proteases in membrane protein homeostasis.
Signal peptide peptidase (SPP) and the four SPP-like proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 constitute a family of aspartyl intramembrane proteases with homology to presenilins. The different members reside in distinct cellular localisations within the secretory pathway and the endo-lysosomal system. Despite individual cleavage characteristics, they all cleave single-span transmembrane proteins with a type II orientation exhibiting a cytosolic N-terminus. Though the identification of substrates is not complete, SPP/SPPL-mediated proteolysis appears to be rather selective. Therefore, according to our current understanding cleavage by SPP/SPPL proteases rather seems to serve a regulatory function than being a bulk proteolytic pathway. In the present review, we will summarise our state of knowledge on SPP/SPPL proteases and in particular highlight recently identified substrates and the functional and/or (patho)-physiological implications of these cleavage events. Based on this, we aim to provide an overview of the current open questions in the field. These are connected to the regulation of these proteases at the cellular level but also in context of disease and patho-physiological processes. Furthermore, the interplay with other proteostatic systems capable of degrading membrane proteins is beginning to emerge.


[Visual episodes in non-ketonic hyperglycemia: contribution of 1 case with alteration in diffusion magnetic resonance imaging].
Non-ketonic hyperglycemia (NKH) is recognized both as a direct cause of a precipitating factor of many types of epileptic seizures-partial motor, including partial motor status, complex partial, as well as reflex motor seizures and hemichorea-hemibalism syndrome. Less known is its association with isolated visual alterations. We present a clinical case with visual episodes and diffusion restriction. Clinical description together with the results of complementary tests: magnetic resonance imaging (MRI) brain sequences: T1W, T2W, FLAIR, DP, diffusion; and magnetic resonance angio-imaging, electroencephalogram (EEG), visual campimetry, carotid echodoppler. 51 year old male with visual complaints manifested as bright light in left lower quadrant with progressive increase in frequency until it becomes constant. Polyuria, polydipsia and weight loss are associated. No personal background of diabetes mellitus, epilepsy or migraine. Among the laboratory tests, non-ketonic hyperglycemia of 569 mg/dl stands out. The visual campimetry showed left lower quadrantanopsia, the intercritical EEG was reported as normal and the brain MRI showed alteration in right cortical-subcortical occipital diffusion sequence. Interpreted as simple partial seizure in the occipital region secondary to hyperglycemia, it was corrected, and the subject was asymptomatic at one month of discharge. We stress that isolated visual episodes may be the initial manifestation of NKH. The rareness of the alteration in the diffussion MRI (interpretable as artifact) orients to a possible mechanism of simple partial status. A high index of suspicion of this entity is important since adequate hypoglycemic treatment permits a perfect control of the seizure.


Association of Dietary Nitrate and a Mediterranean Diet With Age-Related Macular Degeneration Among US Adults: The Age-Related Eye Disease Study (AREDS) and AREDS2.
Low dietary nitrate intake has previously been suggested to be a risk factor for age-related macular degeneration (AMD) progression; however, this finding has not been replicated in other cohorts or adjusted for dietary patterns. To determine whether there is an association between dietary nitrate intake and AMD progression. This cohort study analyzed data from the prospective Age-Related Eye Disease Study (AREDS) and AREDS2 randomized clinical trial cohorts and their extended follow-up studies, which were conducted in multicenter outpatient retinal practices. Participants in both trials had non-late AMD in at least 1 eye. Data were analyzed from March 1, 2020, to September 30, 2022. Dietary nitrate intake. Association between dietary nitrate intake and development of late AMD (neovascular AMD [nAMD] or geographic atrophy [GA]) or large drusen. The interactions of dietary patterns, with common at-risk single-nucleotide polymorphisms, were also assessed. In the combined AREDS/AREDS2 cohort of 7788 participants (4288 AREDS participants and 3610 AREDS2 participants [110 of whom participated in both studies]), there were 13 511 eligible eyes. The combined cohort comprised 4396 women (56%) and 3392 men (44%), and the combined mean (SD) age was 71.1 (6.6) years. Dietary nitrate intake was associated with a decreased risk of progression to late AMD in the combined AREDS/AREDS2 cohort (hazard ratio [HR], 0.77 [95% CI, 0.69-0.86] for quartile 4 vs quartile 1 of intake) and a decreased risk of GA (HR, 0.71 [95% CI, 0.61-0.83]) and nAMD (HR, 0.85 [95% CI, 0.73-0.99]). In AREDS, increased nitrate intake (quartile 4 vs quartile 1) was associated with a decreased risk of late AMD (HR, 0.77 [95% CI, 0.65-0.91]) and GA (HR, 0.80 [95% CI, 0.65-0.97]) but not nAMD; in AREDS2, there was no association between nitrate intake (quartile 4 vs quartile 1) and late AMD (HR, 0.90 [95% CI, 0.80-1.02]) or nAMD (HR, 0.93 [95% CI, 0.78-1.11]). There was a correlation between Mediterranean dietary patterns and dietary nitrate intake (r = 0.52, P < .001). The findings of this cohort study suggest that dietary nitrate intake was associated with lower AMD risk. However, this association disappeared after adjusting for Mediterranean dietary patterns. These results are subject to potential bias and are hypothesis-generating in nature; therefore, they are insufficient to support new clinical recommendations. Previously described associations between dietary nitrate intake and AMD may in fact represent overall dietary patterns. Further research is needed before dietary nitrate intake can be recommended as a therapy for AMD.


