Study on growth of Rickettsia. V. Penetration of Rickettsia tsutsugamushi into mammalian cells in vitro.
A system has been described in which the penetration of Rickettsia tsutsugamushi (Karp strain) into tissue culture cells can be quantitated, and the factors affecting this process studied. The results indicated that rickettsial penetration in vitro depended largely on the viability of the organisms. Certain components of the fluid environment such as the divalent cations and protein were found to be of importance. The temperature dependence of the penetration process was found to vary with the nature of the suspending medium. A number of compounds related to L-glutamic acid enhanced penetration, whereas metabolic inhibitors depressed this process. Aureomycin at concentrations between 50 and 250 microg./ml. inhibited the penetration of rickettsiae while chloramphenicol at similar concentrations was ineffective. The results are discussed in terms of the biological and biochemical properties of this group of agents.


Pneumocephalus With Stroke-Like Symptoms: A Rare Complication of Mastoiditis.
Pneumocephalus is defined as the presence of gas or air in the intracranial space and typically arises as a result of neurotrauma. Clinically, pneumocephalus most often presents asymptomatically but may cause headache, nausea, vomiting, and confusion. Pneumocephalus arising from mastoiditis is an unforeseen complication with only a handful of cases reported. We report a case of an elderly male who presented with stroke-like symptoms in the setting of erosive mastoiditis with pneumocephalus.


Drivers and barriers to sustained use of Blair ventilated improved pit latrine after nearly four decades in rural Zimbabwe.
Some latrines remain unused even under conditions of high coverage in rural areas of low- and middle-income countries. Not much is known on household latrine use in the long term in the absence of an intervention. The current work assesses drivers and barriers to sustained use of a ventilated improved pit latrine (Blair VIP) design where it originated and how rural households adapt it to climate change. A mixed methods study was conducted from November 2020 to May 2021 among rural households of Mbire district, Zimbabwe. A cross sectional survey of 238 households with Blair ventilated improved pit (BVIP) latrines was conducted using a questionnaire and a latrine observation checklist. Data were analysed using logistic regression. Qualitative data were collected using six focus groups among house heads and analysed by thematic analysis. The latrine has perceived health, non-health and hygiene benefits for its sustained use. However, there are design, environmental and social barriers. The quantitative study indicated that determinants of latrine use were contextual (individual and household levels) and technology (individual level) factors. Focus groups indicated that latrine use was influenced by social, technology and contextual factors at multiple level factors. Interplay of factors influenced the intention to adapt the BVIP latrine to climate change. Local climate change adaptation strategies for the latrine were odour and erosion control, construction of the conventional latrine design and raised structures. The conventional BVIP latrine design is durable and relatively resilient to climate change with high local household use. High construction cost of the latrine causes households to build incomplete and poor quality designs which affect odour and fly control. These are barriers to sustained latrine use. The government should implement the new sanitation policy which considers alternative sanitation options and offer community support for adapting sanitation to climate change.


Working with anxious or fearful patients: a training tool for the medical practice staff.
Every medical practice must work with at least some patients who are fearful or anxious. This article explores the seven most common root causes of patient fear and anxiety and offers practical suggestions for avoiding or minimizing these fears. It addresses, in particular, the patient's fear of the unknown, of being humiliated, of reenacting a medical legend or horror story, and of being vulnerable and helpless. It also explores the patient's fear of high medical fees or being unable to pay, a poor or disappointing clinical outcome, and being in a hospital-like environment. The author suggests that the medica lpractice staff may wish to seek training in fear control and includes practical how-to strategies for minimizing fear when working with children.


Third European Stroke Science Workshop.
Lake Eibsee, Garmisch-Partenkirchen, November 19 to 21, 2015: The European Stroke Organization convened >120 stroke experts from 27 countries to discuss latest results and hot topics in clinical, translational, and basic stroke research. Since its inception in 2011, the European Stroke Science Workshop has become a cornerstone of European Stroke Organization's academic activities and major highlight for researchers in the field. Participants include stroke researchers at all career stages who convene for plenary lectures and discussions, thus facilitating crosstalk among researchers from different fields. As in previous years, the workshop was organized into 7 scientific sessions each focusing on a major research topic. All sessions started with a keynote lecture that provided an overview on current developments and set the scene for the following presentations. The latter were short focused talks on a timely topic and included the most recent findings, including unpublished data. A new element at this year's meeting was a hot topic session in which speakers had to present a provocative concept or update sharply within 5 minutes. In the following, we summarize the key contents of the meeting. The program is provided in the online-only Data Supplement.


Unimpaired osmotic water permeability and fluid secretion in bile duct epithelia of AQP1 null mice.
The mechanisms by which fluid moves across the luminal membrane of cholangiocyte epithelia are uncertain. Previous studies suggested that aquaporin-1 (AQP1) is an important determinant of water movement in rat cholangiocytes and that cyclic AMP mediates the movement of these water channels from cytoplasm to apical membrane, thereby increasing the osmotic water permeability. To test this possibility we measured agonist-stimulated fluid secretion and osmotically driven water transport in isolated bile duct units (IBDUs) from AQP1 wild-type (+/+) and null (-/-) mice. AQP1 expression was confirmed in a mouse cholangiocyte cell line and +/+ liver. Forskolin-induced fluid secretion, measured from the kinetics of IBDU luminal expansion, was 0.05 fl/min and was not impaired in -/- mice. Osmotic water permeability (P(f)), measured from the initial rate of IBDU swelling in response to a 70-mosM osmotic gradient, was 11.1 x 10(-4) cm/s in +/+ mice and 11.5 x 10(-4) cm/s in -/- mice. P(f) values increased by approximately 50% in both +/+ and -/- mice following preincubation with forskolin. These findings provide direct evidence that AQP1 is not rate limiting for water movement in mouse cholangiocytes and does not appear to be regulated by cyclic AMP in this species.


Near IR-emitting DNA-probes exploiting stepwise energy transfer processes.
The synthesis and characterisation of two new cyclen-based near IR-emitting lanthanide complexes is reported; the lanthanides are sensitised by rhodamine, which in turn is excited by energy transfer from a coumarin 2 moiety. The three lumophores function as an energy transfer cascade spanning the UV-visible-near IR region of the spectrum, resulting in large Stokes shifts. Double stranded DNA selectively switches one of the two energy transfer processes off, enabling luminescent DNA-sensing in the near IR region. The regioselective di-alkylation of the cyclen scaffold is explained with the help of DFT calculations.


High transvalvular pressure gradients on intraoperative transesophageal echocardiography after aortic valve replacement: what does it mean?
Intraoperative transesophageal echocardiography is now used routinely during aortic valve replacement, allowing immediate evaluation of replaced or repaired valves. It is well recognised that high transvalvular pressure gradients can be detected immediately after implantation of a prosthetic aortic valve which may be due to multifactorial confounding variables, including functional phenomena, pressure recovery and prosthesis-patient mismatch. This review article explores the variety of methods available for assessing prosthetic aortic valve function and considers causative factors which may contribute to high transvalvular gradients in an attempt to determine whether a physical problem with the valve exists, or whether the valve is functioning normally.


A survey of the practice and management of radiotherapy linear accelerator quality control in the UK.
The objective of this study was to determine current radiotherapy linear accelerator quality control (QC) practice in the UK, as a comparative benchmark and indicator of development needs, and to raise awareness of QC as a key performance indicator. All UK radiotherapy centres were invited to complete an online questionnaire regarding their local QC processes, and submit their QC schedules. The range of QC tests, frequency of measurements and acceptable tolerances in use across the UK were analysed, and consensus and range statistics determined. 72% of the UK's 62 radiotherapy centres completed the questionnaire and 40% provided their QC schedules. 60 separate QC tests were identified from the returned schedules. There was a large variation in the total time devoted to QC between centres: interquartile range from 13 to 26 h per linear accelerator per month. There has been a move from weekly to monthly testing of output calibration in the last decade, with reliance on daily constancy testing equipment. 33% of centres thought their schedules were in need of an update and only 30% used risk-assessment approaches to determine local QC schedule content. Less than 30% of centres regularly complete all planned QC tests each month, although 96% achieve over 80% of tests. A comprehensive "snapshot" of linear accelerator QC testing practice in the UK has been collated, which demonstrates reasonable agreement between centres in their stated QC test frequencies. However, intelligent design of QC schedules and management is necessary to ensure efficiency and appropriateness.


Factors associated with hearing loss among workers of the airline industry in Korea.
Noise-induced hearing loss (NIHL) is a major occupational hazard. Occupational noise exposure threatens the hearing of many workers. In addition to noise exposure in the workplace, multiple factors affect individual susceptibility to NIHL. The purpose of this study was to identify factors associated with hearing loss in airport workers. A cross-sectional epidemiological design was used. Study subjects were 255 high noise-exposed full-time male workers at a large metropolitan airport in Korea. Data were collected by a self-administered questionnaire, blood pressure measurement, audiological assessment, and a record review of baseline hearing and noise levels of locations where the employee worked. The result of multivariate analysis showed that both occupational noise exposure (noise exposure level, years of noise exposure) and personal risk factors including non-occupational noise exposure, history of ear disease, ototoxic drug use, cigarette smoking, hypertension, and use of hearing protective devices (HPDs) were significantly associated with hearing loss. An aggressive hearing conservation program is needed at the airport, emphasizing both job-related noise exposure and personal risk factors for hearing loss.


Are free radical reactions increased in the diabetic eye?
Reactive oxygen species (ROS) are thought to play a significant role in the development of diabetic retinopathy; however, no direct evidence supports ROS generation in vivo. This study used in vivo electron spin resonance (ESR) spectroscopy with a surface resonator to detect local free radical reactions. The ESR signal decay of carbamoyl-PROXYL was enhanced in the eyes of streptozotocin (STZ)-induced diabetic mice. This enhanced signal decay was suppressed by the administration of SOD or the pretreatment with aminoguanidine. We demonstrate, for the first time, specific free radical reactions in the eyes of mice with STZ-induced diabetes.


Reproducibility of M-mode echocardiographic assessment of left ventricular function. Significance of the temporal range of measurements.
The aim of this study was to evaluate the reproducibility of M-mode echocardiography in sequential studies on left ventricular (LV) function. To that end, 3 to 8 serial recordings were performed on 4 groups of healthy subjects (total number, 49) within dissimilar time periods: (1) within 3 h, (2) within 24 h, (3) within 1 to 2 weeks and (4) within 14 to 18 months. The examination technique was carefully standardized and the recordings were analysed by digitization and computer assistance; the data were averaged from 5 beats on each recording. The random variation of LV transverse dimensions and performance was roughly equal in the first 3 of these studies; the mean coefficients of variation ranged from 1.2 to 1.8% for end-diastolic dimension, from 1.7 to 2.3% for end-systolic dimension, and from 2.5 to 3.6% for ejection fraction. In the long-term study the respective mean coefficients were larger: 3.2%, 3.7% and 5.5%. The coefficients of variation for estimates of stroke volume, cardiac output, and peripheral vascular resistance ranged from 4.1 to 8.4% in studies completed within 1 day or inside 1 or 2 weeks but increased to 10-15.5% in the long-term study. The beat-to-beat variability of LV dimensions and performance, as assessed in a subgroup of 11 subjects, was roughly equal in quantity to the variability observed in the long-term study.(ABSTRACT TRUNCATED AT 250 WORDS)


Modulation of Itch by Conditioning Itch and Pain Stimulation in Healthy Humans.
Little is known about endogenous descending control of itch. In chronic pain, descending pain inhibition is reduced as signified by lowered conditioned pain modulation. There are indications that patients with chronic itch may also exhibit reduced endogenous descending inhibition of itch and pain. This study aimed to investigate whether and the extent to which itch can be modulated by conditioning itch and pain stimuli. Twenty-six healthy volunteers participated. The study consisted of 5 conditions designed to systematically assess endogenous modulation of itch or pain: 1) itch-induced modulation of contralateral itch, 2) pain-induced modulation of contralateral itch, 3) pain-induced modulation of ipsilateral itch, 4) pain-induced modulation of contralateral pain, and 5) itch-induced modulation of contralateral pain. Conditioning stimuli were cold pressor-induced pain and histamine-evoked itch, whereas the test stimuli were electrical stimulation paradigms designed to evoke itch or pain. Pain was significantly reduced (conditioned pain modulation-effect) by the conditioning pain stimulus (P < .001), but not by the conditioning itch stimulus (negative control condition). Itch was significantly reduced (conditioned itch modulation-effect) by contra- as well as ipsilateral applied conditioning pain (both P < .001), whereas conditioning itch stimulation only marginally reduced itch. Endogenous descending itch inhibition through mechanisms that are independent of segmental gating can be readily evoked by heterotopic conditioning pain stimulation. However, robust descending inhibition of itch cannot be evoked with conditioning itch stimulation. The study showed a hierarchical prioritization favoring pain-induced central descending modulation of itch as well as pain in humans. Future studies addressing potential aberrations in pain-evoked descending modulation of itch in chronic itch patients are warranted.


Acute hemorrhagic pancreatitis and diabetes mellitus following endoscopic retrograde cholangiopancreatography.
Endoscopic retrograde cholangiopancreatography (ERCP) is generally regarded as a safe and useful investigation in the diagnosis and management of diseases of the pancreas and biliary tree. Asymptomatic hyperamylasemia is not uncommon following this procedure, but clinical pancreatitis is rare. The authors report on a 56-year-old man who suffered life-threatening acute hemorrhagic pancreatitis 2 weeks after ERCP was performed for the investigation of abdominal pain. He subsequently had insulin-dependent diabetes mellitus, which was not present at the time of ERCP.


Relating retinal nerve fiber layer thickness and functional estimates of ganglion cell sampling density in healthy eyes and in early glaucoma.
To investigate the relationship between retinal nerve fiber layer (RNFL) thickness and peripheral grating resolution acuity (PGRA) as well as differential light sensitivity (DLS) in healthy subjects and patients with early glaucoma. The agreement between estimates of retinal ganglion cell (GC) density from each functional test is explored. PGRA was measured in 24 patients with early glaucoma (mean deviation [MD] > -8 dB) and 26 healthy subjects using achromatic Gabor stimuli in 4 diagonal visual field locations at 10° eccentricity. DLS for a Goldmann size III equivalent was obtained from individual spatial summation functions and expressed in Humphrey Field Analyzer-equivalent decibel values. RNFL thickness was measured around the optic nerve head using Zeiss Stratus optical coherence tomography and related to functional measures using a retinotopic map. Functional GC density was estimated using structure/function models for both tests. Passing-Bablok regression was used to investigate the structure/function relationships. A positive and statistically significant association was found between PGRA and RNFL thickness, and separately between DLS and RNFL thickness, for combined glaucoma and healthy data (both P < 0.05). The slope of the structure/function association in healthy subjects was not significantly different to that in glaucoma patients using either functional measure (both P > 0.05). Agreement between estimates of GC density from psychophysical data was moderate. The relationship between PGRA and RNFL thickness is at least as great in magnitude as that between DLS and RNFL thickness; a significant structure/function association is also observed in healthy subjects alone.