Sensing fabrication errors in diffraction gratings using high dynamic range imaging.
We describe the design of a simple instrument for the identification and characterization of fabrication errors in diffraction gratings. The instrument uses an uncooled charge-coupled device (CCD) camera and a high dynamic range imaging process to detect the light scattered off a grating under test in the focal plane of a lens. We demonstrate that the instrument can achieve a dynamic range around nine orders of magnitude and we show that we are able to clearly identify small, periodic fabrication errors in two test gratings that could not be detected with microscopic techniques.


cERBB-2/Her-2 Neu Overexpression and Prognostic Significance in Uterine Carcinosarcoma.
There is not enough data in the literature regarding Her-2 overexpression in uterine carcinosarcomas or its association with the prognosis. The aim of this study was to determine the Her-2 overexpression rate in uterine carcinosarcoma and to evaluate its relationship with the prognosis. Her-2 protein and gene status were evaluated by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively, in hysterectomy specimens from 51 patients with uterine carcinosarcoma. Her-2 protein expression in the epithelial component was negative in 42 patients (score 0 in 33 cases, score (+1) in 9 cases), score (+2) in 7 patients and score (+3) in 2 patients. None of the patients had Her-2 protein expression within the sarcomatous component of the tumors. Her-2 gene was not amplified in epithelial or mesenchymal tumor areas according to the FISH method. There was no difference between the Her-2 overexpression negative and positive groups in terms of disease-free survival (DFS) and overall survival (OS). Her-2 overexpression was significantly higher in tumors of patients diagnosed at 65 years or older (p=0.046). In our study, no relationship could be shown between Her-2 overexpression and prognosis in uterine carcinosarcoma. More comprehensive studies are needed to illustrate the relationship between Her-2 overexpression and carcinosarcoma prognosis.


Isolation from bovine elastic tissues of collagen type VI and characterization of its form in vivo.
Foetal-bovine nuchal ligament and aorta, together with adult-bovine aorta and pregnant uterus, were extracted under dissociative conditions in the absence and in the presence of a reducing agent. A collagenous glycoprotein of Mr 140000 [designated component 140K(VI)], identified in these extracts as the major periodate/Schiff-positive component, was shown to be related to collagen type VI. Digestion of non-reduced extracts with pepsin yielded periodate/Schiff-positive peptides that, on the basis of their electrophoretic mobilities, amino acid analyses and peptide 'maps', were identical with type VI collagen fragments prepared by standard procedures. It is concluded that collagen type VI occurs in vivo as molecule comprising three chains of Mr 140000 in which the helical domains account for about one-third of each polypeptide. Biosynthetic experiments with nuchal-ligament fibroblasts in culture demonstrated that a bacterial-collagenase-sensitive [3H]fucose-labelled glycoprotein, Mr 140000, was immunoprecipitated from culture medium by a specific antibody to the pepsin-derived form of collagen type VI. This result suggests that the collagenous polypeptides [140K(VI) components] represent the biosynthetic precursors of type VI collagen that do not undergo processing to smaller species before deposition in the extracellular matrix. Analyses of 5M-guanidinium chloride extracts of tissues with markedly different elastin contents and at different stages of development suggested that there was no relationship between collagen type VI and elastic-fibre microfibrils, a conclusion supported by the observation that the immunoprecipitated glycoprotein, Mr 140000, was distinct from the glycoprotein MFPI, Mr 150000, believed to be a constituent of these microfibrils [Sear, Grant & Jackson (1981) Biochem. J. 194, 587-598].