Prevalence of genital human papillomavirus among rural and urban populations in southern Yunnan province, China.
This study was designed to investigate and compare the HPV prevalence, genotypes distribution and associated risk factors in rural and urban women living in Xishuang Banna district, in the province of Yunnan. A total of 177 and 190 women from rural and urban areas were engaged, respectively. HPV DNA was amplified using the L1 consensus primers system (MY09/11 and GP5/6) and HPV GenoArray test was conducted for genotyping. Proportions were compared by chi-square test, and logistic regression was used to evaluate risk factors. A total of 54 women were positive for HPV DNA. Among rural women, 23 women were positive for HPV infection, of which 21 showed a single infection and 2 had a multiple infection. HPV-16 (10/23) was the most prevalent genotype followed by HPV-52 (5/23), and HPV-58 (5/23). Urban women had a higher infection rate for overall HPV (31/54) and for multiple genotype infection (8/31). HPV-52 (9/31) was the most prevalent genotype followed by HPV-39 (7/31) and HPV-68 (5/31). The age-specific HPV prevalence was also different between rural and urban women. In urban area, women with age <35 years had the highest HPV prevalence, which declined thereafter as age advanced. However, in rural women the highest HPV prevalence was observed in an older age group (>56 years). Ethnicity, smoking and parity were significantly associated with HPV infection among urban women. Our study demonstrates that HPV prevalence and genotype distribution varies among women from rural and urban areas in the south of Yunnan.


Comparison of the genomic organization and promoter function for human interleukin-8 receptors A and B.
Human neutrophils are highly responsive to the chemokine interleukin-8 (IL-8) owing to high levels of expression of two related receptors encoded by the single copy genes il8ra and il8rb located on chromosome 2q34-q35. To identify nuclear factors that regulate the expression of IL-8 receptors, we have first defined the organization of both genes and characterized their functional promoters. il8ra and il8rb span approximately 4 and 12 kilobase pairs of genomic DNA, respectively. In both cases, the open reading frame resides on a single exon. In contrast, the 5'-untranslated regions are more complex. For il8ra, it is formed from two exons, whereas for il8rb, seven distinct neutrophil mRNAs are formed by alternative splicing of 11 exons. One of the splice variants, designated IL8RB3, is the predominant form for il8rb. Two equally abundant mRNAs for il8ra, 2.0 and 2.4 kilobases in length, are expressed in neutrophils and arise from usage of two alternative polyadenylation signals. Primer extension analysis identified two major transcription start points for il8ra and 11 for il8rb. Regions extending 300 base pairs (bp) upstream from exon 1 of il8ra and 81 bp upstream from exon 3 of il8rb have limited sequence similarity but had strong constitutive promoter activity when cloned upstream from a chloramphenicol acetyltransferase-encoding reporter gene and transiently transfected into surrogate myeloid (HL-60, and U-937) and lymphoid (Jurkat) cell lines. Neither of these regions has sequences corresponding to classic promoter elements. In contrast, a region 643 base pairs upstream from exon 1 of il8rb had relatively low levels of constitutive promoter activity in all three cell environments, and a conserved TATA element is located 47 bp upstream of the 5'-end of exon 1. Thus, despite marked differences in the complexity of their genomic organization, il8ra and il8rb encode products that are similar in structure, function, and the major cell type of expression.


Radiological aspects of cancer of the esophagus.
The role radiology plays in the study of cancer of the esophagus (CE) involves three different diagnostic steps: identification of CE; staging of CE and postoperative follow-up. This study comprises 117 cases of CE, all surgically confirmed: in 57/117 CE, resection of the neoplasm with subsequent reconstruction of the digestive tract proved possible; in the remaining 60/117 non-resectable CE, a Celestin prosthetic tube was positioned. The radiological diagnosis was correct in 92.3% of the cases, the diagnostic error (9/117: 7.7%) being mainly due to under-diagnoses (6/9) rather than false negatives (3/9). Conventional radiology provided satisfactory results in the staging of CE. Among the postoperative complications in the 57 resected patients, fistulae (13/57) and mediastinal abscesses (1/57) were early complications, whereas stenosis of the anastomosis (5/57) and neoplastic recurrences (3/57) were late complications. The possible role of diagnostic delay in the poor prognosis of CE, as well as its possible causes, are discussed in this paper.


Magneto-Structural Properties and Theoretical Studies of a Family of Simple Heterodinuclear Phenoxide/Alkoxide Bridged MnIIILnIII Complexes: On the Nature of the Magnetic Exchange and Magnetic Anisotropy.
A family of MnIIILnIII strictly dinuclear complexes of general formula [MnIII(μ-L)(μ-OMe)(NO3)LnIII(NO3)2(MeOH)] (LnIII = Gd, Dy, Er, Ho) has been assembled in a one pot synthesis from a polydentate, multipocket aminobis(phenol)ligand [6,6'-{(2-(1-morpholyl)ethylazanediyl)bis(methylene)}bis(2-methoxy-4-methylphenol)], Mn(NO3)2·4H2O, Ln(NO3)3· nH2O, and NEt3 in MeOH. These compounds represent the first examples of fully structurally and magnetically characterized dinuclear MnIIILnIII complexes. Single X-ray diffraction studies reveal that all complexes are isostructural, consisting of neutral dinuclear molecules where the MnIII and LnIII metal ions, which exhibit distorted octahedral MnN2O4 and distorted LnO9 coordination spheres, are linked by phenoxide/methoxide double bridging groups. Static magnetic studies show that the MnIIIGdIII derivative exhibits a weak antiferromagnetic interaction between the metal ions, with a negative axial zero-field splitting D parameter. The MnIIIGdIII complex shows a notable magnetocaloric effect with magnetic entropy change at 5 T and 3 K of -Δ Sm = 16.8 J kg-1 K-1. Theoretical studies were performed to support the sign and magnitude of the magnetic anisotropy of the MnIII ion ( ab initio), to predict the value and nature of JMnGd, to disclose the mechanism of magnetic coupling, and to establish magneto-structural correlations (DFT calculations). The results of these calculations are corroborated by quantum theory of atoms in molecule analysis (QTAIM). Finally, MnIII-DyIII and MnIII-ErIII complexes show field-induced slow relaxation of the magnetization but without reaching a maximum above 2 K in the out-of-phase ac susceptibility. Ab initio calculations were also performed on MnIII-DyIII/HoIII systems to unravel the origin behind the weak SMM characteristics of the molecules possessing two strongly anisotropic ions. The mechanism of magnetic relaxation was developed, revealing a large QTM/tunnel splitting at the single-ion level. Furthermore, the anisotropy axes of the MnIII and LnIII ions were calculated to be noncollinear, leading to reduction of the overall anisotropy in the molecules. Hence, the herein reported complexes demonstrate that a combination of two anisotropic metal ions does not guarantee SMM behavior.


Space and attention in parietal cortex.
The space around us is represented not once but many times in parietal cortex. These multiple representations encode locations and objects of interest in several egocentric reference frames. Stimulus representations are transformed from the coordinates of receptor surfaces, such as the retina or the cochlea, into the coordinates of effectors, such as the eye, head, or hand. The transformation is accomplished by dynamic updating of spatial representations in conjunction with voluntary movements. This direct sensory-to-motor coordinate transformation obviates the need for a single representation of space in environmental coordinates. In addition to representing object locations in motoric coordinates, parietal neurons exhibit strong modulation by attention. Both top-down and bottom-up mechanisms of attention contribute to the enhancement of visual responses. The saliance of a stimulus is the primary factor in determining the neural response to it. Although parietal neurons represent objects in motor coordinates, visual responses are independent of the intention to perform specific motor acts.


Osteoclast differentiation gene expression profiling reveals chemokine CCL4 mediates RANKL-induced osteoclast migration and invasion via PI3K pathway.
The migration of osteoclasts (OCs) from circulation and bone marrow into bone surface plays a critical role in the pathogenesis of some bone resorptive diseases, such as rheumatoid arthritis and osteoporosis. To date, how the migration of OCs remains unclear. We investigated gene expression profiling in osteoclastic differentiation of bone marrow-derived macrophages (BMMs) into OCs by microarray analysis. We identified 387 genes overexpressed in osteoclastic differentiation of BMMs. Among them, chemokine CCL4 showed a robust up-regulation signal. High expression of CCL4 was validated in primary BMMs and OC precursor cell line RAW264.7 during differentiation into OCs. The CCL4 neutralization decreased RANKL-induced OC precursor cell migration and invasion in Matrigel-coated transwell membranes assay and in vitro wound healing assay. However, CCL4 inhibition did not affect OCs differentiation and differentiation associated gene expression. The CCL4 inhibition promoted the PI3K phosphorylation at 45 to 60 minutes after RANKL stimulation in RAW264.7. This study indicated that chemokine CCL4 is an important regulator for OCs migration via PI3K pathway, providing a novel therapy target for bone resorptive diseases.


Pain in older adults: development of a tool for measuring knowledge of residential aged care staff.
To develop a psychometrically sound tool for measuring the knowledge of nursing and care staff about the experience, assessment and management of pain in older people (including people with dementia) for use in the residential aged care setting. The Pain in Older Adults Knowledge Survey (POAKS) was developed and tested in two phases. Phase 1 involved developing an initial item pool with good content validity based on a review of the research literature and a modified Delphi technique involving national and international experts. A pool of 24 items was developed for testing. Initial testing of the psychometric properties of the POAKS with 30 university employees led to refinement and final wording of items. Phase 2 involved testing of the psychometric properties of the POAKS with 279 respondents, including first year (n = 176) and third year (n = 70) nursing students and staff in a residential aged care service (n = 33). Results established the content validity and internal consistency of the POAKS and supported its use as an instrument to measure nursing staff knowledge about the experience, assessment and management of pain in older people. The POAKS will enable residential aged care facilities to measure the level of knowledge among nursing and care staff about pain in older people (including people with dementia). The measure provides a basis for the development and implementation of educational interventions to address knowledge gaps that may impact on the quality of care provided.


Combined regulation of mTORC1 and lysosomal acidification by GSK-3 suppresses autophagy and contributes to cancer cell growth.
There is controversy over the role of glycogen synthase kinase-3 (GSK-3) in cancer progression. Recent work has implicated GSK-3 in the regulation of mammalian target of rapamycin (mTOR), a known player in malignant transformation. Autophagy, a self-degradation pathway, is inhibited by mTOR and is tightly associated with cell survival and tumor growth. Here we show that GSK-3 suppresses autophagy via mTOR complex-1 (mTORC1) and lysosomal regulation. We show that overexpression of GSK-3 isoforms (GSK-3α and GSK-3β) activated mTORC1 and suppressed autophagy in MCF-7 human breast cancer cells as indicated by reduced beclin-1 levels and upregulation of sequestosome 1 (p62/SQSTM1). Further, overexpression of GSK-3 increased the number of autophagosomes and inhibited autophagic flux. This activity was directly related to reduced lysosomal acidification triggered by GSK-3 (in which GSK-3β has a stronger impact). We found that lysosomal acidification is reduced in MCF-7 cells that also exhibit increased levels of autophagosomes and p62/SQSTM1 and increased activity of mTORC1. Subsequently, treating cells with GSK-3 inhibitors restored lysosomal acidification, enhanced autophagic flux and inhibited mTORC1. Furthermore, GSK-3 inhibitors inhibited cell proliferation. We provide evidence that GSK3-mediated mTORC1 activity and GSK-3-mediated lysosomal acidification occur via distinct pathways, yet both mTORC1 and lysosomes control cell growth. Finally, we show that GSK-3-reduced lysosomal acidification inhibits endocytic clearance as demonstrated by reduced endocytic degradation of the epidermal growth factor receptor. Taken together, our study places GSK-3 as a key regulator coordinating cellular homeostasis. GSK-3 inhibitors may be useful in targeting mTORC1 and lysosomal acidification for cancer therapy.


Brain changes and survival of animals with tumors implanted in the brain.
The effects of tumor cells implanted into the brain of animals on survival rates and gross and microscopic brain changes have been ascertained. Walker carcinosarcoma 256 cell suspensions were injected at several brain sites in rats and leukemia L1210 and P388 and Ehrlich ascites tumor cells, intracerebrally into BDF1 mice. Such neoplasms provided for rapid and rather predictable growth increments. The survival rates were dependent on the number of cells introduced, those receiving the higher counts succumbing first. The brains of the animals revealed fairly discrete space-occupying lesions and neurological symptoms became apparent only hours before death due to intracranial pressure.


Defect detection in anisotropic plates based on the instantaneous phase of signals.
Anisotropic materials are widely employed in industry and engineering, and efficient nondestructive testing techniques are important to guarantee the structural integrity of the involved parts. A simple technique is proposed to detect defects in anisotropic plates using ultrasonic guided waves and arrays. The technique is based on the application of an objective threshold to a synthetic aperture image obtained from the instantaneous phase (IP) of the emitter-receiver signal combinations. In a previous work the method was evaluated for isotropic materials, and in this paper it is shown that with some considerations the technique can also be applied to anisotropic plates. These considerations, which should be taken into account in beamforming, are (1) group velocity dependence with propagation direction, and (2) elastic focusing, which results in energy concentration in some propagation directions, with the practical consequence that not all aperture signals effectively contribute to the image. When compared with conventional delay-and-sum image beamforming techniques, the proposed IP technique results in significant improvements relative to defect detection and artifacts/dead zone reduction.


Monte Carlo modeling of photon migration in realistic human thoracic tissues for noninvasive monitoring of cardiac hemodynamics.
Noninvasive monitoring of cardiac hemodynamics remains challenging in cardiovascular medicine. The possibility of noninvasive optical monitoring of cardiac hemodynamics was theoretically investigated in this study. By utilizing the Monte Carlo simulation method for voxelized media (MCVM) and Visible Chinese Human dataset, we quantified and visualized the photon migration in human thoracic region. The light fluence distribution was showed to reach heart tissue (∼3 cm depth underbody surface) and 12% of the total fluence was absorbed by the myocardium. The proportion of spatial sensitivity distribution (SSD) in cardiac tissue to the total SSD reached 0.0195%. The portion of SSD increased following with cardiac diastole and diffuse reflectance deceased linearly with increasing cardiac volume. The optimal separation between the light source and detector was provided to be 3.5 to 4.0 cm for future development of noninvasive cardiac hemodynamics monitoring. A pilot experimental study was conducted to measure the diffuse reflectance light and fingertip photoplethysmography. These data suggest that the fluctuation period of near-infrared (NIR) diffuse reflectance was consistent with the cardiac cycle, while the fluctuation features of the NIR signal was not consistent with that of photoplethysmography. All results indicate the great potential of noninvasive optical monitoring of myocardial hemodynamics.


Optic chiasmatic-hypothalamic glioma.
Optic chiasmatic-hypothalamic gliomas (OCHGs) have been considered benign tumors and self-limiting in growth potential because of their histological appearance. Unfortunately, most clinical series have reported significant morbidity and mortality especially with the more extensive, posteriorly positioned tumors. The biological behavior of OCHGs is age-dependent, with patients younger than five years and older than 20 years typically having tumors that exhibit aggressive growth. There are no specific pathological features to help differentiate the clinical behavior of such tumors. The emergence of modern imaging techniques, including magnetic resonance imaging (MRI), has facilitated the monitoring of the natural history of the disease and the determination of the effects of therapy. Most patients with OCHGs survive for many years. While the natural history of an OCHG for any individual may be indeterminate, enough data are now available from large series to make recommendations for treatment. Our current treatment policy for patients with OCHGs in the context of NF-I without visual failure is a conservative one involving CSF shunting for hydrocephalus if present and medical therapy for endocrinologic dysfunction. Patients with or without NF-I with visual deterioration or progressive neurological deficits and a rapidly expanding suprasellar mass lesion are treated surgically. After tumor resection, patients whose vision is significantly compromised or who show progression of their disease on serial neuroimaging scans receive chemotherapy. If chemotherapy proves ineffective in disease stabilization, then considerations of radiation therapy are given to children over five years old.