Health-related quality of life in adults with epilepsy: the effect of age, age at onset and duration of epilepsy in a multicentre Italian study.
The potential effect of age-related factors on health-related quality of life (HRQOL) of patients with epilepsy has rarely been analyzed in the literature. We examined this association in a selected population of 815 adults with epilepsy recruited in the context of a multicentre study for the evaluation of Epi-QoL, one of the first Italian epilepsy-specific measures of HRQOL for adults with epilepsy. The Epi-QoL is a 46-item self-administered questionnaire focusing on six domains, which was successfully tested for reproducibility and validity. Ordinary least-squares regression models were used to assess the relationships between age-related factors (patient's age, age at seizure onset, and duration of epilepsy) and overall Epi-QoL score, controlling for the effect of potential confounders. We fitted simple regression models including each age-related factor alone to assess the independent role of each factor on the overall Epi-QoL score. We also fitted multiple regression models including pairs of age-related factors solely, as well as one or two age-related factors together with the same set of confounders. Simple regression models showed that age and duration of epilepsy were significant negative predictors of the overall Epi-QoL score: the higher was each age-related factor, the lower was the overall Epi-QoL score; age at onset alone was a nonsignificant predictor of the overall Epi-QoL score. Multiple regression models including two age-related factors solely showed that duration of epilepsy was still a significant negative predictor of the overall Epi-QoL score in both pairwise models, whereas age was a significant negative predictor only in the model including age at onset. Age at onset emerged as a significant positive predictor of the overall Epi-QoL score only in the model including age: the higher was age at onset, the higher was the overall Epi-QoL score. Adjusted regression models including either one or two age-related factors and controlling for the selected confounding variables showed that the age-related factors had no significant effect on the overall Epi-QoL score anymore. If no other known correlates of the overall Epi-QoL score are considered, age and duration of epilepsy can be expected to have a significant negative association with HRQOL in epilepsy (with the effect of duration being stronger and more consistent across models than the one of age), whereas age at onset is a positive predictor of the overall HRQOL of limited significance. However, demographic and clinical factors, such as seizure frequency in the preceding 12 months, may provide a better explanation of HRQOL in epilepsy.


Multi-drug resistance Trichophyton indotineae in a stray dog.
A 2.5-year-old stray dog showed signs of hair loss, mild skin crusting, and redness on extremities and trunk. The etiologic agent was confirmed as Trichophyton indotineae by sequencing of ITS region. Using the Clinical and Laboratory Standards Institute (CLSI M38-A3) guideline, antifungal susceptibility testing showed multidrug resistance phenotype against terbinafine (16 μg/mL-1), itraconazole, and some other tested antifungals (minimum inhibitory concentration, MIC≥16 μg/mL-1). However, luliconazole was found to be active in- vitro (0.016 μg/mL-1). Upon further studies, sequencing of SQLE gene showed an amino acids substitution of Phe397Leu and Ala448Thr, which is potentially linked to terbinafine resistance in Trichophyton species.


Does video e-learning improve pharmacy teams' attitudes and preparedness towards suicide prevention?
Evidence on the role of pharmacy teams in suicide prevention is growing. To support pharmacy teams, a video e-learning was produced by the Centre for Pharmacy Postgraduate Education (CPPE) involving an 'on-the-sofa' style group interview with people with personal and professional experience of suicide and suicide research. The objective was to measure any change in attitudes and preparedness for suicide prevention, following a video e-learning produced for pharmacy staff. People working in any sector of pharmacy in England and who accessed the training video were invited to complete a pre- and post- training questionnaire, between September 2019 and March 2021. Question types included demographics, experiences, attitudes as measured by the Attitudes to Suicide Prevention (ASP) scale, and preparedness. Descriptive statistics were used to summarize demographics and experience and paired t-tests were used to compare pre- and post- questionnaire responses. Both questionnaires were completed by 147 people. Most worked in community pharmacy (88%) and were pharmacists (64%) or pharmacy technicians (20%). Attitudes to suicide prevention improved significantly (pre:31.20 (SD 6.04); post:28.40 (SD 6.50), p < 0.0001) after watching the video, as did self-reported preparedness. Pharmacy teams' self-reported attitudes and preparedness for suicide prevention improved after watching this suicide awareness video compared to baseline. Suicide awareness training tailored to pharmacy teams may be valuable, but the longitudinal impact of any suicide prevention training requires further exploration.


[The uptake of potassium iodide and its effect as an antioxidant in isolated rabbit eyes].
Potassium iodide (KI) passes the cornea of isolated rabbit eyes with kinetics of approximately 0.25 mumol/h/ml aqueous humor. In photodynamic reactions, simulated as light-dependent decay of S-methyl-alpha-ketobutyric acid in the presence of riboflavin, KI acts as an antioxidant cooperating with internal scavengers such as ascorbate. With the simple model reactions applied it may be possible to study mechanism and functions in vivo of eye-protecting factors or combinations of compounds.