Q-Switched Alexandrite Laser-induced Chrysiasis.
Chyriasis is an uncommon side effect that occurs in patients who are receiving prolonged treatment with either intravenous or intramuscular gold as a distinctive blue-gray pigmentation of light-exposed skin. Laser-induced chrysiasis is a rarely described phenomenon in individuals who have received systemic gold and are subsequently treated with a Q-switched laser. To describe the characteristics of patients with laser-induced chrysiasis. The authors describe a 60-year-old woman who developed chrysiasis at Q-switched alexandrite laser treatment sites. They also reviewed the medical literature using PubMed, searching the terms chrysiasis, gold, and laser-induced. Patient reports and previous reviews of these subjects were critically assessed and the salient features are presented. Including the authors' patient, laser-induced chrysiasis has been described in five Caucasian arthritis patients (4 women and 1 man); most of the patients had received more than 8g of systemic gold therapy during a period of 3 to 13 years. Gold therapy was still occurring or had been discontinued as long as 26 years prior to laser treatment. All of the patients immediately developed blue macules at the Q-switched laser treatment site. Resolution of the dyschromia occurred in a 70-year-old woman after two treatment sessions with a long-pulsed ruby laser and the authors' patient after a sequential series of laser sessions using a long-pulsed alexandrite laser, followed by a nonablative fractional laser and an ablative carbon dioxide laser. Laser-induced chrysiasis has been observed following treatment with Q-switched lasers in patients who are receiving or have previously been treated with systemic gold. It can occur decades after treatment with gold has been discontinued. Therefore, inquiry regarding a prior history of treatment with gold-particularly in older patients with arthritis-should be considered prior to treatment with a Q-switched laser. Also, treatment with a long-pulsed laser should be entertained in patients with either idiopathic or laser-induced chrysiasis. (JClinAesthetDermatol. 2015;8(9):48-53.) Chrysiasis is a distinctive blue-gray pigmentation of light exposed skin occurring in individuals who are receiving parenteral gold therapy.1 The 755nm Q-switched alexandrite laser is effective for the treatment of facial lentigines since the melanin granules absorb a high proportion of the laser energy and other chromophores offer little competitive absorption.2 The authors describe a woman who developed chrysiasis at Q-switched alexandrite laser treatment sites and whose dyschromia was successfully treated with a sequential series of laser sessions using a long-pulsed alexandrite laser, followed by a nonablative fractional laser and an ablative carbon dioxide laser.


Secretion of alpha and TSH-beta subunits in patients with acromegaly: an in vivo study.
Plasma alpha and TSH-beta subunit responses to iv administration of GHRH were examined in 19 patients with active acromegaly. In 4 patients (21%), plasma alpha subunit levels were increased over 50% of basal levels after administration of GHRH, whereas plasma TSH-beta subunit levels were increased in response to GHRH in another 5 patients (26%). No patient showed simultaneous increases of alpha and beta subunits. After successful surgery, alpha and TSH-beta subunits did not respond to GHRH. These findings support the idea that some pituitary adenomas in acromegaly cosecrete GH and either alpha subunit or TSH-beta subunit.


Blood supply of the facial nerve in the middle fossa: the petrosal artery.
To define the arterial supply to the facial nerve that crosses the floor of the middle cranial fossa. Twenty-five middle fossae from adult cadaveric-injected specimens were examined under 3 to 40x magnification. The petrosal branch of the middle meningeal artery is the sole source of supply that crossed the floor of the middle fossa to irrigate the facial nerve. The petrosal artery usually arises from the first 10-mm segment of the middle meningeal artery after it passes through the foramen spinosum, but it can arise within or just below the foramen spinosum. The petrosal artery is commonly partially or completely hidden in the bone below the middle fossa floor. It most commonly reaches the facial nerve by passing through the bone enclosing the geniculate ganglion and tympanic segment of the nerve and less commonly by passing through the hiatus of the greater petrosal nerve. The petrosal artery frequently gives rise to a branch to the trigeminal nerve. The middle meningeal artery was absent in one of the 25 middle fossae, and a petrosal artery could not be identified in four middle fossae. The petrosal arteries were divided into three types based on their pattern of supply to the facial nerve. The petrosal artery is at risk of being damaged during procedures in which the dura is elevated from the floor of the middle fossa, the middle fossa floor is drilled, or the middle meningeal artery is embolized or sacrificed. Several recommendations are offered to avoid damaging the facial nerve supply while performing such interventions.


Intracellular delivery of 10-hydroxycamptothecin with targeted nanostructured lipid carriers against multidrug resistance.
10-Hydroxycamptothecin (HCPT) is a clinical therapy agent against hepatoma. The chemotherapy of HCPT is strongly obstructed by the emergence of multidrug resistance (MDR), serious systemic toxicity, malfunction of rapid phagocytic and renal clearance disorder which are undesirable for its chemotherapy. In this paper, a drug delivery system (DDS) for HCPT has been developed in order to overcome MDR. Nanostructured lipid carriers (NLC) coated with xyloglucan (XG) was prepared by soya oil and XG consisting of side chains with galactose residues, a terminal moiety that can be used to target HCPT to hepatoma. The therapeutic potential of XG-NLC/HCPT was investigated on HepG2/HCPT cells and on human tumor xenograft nude mouse model (implanted with HepG2/HCPT cells). XG-NLC/HCPT not only indicated superior cytotoxicity against the drug resistant HepG2 cells but also in vivo, generated a higher therapeutic effect. Systemic toxicity study demonstrated that the carrier reduced systemic toxicity. XG-NLC/HCPT proved a great potential to serve as DDS to overcome MDR of HepG2/HCPT cells. These results suggested that XG NLC/HCPT represent a promising carrier for drug delivery to the hepatoma and reverse the drug resistant of HepG2 cells and XG could be exploited as a potential targeting device for liver tissue.


Nanoparticle-Enhanced Radiotherapy to Trigger Robust Cancer Immunotherapy.
External radiotherapy is extensively used in clinic to destruct tumors by locally applied ionizing-radiation beams. However, the efficacy of radiotherapy is usually limited by tumor hypoxia-associated radiation resistance. Moreover, as a local treatment technique, radiotherapy can hardly control tumor metastases, the major cause of cancer death. Herein, core-shell nanoparticles based poly(lactic-co-glycolic) acid (PLGA) are fabricate, by encapsulating water-soluble catalase (Cat), an enzyme that can decompose H2 O2 to generate O2 , inside the inner core, and loading hydrophobic imiquimod (R837), a Toll-like-receptor-7 agonist, within the PLGA shell. The formed PLGA-R837@Cat nanoparticles can greatly enhance radiotherapy efficacy by relieving the tumor hypoxia and modulating the immune-suppressive tumor microenvironment. The tumor-associated antigens generated postradiotherapy-induced immunogenic cell death in the presence of such R837-loaded adjuvant nanoparticles will induce strong antitumor immune responses, which together with cytotoxic T-lymphocyte associated protein 4 (CTLA-4) checkpoint blockade will be able to effectively inhibit tumor metastases by a strong abscopal effect. Moreover, a long term immunological memory effect to protect mice from tumor rechallenging is observed post such treatment. This work thus presents a unique nanomedicine approach as a next-generation radiotherapy strategy to enable synergistic whole-body therapeutic responses after local treatment, greatly promising for clinical translation.


Atypical femur fracture in an adolescent boy treated with bisphosphonates for X-linked osteoporosis based on PLS3 mutation.
Long-term use of bisphosphonates has raised concerns about the association with Atypical Femur Fractures (AFFs) that have been reported mainly in postmenopausal women. We report a case of an 18-year-old patient with juvenile osteoporosis based on X-linked osteoporosis due to a PLS3 mutation who developed a low trauma femoral fracture after seven years of intravenous and two years of oral bisphosphonate use, fulfilling the revised ASBMR diagnostic criteria of an AFF. The occurrence of AFFs has not been described previously in children or adolescents. The underlying monogenetic bone disease in our case strengthens the possibility of a genetic predisposition at least in some cases of AFF. We cannot exclude that a transverse fracture of the tibia that also occurred after a minor trauma at age 16 might be part of the same spectrum of atypical fractures related to the use of bisphosphonates. In retrospect our patient experienced prodromal pain prior to both the tibia and the femur fracture. Case reports of atypical fractures in children with a monogenetic bone disease such as Osteogenesis Imperfecta (OI) or juvenile osteoporosis are important to consider in the discussion about optimal duration of bisphosphonate therapy in growing children. In conclusion, this case report 1) highlights that AFFs also occur in adolescents treated with bisphosphonates during childhood and pain in weight-bearing bones can point towards this diagnosis 2) supports other reports suggesting that low trauma fractures of other long bones besides the femur may be related to long-term use of bisphosphonates 3) strengthens the concept of an underlying genetic predisposition in some cases of AFF, now for the first time reported in X-linked osteoporosis due to a mutation in PLS3 and 4) should be considered in decisions about the duration of bisphosphonate therapy in children with congenital bone disorders.


Amylase-producing plasmacytoma cell lines, AD3 and FR4, with der(14)t(8;14) and dic(8)t(1;8) established from ascites.
We established two human plasma cell lines, FR4 and AD3, from ascitic fluid in a patient with IgA k plasmacytoma (PC). Aberrant amylase production was found in this patient. Both AD3 and FR4 were free of Epstein-Barr virus, and both produced Ig A k in vitro. They produced amylase of the salivary type in vitro. This was confirmed by the demonstration of amylase mRNA comigrating with salivary gland mRNA. These cell lines commonly had unusual chromosomal abnormalities der(14)t(8;14) and dic(8)t(1;8). AD3 had additional chromosomal abnormalities compared with FR4. This suggests that AD3 is a subline of FR4. The oncogene c-myc is rearranged in most case of Burkitt's lymphoma with t(8;14). However, neither rearrangement nor amplification of the c-myc allele was detected in our PC lines. These lines expressed c-myc of 2.4 kb. There were no structural changes in the amylase genes of AD3 and FR4 detectable with Southern blotting analysis. As these lines were authentic PC lines, they would be useful for the future study of the relationship between the mechanism of oncogenesis and the rare tumor aberration, amylase production.


PRMT5 activates KLF5 by methylation to facilitate lung cancer.
The highly expressed oncogenic factor Krüppel-like factor 5 (KLF5) promotes various cancerous processes, such as cell growth, survival, anti-apoptosis, migration and metastasis, particularly in lung cancer. Nevertheless, the modifications to KLF5 after translation are poorly understood. Protein arginine methyltransferase 5 (PRMT5) is considered as an oncogene known to be involved in different types of carcinomas, including lung cancer. Here, we show that the expression levels of PRMT5 and KLF5 are highly expressed lung cancer. Moreover, PRMT5 interacts with KLF5 and facilitates the dimethylation of KLF5 at Arginine 41 in a manner that depends on methyltransferase activity. Downregulation or pharmaceutical suppression of PRMT5 reduces the expression of KLF5 and its downstream targets both in vitro and in vivo. Mechanistically, the dimethylation of KLF5 by PRMT5 promotes the maintenance and proliferation of lung cancer cells at least partially by stabilising KLF5 via regulation of the Akt/GSK3β signalling axis. In summary, PRMT5 methylates KLF5 to prevent its degradation, thereby promoting the maintenance and proliferation of lung cancer cells. These results suggest that targeting PRMT5/KLF5 axis may offer a potential therapeutic strategy for lung cancer.


Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum.
Epigenetic mechanisms regulate programmed gene expression during prenatal neurogenesis and serve as a mediator between genetics and environment in postnatal life. The recent discovery of 5-hydroxymethylcytosine (5-hmC), with highest concentration in the brain, has added a new dimension to epigenetic regulation of neurogenesis and the development of complex behavior disorders. Here, we take a candidate gene approach to define the role 5-hmC in Engrailed-2 (EN-2) gene expression in the autism cerebellum. The EN-2 homeobox transcription factor, previously implicated in autism, is essential for normal cerebellar patterning and development. We previously reported EN-2 overexpression associated with promoter DNA hypermethylation in the autism cerebellum but because traditional DNA methylation methodology cannot distinguish 5-methylcytosine (5-mC) from 5-hmC, we now extend our investigation by quantifying global and gene-specific 5-mC and 5-hmC. Globally, 5-hmC was significantly increased in the autism cerebellum and accompanied by increases in the expression of de novo methyltransferases DNMT3A and DNMT3B, ten-eleven translocase genes TET1 and TET3, and in 8-oxo-deoxyguanosine (8-oxo-dG) content, a marker of oxidative DNA damage. Within the EN-2 promoter, there was a significant positive correlation between 5-hmC content and EN-2 gene expression. Based on reports of reduced MeCP2 affinity for 5-hmC, MeCP2 binding studies in the EN-2 promoter revealed a significant decrease in repressive MeCP2 binding that may contribute to the aberrant overexpression of EN-2. Because normal cerebellar development depends on perinatal EN-2 downregulation, the sustained postnatal overexpression suggests that a critical window of cerebellar development may have been missed in some individuals with autism with downstream developmental consequences. Epigenetic regulation of the programmed on-off switches in gene expression that occur at birth and during early brain development warrants further investigation.


[Non tumoural cerebral biopsies: an analysis of 50 cases].
Cerebral biopsy is an invasive technique with limited, specific indications in view of the potential risk of complications. We reviewed a series of 50 cases of biopsies via craniotomy done over a period of 10 years to investigate suspected non tumoural disorders. There was a predominance of space occupying lesions and treatable infections (36%) and of non diagnostic biopsies (40%). There was poor correlation between the initial clinical diagnosis and the histological findings (50 71%). There were no complications. We conclude that the indications should be better defined and, in view of the satisfactory tolerance of this procedure, the study group should be increased by using other less invasive procedures (stereotaxia).


COX-2 inhibitors block chemotherapeutic agent-induced apoptosis prior to commitment in hematopoietic cancer cells.
Enzymatic inhibitors of pro-inflammatory cyclooxygenase-2 (COX-2) possess multiple anti-cancer effects, including chemosensitization. These effects are not always linked to the inhibition of the COX-2 enzyme. Here we analyze the effects of three COX-2 enzyme inhibitors (nimesulide, NS-398 and celecoxib) on apoptosis in different hematopoietic cancer models. Surprisingly, COX-2 inhibitors strongly prevent apoptosis induced by a panel of chemotherapeutic agents. We selected U937 cells as a model of sensitive cells for further studies. Here, we provide evidence that the protective effect is COX-independent. No suppression of the low basal prostaglandin (PG)E(2) production may be observed upon treatment by COX-2 inhibitors. Besides, the non-active celecoxib analog 2,5-dimethyl-celecoxib is able to protect from apoptosis as well. We demonstrate early prevention of the stress-induced apoptotic signaling, prior to Bax/Bak activation. This preventive effect fits with an impairment of the ability of chemotherapeutic agents to trigger apoptogenic stress. Accordingly, etoposide-induced DNA damage is strongly attenuated in the presence of COX-2 inhibitors. In contrast, COX-2 inhibitors do not exert any anti-apoptotic activity when cells are challenged with physiological stimuli (anti-Fas, TNFα or Trail) or with hydrogen peroxide, which do not require internalization and/or are not targeted by chemoresistance proteins. Altogether, our findings show a differential off-target anti-apoptotic effect of COX-2 inhibitors on intrinsic vs. extrinsic apoptosis at the very early steps of intracellular signaling, prior to commitment. The results imply that an exacerbation of the chemoresistance phenomena may be implicated.