CHMP4C Disruption Sensitizes the Human Lung Cancer Cells to Irradiation.
Human lung cancer is highly invasive and the most malignant among human tumors. Adenocarcinoma as a specific type of non-small cell lung cancer occurs with high frequency and is also highly resistant to radiation therapy. Thus, how to avoid radiation resistance and improve radiotherapy effectiveness is a crucial question. In the present study, human lung cancer A549 and H1299 cells were irradiated using γ-rays from a Co60 irradiator. Protein expression was detected by Western blotting. Cell cycle and apoptosis were measured by flow cytometry. Surviving fraction was determined by colony formation assay. γH2AX and 53BP1 foci formation were examined by fluorescence microscopy. In the results, we show that CHMP4C, a subunit of Endosomal sorting complex-III (ESCRT-III), is involved in radiation-induced cellular response. Radiation-induced Aurora B expression enhances CHMP4C phosphorylation in non-small cell lung cancer (NSCLC) cells, maintaining cell cycle check-point and cellular viability as well as resisting apoptosis. CHMP4C depletion enhances cellular sensitivity to radiation, delays S-phase of cell cycle and reduces ionizing radiation (IR)-induced γH2AX foci formation. We found that Aurora B targets CHMP4C and inhibition of Aurora B exhibits similar effects with silencing of CHMP4C in radioresistance. We also confirm that CHMP4C phosphorylation is elevated after IR both in p53-positive and-negative cells, indicating that the close correlation between CHMP4C and Aurora B signaling pathway in mediating radiation resistance is not p53 dependent. Together, our work establishes a new function of CHMP4C in radiation resistance, which will offer a potential strategy for non-small cell lung cancer by disrupting CHMP4C.


Effects of age on pulmonary perfusion heterogeneity measured by magnetic resonance imaging.
Normal aging is associated with a decline in pulmonary function and efficiency of gas exchange, although the effects on the spatial distribution of pulmonary perfusion are poorly understood. We hypothesized that spatial pulmonary perfusion heterogeneity would increase with increasing age. Fifty-six healthy, nonsmoking subjects (ages 21-76 yr) underwent magnetic resonance imaging with arterial spin labeling (ASL) using a Vision 1.5-T whole body scanner (Siemens Medical Systems, Erlangen, Germany). ASL uses a magnetically tagged bolus to generate perfusion maps where signal intensity is proportional to regional pulmonary perfusion. The spatial heterogeneity of pulmonary blood flow was quantified by the relative dispersion (RD = SD/mean, a global index of heterogeneity) of signal intensity for voxels within the right lung and by the fractal dimension (D(s)). There were no significant sex differences for RD (P = 0.81) or D(s) (P = 0.43) when age was considered as a covariate. RD increased significantly with increasing age by approximately 0.1/decade until age 50-59 yr, and there was a significant positive relationship between RD and age (R = 0.48, P < 0.0005) and height (R = 0.39, P < 0.01), but not body mass index (R = 0.07, P = 0.67). Age and height combined in a multiple regression were significantly related to RD (R = 0.66, P < 0.0001). There was no significant relationship between RD and spirometry or arterial oxygen saturation. D(s) was not related to age, height, spirometry, or arterial oxygen saturation. The lack of relationship between age and D(s) argues against an intrinsic alteration in the pulmonary vascular branching with age as being responsible for the observed increase in global spatial perfusion heterogeneity measured by the RD.


[Free and protein-bound corticosterone levels of the plasma of white rats under hypoxia and after administration of adrenocorticotropic hormone].
It has been demonstrated that the plasma ratio of free and protein-bound corticosterone depends on the hypoxic degree. After two-hour hypoxia at an altitude of 10,000 m the trained and untrained animals exhibit a similar content of the free hormone (21%), with the content of total corticosterone being significantly lower in the trained rats. A study of variations in the content of free corticosterone in rats exposed to hypoxia for 20, 45 and 120 min and ACTH administration during the same time intervals has shown that the binding capacity of transcortine changes 45 min after the beginning of hypoxia.


Antimicrobial resistance patterns and plasmid profiles of Staphylococcus aureus isolated from milk and meat.
To determine the frequency of resistance of Staphylococcus aureus to various antimicrobial agents, and the relationship between antimicrobial resistance of the isolates and carriage of plasmids. A random sampling of milk and meat samples was carried out. Milk was collected from various dairy co-operative societies in Nairobi and Kiambu districts. Minced meat samples were purchased from various outlets in the city of Nairobi. Ninety six Staphylococcus aureus isolates from milk (seventy five isolates) and minced meat (twenty one isolates) samples. Plasmid profiles and antimicrobial susceptibility tests to ampicillin, lincomycin, penicillin, erythromycin, methicillin, minocycline, cotrimoxazole and chloramphenicol. Seventy one per cent of the isolates carried between one and six plasmids of molecular sizes ranging from 0.1 to 14.5 kilobases. High frequency of resistance was observed with lincomycin (67.7%), penicillin (66.7%) and cotrimoxazole (51%). A high percentage (76%) of isolates were susceptible to minocycline followed by erythromycin (57.3%). Most (80.2%) of isolates were multiply resistant to between two and six antibiotics. Most Staphylococcus aureus isolates were multiply resistant to various antimicrobial agents, but there was no apparent relationship between carriage of plasmids and antimicrobial resistance. Milk and meat may contain resistant Staphylococcus aureus posing a potential risk to consumers.


Glucose dissimilation in Entamoeba histolytica.
The endproducts of glucose dissimilation in Entamoeba histolytica were found to be carbon dioxide, acetate and ethanol. This organism possesses the same Embden-Meyerhof intermediates as found in most investigated organisms. In its glucose-to-pyruvate pathway several unusual features were demonstrated. The pyruvate-to-acetate and pyruvate-to-ethanol pathways are also unique as compared with that of other organisms. The distinctive properties of the metabolic enzymes provide the basis for the development of new chemotherapeutic agents against this parasitic protozoa.


SARS-CoV-2 spike-specific memory B cells express higher levels of T-bet and FcRL5 after non-severe COVID-19 as compared to severe disease.
SARS-CoV-2 infection elicits a robust B cell response, resulting in the generation of long-lived plasma cells and memory B cells. Here, we aimed to determine the effect of COVID-19 severity on the memory B cell response and characterize changes in the memory B cell compartment between recovery and five months post-symptom onset. Using high-parameter spectral flow cytometry, we analyzed the phenotype of memory B cells with reactivity against the SARS-CoV-2 spike protein or the spike receptor binding domain (RBD) in recovered individuals who had been hospitalized with non-severe (n = 8) or severe (n = 5) COVID-19. One month after symptom onset, a substantial proportion of spike-specific IgG+ B cells showed an activated phenotype. In individuals who experienced non-severe disease, spike-specific IgG+ B cells showed increased expression of markers associated with durable B cell memory, including T-bet and FcRL5, as compared to individuals who experienced severe disease. While the frequency of T-bet+ spike-specific IgG+ B cells differed between the two groups, these cells predominantly showed an activated switched memory B cell phenotype in both groups. Five months post-symptom onset, the majority of spike-specific memory B cells had a resting phenotype and the percentage of spike-specific T-bet+ IgG+ memory B cells decreased to baseline levels. Collectively, our results highlight subtle differences in the B cells response after non-severe and severe COVID-19 and suggest that the memory B cell response elicited during non-severe COVID-19 may be of higher quality than the response after severe disease.


A countrywide analysis of 27 solar power plants installed at different climates.
The world is still heavily using nonconventional energy sources, which are worryingly based on carbon. The step is now alternative energy sources hoping that they will be more environmentally friendly. One of the important energy conversion forms by using these sources is photovoltaic solar systems. These type of power plants is on the increase in everyday on the world. Before investment a solar power plant in a specified region, a techno-economic analyse is performed for that power plant by using several meteorological data like solar irradiance and ambient temperature. However, this analyses generally lacks evaluation on effects of climatic and geographical conditions. In this work, 5 years of data of 27 grid-connected photovoltaic power plants are investigated, which are installed on seven different climate types in Turkey. Firstly, the power plants are categorized considering the tilt angles and Köppen-Gieger climate classification. The performance evaluations of the plants are mainly conducted using monthly average efficiencies and specific yields. The monthly average efficiencies, which were classified using the tilts and climate types were from 12 to 17%, from 12 to 16% and from 13 to 15% for tilts 30°/10°, 25° and 20°, respectively. The variation in the specific yields decrease with elevation as y(x) = - 0.068x + 1707.29 (kWh/kWp). As the performances of photovoltaic systems for some locations within the Csb climatic regions may relatively lower than some other regions with same climate type. Thus, techno-economic performance for PVPP located in this climate classification should be carefully treated.


Yantar, a conserved arginine-rich protein is involved in Drosophila hemocyte development.
To identify novel factors involved in Drosophila hematopoiesis, we screened a collection of lethal recessive mutations that also affected normal hemocyte composition in larvae. We present the characterization of the gene yantar (ytr) for which we isolated null and hypomorphic mutations that were associated with severe defects in hemocyte differentiation and proliferation; ytr is predominantly expressed in the hematopoietic tissue during larval development and encodes an evolutionary conserved protein which is predominantly localized in the nucleus. The hematopoietic phenotype in ytr mutants is consistent with a defect or block in differentiation of precursor hemocytes: mutant larvae have enlarged lymph glands (LGs) and have an excess of circulating hemocytes. In addition, many cells exhibit both lamellocyte and crystal cell markers. Ytr function has been preserved in evolution as hematopoietic specific expression of the Drosophila or mouse Ytr proteins rescue the differentiation defects in mutant hemocytes.