Ignatzschineria cameli sp. nov., isolated from necrotic foot tissue of dromedaries (Camelus dromedarius) and associated maggots (Wohlfahrtia species) in Dubai.
Five bacterial strains, UAE-HKU57T, UAE-HKU58, UAE-HKU59, UAE-HKU60 and UAE-HKU61, were isolated in Dubai, UAE, from necrotic foot tissue samples of four dromedaries (Camelus dromedarius) and associated maggots (Wohrlfartia species). They were non-sporulating, Gram-negative, non-motile bacilli. They grew well under aerobic conditions at 37 °C, but not anaerobically. The pH range for growth was pH 7.0-9.0 (optimum, pH 7.5-8.0) and the strains could tolerate NaCl concentrations (w/v) up to 2 % (optimum, 0.5 %). They were catalase- and cytochrome oxidase-positive, but caseinase-, gelatinase- and urease-negative. Their phenotypic characters were distinguishable from other closely related species. Phylogenetic analyses of the almost-complete 16S rRNA gene and partial 23S rRNA gene, gyrB, groEL and recA sequences revealed that the five isolates were most closely related to undescribed Ignatzschineria strain F8392 and Ignatzschineria indica, but in most phylogenies clustered separately from these close relatives. Average nucleotide identity analysis showed that genomes of the five isolates (2.47-2.52 Mb, G+C content 41.71-41.86 mol%) were 98.00-99.97% similar to each other, but ≤87.18 % similar to other Ignatzschineriaspecies/strains. Low DNA relatedness between the five isolates to other Ignatzschineriaspecies/strains was also supported by Genome-to-Genome Distance Calculator analysis. The chemotaxonomic traits of the five strains were highly similar. They were non-susceptible (intermediate or resistant) to tetracycline and resistant to trimethoprim/sulphamethoxazole. The name Ignatzschineria cameli sp. nov. is proposed to accommodate these five strains, with strain UAE-HKU57T (=CCOS1165T=NBRC 113042T) as the type strain.


Epidemiology of multidrug-resistant Gram-negative pathogens isolated from ventilator-associated pneumonia in ICU patients.
Antibiotic resistance among Gram-negative pathogens isolated from ventilator-associated pneumonia (VAP) poses a grave threat in intensive care unit (ICU) patients. The aim of this study was to assess the prevalence of pathogens in ICU patients and their drug resistance profile. The prevalence of extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases and metallo-β-lactamases (MBLs) was also assessed. Tracheal aspirates were collected aseptically from 87 ICU patients between May 2012 and January 2014. Cultured isolates were identified by standard microbiological techniques. Antimicrobial susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. ESBLs and AmpC β-lactamases were detected both phenotypically and genotypically; MBLs were detected phenotypically. A total of 77 isolate were cultured. Gram-negative bacteria comprised 68 (88.3%) of the total isolates, among which 49 (72.1%) were multidrug-resistant (MDR). Gram-positive organisms comprised four (5.2%) of the total isolates and all four (100%) were MDR. Aspergillus fumigatus (6.4%) was the only fungal pathogen identified. Pseudomonas aeruginosa was the predominant pathogen associated with VAP. The rising trend of antibiotic resistance in Gram-negative organisms is alarming. Regular monitoring of the pattern of resistance in ICUs is critical in effective management of VAP patients.


Novel characterization of landscape-level variability in historical vegetation structure.
We analyzed historical timber inventory data collected systematically across a large mixed-conifer-dominated landscape to gain insight into the interaction between disturbances and vegetation structure and composition prior to 20th century land management practices. Using records from over 20 000 trees, we quantified historical vegetation structure and composition for nine distinct vegetation groups. Our findings highlight some key aspects of forest structure under an intact disturbance regime: (1) forests were low density, with mean live basal area and tree density ranging from 8-30 m2 /ha and 25-79 trees/ha, respectively; (2) understory and overstory structure and composition varied considerably across the landscape; and (3) elevational gradients largely explained variability in forest structure over the landscape. Furthermore, the presence of large trees across most of the surveyed area suggests that extensive stand-replacing disturbances were rare in these forests. The vegetation structure and composition characteristics we quantified, along with evidence of largely elevational control on these characteristics, can provide guidance for restoration efforts in similar forests.


Use of standardised assessment forms in referrals to hepatology outpatient services: implications for accurate triaging of patients with chronic hepatitis C.
To determine the spectrum of disease among non-urgent referrals to a tertiary hospital hepatology outpatient clinic, assess the adequacy of referral information in terms of risk stratification and determine whether a specifically designed referral template altered urgency for specialist assessment. A snapshot of the waiting list of a hepatology clinic at a tertiary hospital was taken from the scheduling database. Information was retrieved from referrals and attached investigations. Updated information was requested from subjects and their current general practitioner. Hepatitis C virus accounted for 68.7% of the 1223 reviewed referrals. Clinical information provided by referring clinicians was often incomplete. Provision of updated information identified the presence of comorbidities (obesity, 'heavy' alcohol consumption, mental health issues) and altered the need or urgency for specialist assessment in 22% of cases. Hepatitis C virus accounts for the majority of non-urgent referrals waiting to access hepatology outpatient consultations. Using a standardised assessment form as part of the referral process provides more information on comorbidities and risk factors and facilitates more accurate triaging of clinical urgency. Wider adoption of this strategy may increase appropriate access to hepatology services and reduce the future burden of cirrhosis and hepatocellular cancer. WHAT IS KNOWN ABOUT THE TOPIC? Little published data are available that describe the content and standard of hepatology referrals, or the urgency with which these patients need to be reviewed. Inadequate clinical information impairs the ability to accurately triage referrals and may lead to delays in access. WHAT DOES THE PAPER ADD? Almost 70% of reviewed referrals were for management of patients with hepatitis C virus infection, confirming this condition remains a major priority area in liver disease. Clinical information provided by referring clinicians was often incomplete, impairing the ability to accurately triage referrals. Only a minority of referrals provided information about relevant comorbidities (alcohol intake, injecting drug use, mental health issues and obesity) that negatively impact on the progression of liver disease or the response to antiviral treatment. WHAT ARE THE IMPLICATIONS FOR PRACTITIONERS? Hepatitis C virus remains a major health priority area in liver disease, increasing the future burden of cirrhosis and hepatocellular cancer. Many referred patients have comorbidities that increase their risk of progressive liver disease and related complications. Strategies to increase recognition and management of liver disease and its comorbidities in the community are required. The use of a standardised assessment form in referrals to hepatology outpatient services may assist with triaging of patients and improve access to appropriate care.


Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition.
Cardiovirus Leader proteins (LX) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus LM structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus LE, and also larger complexes with LE:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant LS and LT from Theiloviruses. When mutations were introduced to alter the LE zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on LE must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by LE.


Marketing genetic tests: empowerment or snake oil?
Genetic tests are currently being offered to the general public with little oversight and regulation as to which tests are allowed to be sold clinically and little control over the marketing and promotion of sales and use. This article provides discussion and data to indicate that the general public holds high opinions of genetic testing and that current media outlets for public education on genetic testing are not adequate to increase accurate knowledge of genetics. The authors argue that more regulation is needed to control and correct this problem in the United States.


Quality indicators used by trauma centers for performance measurement.
To describe the quality indicators (QIs) that trauma centers use for quality measurement and performance improvement. Measuring and reporting quality of care is a critical step to improve the quality of care. QIs compare actual trauma care against ideal criteria and identify patients in whom care may have been suboptimal and should be further reviewed. Three hundred thirty verified trauma centers in the United States, Canada, Australia, and New Zealand had their websites reviewed and leadership surveyed regarding QI use. The indicators identified were classified according to definition specifications, phase of care, Institute of Medicine aims, and contents. Two hundred fifty-one centers responded to the survey (76%) and the majority (97%) indicated that they use QIs. We obtained 10,587 QIs from 262 centers (survey responses and website review) of which 1,102 were unique indicators. The QIs primarily assessed the safety (49%), effectiveness (32%), efficiency (27%), and timeliness (22%) of hospital processes (64%) and outcomes (24%). The majority of indicators were used by a small number of centers (551 of 1,102 unique indicators used by single centers). Our study provides the first description of the QIs used by verified trauma centers in four high-income countries with similar systems of trauma care. The majority of trauma centers measure QIs designed to examine the safety, effectiveness, efficiency, and timeliness of hospital processes and outcomes. Opportunities exist to standardize existing QIs to allow broader implementation and develop new QIs to examine patient-centered care and equality of care.