Spatial clustering of childhood leukaemia with the integration of the Paediatric Environmental History.
Leukaemia remains the most common type of paediatric cancer and its aetiology remains unknown, but considered to be multifactorial. It is suggested that the initiation in utero by relevant exposures and/or inherited genetic variants and, other promotional postnatal exposures are probably required to develop leukaemia. This study aimed to map the incidence and analyse possible clusters in the geographical distribution of childhood acute leukaemia during the critical periods and to evaluate the factors that may be involved in the aetiology by conducting community and individual risk assessments. We analysed all incident cases of acute childhood leukaemia (<15 years) diagnosed in a Spanish region during the period 1998-2013. At diagnosis, the addresses during pregnancy, early childhood and diagnosis were collected and codified to analyse the spatial distribution of acute leukaemia. Scan statistical test methodology was used for the identification of high-incidence spatial clusters. Once identified, individual and community risk assessments were conducted using the Paediatric Environmental History. A total of 158 cases of acute leukaemia were analysed. The crude rate for the period was 42.7 cases per million children. Among subtypes, acute lymphoblastic leukaemia had the highest incidence (31.9 per million children). A spatial cluster of acute lymphoblastic leukaemia was detected using the pregnancy address (p<0.05). The most common environmental risk factors related with the aetiology of acute lymphoblastic leukaemia, identified by the Paediatric Environmental History were: prenatal exposure to tobacco (75%) and alcohol (50%); residential and community exposure to pesticides (62.5%); prenatal or neonatal ionizing radiation (42.8%); and parental workplace exposure (37.5%) CONCLUSIONS: Our study suggests that environmental exposures in utero may be important in the development of childhood leukaemia. Due to the presence of high-incidence clusters using pregnancy address, it is necessary to introduce this address into the childhood cancer registers. The Paediatric Environmental History which includes pregnancy address and a careful and comprehensive evaluation of the environmental exposures will allow us to build the knowledge of the causes of childhood leukaemia.


Molecular diversity of native rhizobia trapped by five field pea genotypes in Indian soils.
Five pea cultivars; HFP 4, HVP 3-5, HFP 9426, Jayanti and Hariyal, being grown in CCS Haryana Agricultural University farm were used to isolate native rhizobia. Selected 54 rhizobia, from all cultivars, were authenticated as rhizobia by plant infectivity test. Along with nodulation, symbiotic effectiveness in terms of symbiotic ratios showed wide range of effectiveness of pea rhizobia from 1.11 to 5.0. DNA of all the 54 rhizobia was extracted and amplified by PCR, using ERIC and 16S rDNA primers. Dendrogram based on ERIC profiles of these 54 rhizobia showed the formation of 13 subclusters at 80% level of similarity. Dendrogram based on RFLP of 16S rDNA by three restriction endonucleases; Msp I, Csp 6I and Rsa I; also formed 13 subclusters at 80% level of similarity. However, positioning of subclusters was different from that of ERIC based dendrogram. Majority of the isolates i.e. 64.8% by ERIC profiles and 44.4% by RFLP of 16S rDNA formed one cluster. Isolates from same nodule were not 100% similar. Considering each cluster representing a rhizobial genotype, both techniques used to assess molecular diversity indicated the presence of 13 genotypes of field pea rhizobia in CCS Haryana Agricultural University farm soil. Two pea rhizobial genotypes were able to nodulate all the five pea cultivars. Furthermore, high strain richness index (0.43-0.5) of field pea rhizobia was observed by both the techniques.


Cognitive Diagnostic Models With Attribute Hierarchies: Model Estimation With a Restricted Q-Matrix Design.
Attribute hierarchy is a common assumption in the educational context, where the mastery of one attribute is assumed to be a prerequisite to the mastery of another one. The attribute hierarchy can be incorporated through a restricted Q matrix that implies the specified structure. The latent class-based cognitive diagnostic models (CDMs) usually do not assume a hierarchical structure among attributes, which means all profiles of attributes are possible in a population of interest. This study investigates different estimation methods to the classification accuracy for a family of CDMs when they are combined with a restricted Q-matrix design. A simulation study is used to explain the misclassification caused by an unrestricted estimation procedure. The advantages of the restricted estimation procedure utilizing attribute hierarchies for increased classification accuracy are also further illustrated through a real data analysis on a syllogistic reasoning diagnostic assessment. This research can provide guidelines for educational and psychological researchers and practitioners when they use CDMs to analyze the data with a restricted Q-matrix design and make them be aware of the potentially contaminated classification results if ignoring attribute hierarchies.


Electrically controlled drug delivery from graphene oxide nanocomposite films.
On-demand, local delivery of drug molecules to target tissues provides a means for effective drug dosing while reducing the adverse effects of systemic drug delivery. This work explores an electrically controlled drug delivery nanocomposite composed of graphene oxide (GO) deposited inside a conducting polymer scaffold. The nanocomposite is loaded with an anti-inflammatory molecule, dexamethasone, and exhibits favorable electrical properties. In response to voltage stimulation, the nanocomposite releases drug with a linear release profile and a dosage that can be adjusted by altering the magnitude of stimulation. No drug passively diffuses from the composite in the absence of stimulation. In vitro cell culture experiments demonstrate that the released drug retains its bioactivity and that no toxic byproducts leach from the film during electrical stimulation. Decreasing the size and thickness of the GO nanosheets, by means of ultrasonication treatment prior to deposition into the nanocomposite, alters the film morphology, drug load, and release profile, creating an opportunity to fine-tune the properties of the drug delivery system to meet a variety of therapeutic needs. The high level of temporal control and dosage flexibility provided by the electrically controlled GO nanocomposite drug delivery platform make it an exciting candidate for on-demand drug delivery.


Pharmacological inhibition of PAI-1 alleviates cardiopulmonary pathologies induced by exposure to air pollutants PM2.5.
Numerous studies have established that acute or chronic exposure to environmental pollutants like particulate matter (PM) leads to the development of accelerated aging related pathologies including pulmonary and cardiovascular diseases, and thus air pollution is one of the major global threats to human health. Air pollutant particulate matter 2.5 (PM2.5)-induced cellular dysfunction impairs tissue homeostasis and causes vascular and cardiopulmonary damage. To test a hypothesis that elevated plasminogen activator inhibitor-1 (PAI-1) levels play a pivotal role in air pollutant-induced cardiopulmonary pathologies, we examined the efficacy of a drug-like novel inhibitor of PAI-1, TM5614, in treating PM2.5-induced vascular and cardiopulmonary pathologies. Results from biochemical, histological, and immunohistochemical studies revealed that PM2.5 increases the circulating levels of PAI-1 and thrombin and that TM5614 treatment completely abrogates these effects in plasma. PM2.5 significantly augments the levels of pro-inflammatory cytokine interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF), and this also can be reversed by TM5614, indicating its efficacy in amelioration of PM2.5-induced increases in inflammatory and pro-thrombotic factors. TM5614 reduces PM2.5-induced increased levels of inflammatory markers cluster of differentiation 107 b (Mac3) and phospho-signal transducer and activator of transcription-3 (pSTAT3), adhesion molecule vascular cell adhesion molecule 1 (VCAM1), and apoptotic marker cleaved caspase 3. Longer exposure to PM2.5 induces pulmonary and cardiac thrombosis, but TM5614 significantly ameliorates PM2.5-induced vascular thrombosis. TM5614 also reduces PM2.5-induced increased blood pressure and heart weight. In vitro cell culture studies revealed that PM2.5 induces the levels of PAI-1, type I collagen, fibronectin (Millipore), and sterol regulatory element binding protein-1 and 2 (SREBP-1 and SREBP-2), transcription factors that mediate profibrogenic signaling, in cardiac fibroblasts. TM5614 abrogated that stimulation, indicating that it may block PM2.5-induced PAI-1 and profibrogenic signaling through suppression of SREBP-1 and 2. Furthermore, TM5614 blocked PM2.5-mediated suppression of nuclear factor erythroid related factor 2 (Nrf2), a major antioxidant regulator, in cardiac fibroblasts. Pharmacological inhibition of PAI-1 with TM5614 is a promising therapeutic approach to control air pollutant PM2.5-induced cardiopulmonary and vascular pathologies.


Genotypic variations of Shiga toxin-converting phages from enterohaemorrhagic Escherichia coli O157: H7 isolates.
Pulsed-field gel electrophoresis (PFGE) analysis revealed that enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains had considerable variations in their genomes. This study investigated whether or not the molecular profile of Shiga toxin (Stx) 1- and Stx2-converting phages isolated from EHEC O157:H7 strains, derived from various sources in the USA and Japan, corresponded to the variations of host strains' genotypes as determined by PFGE. A total of 51 Stx-converting phages including 12 Stx1-converting phages and 37 Stx2-converting phages was isolated from seven USA isolates and 20 Japanese isolates. The average Dice coefficient values showed 44% similarity between phage DNAs in Stx2-converting phages digested with SmaI and 55% in Stx1-converting phages digested with HindIII, indicating considerable variation among phage DNA. In particular, restriction fragment length polymorphism (RFLP) patterns of Stx2-converting phage DNA varied according to the PFGE type of their host strain, which suggests that the phage genomes have altered their genotypic characteristics with those of host genomes. However, there are several exceptions: the RFLP patterns of some Stx2-converting phages were quite similar irrespective of the different genotypes of the host strains, indicating that horizontal transfer of Stx2-converting phage may also occur under some circumstances.


Ab Externo Imaging of Human Episcleral Vessels Using Fiberoptic Confocal Laser Endomicroscopy.
There is a growing interest in targeting minimally invasive surgery devices to the aqueous outflow system to optimize treatment outcomes. However, methods to visualize functioning, large-caliber aqueous and episcleral veins in-vivo are lacking. This pilot study establishes an ex-vivo system to evaluate the use of a confocal laser microendoscope to noninvasively image episcleral vessels and quantify regional flow variation along the limbal circumference. A fiber-optic confocal laser endomicroscopy (CLE) system with lateral and axial resolution of 3.5 μ m and 15 μ m, respectively, was used on three porcine and four human eyes. Diluted fluorescein (0.04%) was injected into eyes kept under constant infusion. The microprobe was applied to the sclera 1 mm behind the limbus to acquire real-time video. Image acquisition was performed at 15-degree intervals along the limbal circumference to quantify regional flow variation in human eyes. Vascular structures were visualized in whole human eyes without processing. Schlemm's canal was visualized only after a scleral flap was created. Fluorescent signal intensity and vessel diameter variation were observed along the limbal circumference, with the inferior quadrant having a statistically higher fluorescein signal compared to the other quadrants in human eyes ( P < 0.05). This study demonstrates for the first time that the fiber-optic CLE platform can visualize the episcleral vasculature with high resolution ex-vivo with minimal tissue manipulation. Intravascular signal intensities and vessel diameters were acquired in real-time; such information can help select target areas for minimally invasive glaucoma surgery (MIGS) to achieve greater intraocular pressure reduction.


Cold exposure lowers energy expenditure at the cellular level.
Mitochondrial function is intimately involved in various metabolic processes and is therefore essential to maintain cell viability. Of particular importance is the fact that mitochondrial membrane potential (ΔΨm ) is coupled with oxidative phosphorylation to drive adenosine triphosphate (ATP) synthesis. We have examined the effects of cold temperature stress on ΔΨm and the role of cold temperature receptor expression on ΔΨm . Human bronchial endothelial cell line, BEAS-2B, and human embryonic kidney, HEK293, cell line were transfected with the gene for cold temperature responsive receptor protein TRPM8 or TRPA1, and exposed to cold temperature. ΔΨm was monitored using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazoyl carbocyanine iodide derivative (JC-10), a ΔΨm probe. While cold temperatures significantly increased ΔΨm and mitochondrial ATP levels in cells transfected with temperature responsive receptor TRPM8 or TRPA1, no change was noted in wild-type cells. Moreover, the change in ΔΨm and ATP level was a dynamic process. ΔΨm was raised to peak levels within 10 min of cold exposure, followed by a return to baseline levels at 30 min. Our findings suggest that cold temperature exposure increased mitochondrial ΔΨm via a mechanism involving cold temperature receptors.


Zapped assembly of polymeric (ZAP) nanoparticles for anti-cancer drug delivery.
The starting hypothesis for this work was that microwave synthesis could enable the rapid assembly of polymers into size-specific nanoparticles (NPs). The Zapped Assembly of Polymeric (ZAP) NPs was initially realized using poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) block copolymers and distinct microwave reaction parameters. A library of polymeric NPs was generated with sizes ranging from sub-20 nm to 350 nm and low polydispersity. Select ZAP NPs were synthesized in 30 seconds at different scales and concentrations, up to 200 mg and 100 mg mL-1, without substantial size variation. ZAP NPs with diameters of 25 nm, 50 nm, and 100 nm were loaded with the chemotherapeutic paclitaxel (PXL), demonstrated unique release profiles, and exhibited dose-dependent cytotoxicity similar to Taxol. Incorporation of d-alpha tocopheryl polyethylene glycol succinate (TPGS) and PLGA33k allowed for the production of a sub-40 nm NP with an exceptionally high loading of PXL (12.6 wt%, ca. 7 times the original NP) and a slower release profile. This ZAP NP platform demonstrated scalable, flexible, and tunable synthesis with potential toward clinical scale production of size-specific drug carriers.


Attributable risks for colorectal cancer in northern Italy.
Using data from a case-control study conducted between 1985 and 1992 in northern Italy on 828 cases of colon cancer, 498 cases of rectal cancer and 2,024 controls in hospital for acute, non-neoplastic, non-digestive tract disorders, we estimated the percent population attributable risk (PAR) for colorectal cancer in relation to beta-carotene, vitamin C (as markers of a diet rich in fruit and vegetables), red meat and seasoning fat intake, daily meal frequency and family history of the disease. On the basis of multivariate odds ratios, adjusted for total calorie intake, a low intake of beta-carotene accounted for 39% of all the cases and a low intake of vitamin C for 14%. These two micronutrients together explained 43% of all colorectal cancer cases in this population. A high frequency of intake of red meat consumption explained 17% of all cases, and a high score of seasoning fats 4%. A higher daily meal frequency was responsible for 13% of the cases, and these 5 dietary factors together explained 63% of colorectal cancer cases in this population. Family history of colorectal cancer accounted for 4% of all cases. These estimates were similar for colon and rectal cancers separately, in males and females, and in younger and elderly subjects, except for seasoning fats and family history, whose PARs were apparently greater for colon cancer and at younger age. Thus, even though available dietary data were limited in several aspects, and the PAR estimates were based on somewhat arbitrary assumptions regarding the exposure distribution, about two-thirds of all colorectal cancers in this population could be explained in terms of a few risk factors or risk indicators considered. This would correspond to the avoidance of a large proportion of the over 18,000 deaths from colorectal cancer registered per year in the whole of Italy.