A production of monoclonal antibodies by a simple electrofusion technique induced by ac pulses.
A simple and effective technique of electrofusion of the mouse myeloma cells and lymphocytes induced by AC pulses was investigated. Instead of pearl chain formation, we used a mild centrifugation of high-density cell suspensions to enhance the cell-cell contacts. The optimal frequency of the AC field used for cell fusion was 10 kHz. We confirmed the production of monoclonal antibodies against human serum albumin (HSA) by the hybridoma obtained by this method. Under the conditions used in the present work, the efficiency of hybridoma formation is several times higher than those obtained by the chemical method using polyethylene glycol (PEG). Hybridoma colonies were detected in as high as 95% of the wells and 33% of them proved positive against HSA.


Optical-limiting properties of oleylamine-capped gold nanoparticles for both femtosecond and nanosecond laser pulses.
We report strong broad-band optical-limiting properties of oleylamine-capped gold nanoparticles in solution for both femtosecond and nanosecond laser pulses. The nanosecond optical-limiting effects were characterized by using fluence-dependent transmittance measurements with 7 ns laser pulses at 532 and 1064 nm, and the femtosecond optical-limiting effects were characterized with a z-scan technique using 300 fs laser pulses at 780 nm. The oleylamine-capped gold nanoparticles were found to show strong broad-band optical-limiting effects for nanosecond laser pulses at 532 and 1064 nm and femtosecond laser pulses at 780 nm. These oleylamine-capped gold nanoparticles displayed exceptional optical-limiting effects with thresholds lower than that of carbon nanotube suspension, a benchmark optical limiter. Input-fluence- and angle-dependent scattering measurements suggested that nonlinear scattering played an important role in the observed optical-limiting behavior at 532 and 1064 nm.


N-Methyl-N-[(Z)-2-phenylpropenyl]thiobenzamide.
The crystal structure of the title thiobenzamide, C(17)H(17)NS, was determined to investigate the relationship between the photoreactivity in solid state and the structure. The geometry was confirmed to be the Z isomer.


Two-parameter bifurcations in LPA model.
The structured population LPA model is studied. The model describes flour beetle (Tribolium) population dynamics of four stage populations: eggs, larvae, pupae and adults with cannibalism between these stages. We concentrate on the case of non-zero cannibalistic rates of adults on eggs and adults on pupae and no cannibalism of larvae on eggs, but the results can be numerically continued to non-zero cannibalism of larvae on eggs. In this article two-parameter bifurcations in LPA model are analysed. Various stable and unstable invariant sets are found, different types of hysteresis are presented and abrupt changes in dynamics are simulated to explain the complicated way the system behaves near two-parameter bifurcation manifolds. The connections between strong 1:2 resonance and Chenciner bifurcations are presented as well as their very significant consequences to the dynamics of the Tribolium population. The hysteresis phenomena described is a generic phenomenon nearby the Chenciner bifurcation or the cusp bifurcation of the loop.


An H-2 haplotype possibly derived by crossing-over between (A alpha A beta) duplex and the E beta locus.
The B10.STA62 strain carries the H-2w27 haplotype derived from a wild mouse captured in the vicinity of Ann Arbor, Michigan. Products of two class II loci composing this haplotype, A alpha and A beta, are serologically, biochemically (by tryptic peptide mapping), and functionally indistinguishable from products controlled by the Ab alpha and Ab beta genes of the B10.A(5R) strain. In contrast, the polypeptide chain controlled by the third class II locus, E beta, is different from that controlled by the Eb beta gene. This Ew27 beta chain lacks an antigenic determinant present on the Eb molecule and carries determinants lacking on the Eb molecule, the Eb beta and Ew27 beta peptide maps differ in at least six peptides, and cytotoxic T cells specific for the Eb beta chains do not react with B10.STA62 target cells. This great difference between the Eb beta and Ew27 beta chains suggests that the corresponding genes have not been derived from one another by a direct mutational conversion; instead, H-2w27 appears to be a recombinant haplotype derived by crossing-over between the A alpha A beta duplex and the E beta locus. This is the first recombinant discovered separating these class II loci.


The contribution of protein kinases to plastic events in the superior colliculus.
1. The superior colliculus (SC)/optic tectum is a multi-layered midbrain area that harbours representations of visual and auditory space and somatic body surface. The development and maintenance of these sensory maps has been shown to involve activity and experience-dependent mechanisms. 2. The implantation of an extra eye primordium into the developing forebrain of Rana pipens results in the formation of dually innervated tecta that would have normally be solely innervated by the contralateral retina. The retinal projections are arranged in an interdigitating pattern of alternate stripes of terminations from each retina. The establishment of this striped pattern requires retinal activity and depends on N-methyl-D-aspartate (NMDA) receptors. Manipulation of protein kinase activity leads to the formation of an abnormal stripe pattern. 3. Regeneration of the goldfish retinotectal projection, following crush of the optic nerve, occurs in an activity dependent manner involving NMDA receptors. Furthermore, a critical period exists, during which retinal activity is vital for reformation of the visual map. Protein kinase manipulations during this period disrupt normal reformation. The same manoeuvres at other time points have little effect on map reformation. 4. An unusual form of long-term potentiation (SC-LTP) has been demonstrated in the in vitro preparation of the guinea-pig SC. By stimulating the optic layer of the SC, a postsynaptic potentiation can be recorded in the superficial grey layer. The expression of SC-LTP is masked but not prevented by blockade of NMDA receptors. The role of protein kinases in this form of synaptic modification has also been studied using various manipulations and inhibitors with varying substrate specificity. Whereas H7, an inhibitor reputed to be protein kinase C specific, only masks the expression of SC-LTP, K252a which has a broad substrate specificity blocks the induction of SC-LTP. 5. Experience-dependent formation of the auditory space map in the deeper layers of the SC is believed to be under the instruction of the visual representation in the superficial layers. Furthermore, a crucial period exists during which normal auditory and visual experience are required for successful establishment of the auditory map. Chronic exposure to 5-aminophosphonopentanoic acid (AP5) during this time prevents the formation of the map. Chronic exposure to K252a, a broad kinase inhibitor, over the same time period, also disrupts the formation of the auditory space map. 6. Taken together, these models emphasise the role of protein kinases in synaptic plasticity observed in the SC. Furthermore, interference with protein kinase activity at crucial stages of regeneration or development appears to disrupt the sequence of events that lead to the consolidation of SC receptive fields.


Multiplexing potential of NIR resonant and non-resonant Raman reporters for bio-imaging applications.
Multiplexed imaging, which allows for the interrogation of multiple molecular features simultaneously, is vital for addressing numerous challenges across biomedicine. Optically unique surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to serve as a vehicle to achieve highly multiplexed imaging in a single acquisition, which is non-destructive, quantitative, and simple to execute. When using laser excitation at 785 nm, which allows for a lower background from biological tissues, near infrared (NIR) dyes can be used as Raman reporters to provide high Raman signal intensity due to the resonance effect. This class of imaging agents are known as surface-enhanced resonance Raman scattering (SERRS) NPs. Investigators have predominantly utilized two classes of Raman reporters in their nanoparticle constructs for use in biomedical applications: NIR-resonant and non-resonant Raman reporters. Herein, we investigate the multiplexing potential of five non-resonant SERS: BPE, 44DP, PTT, PODT, and BMMBP, and five NIR resonant SERRS NP flavors with heptamethine cyanine dyes: DTTC, IR-770, IR-780, IR-792, and IR-797, which have been extensively used for biomedical imaging applications. Although SERRS NPs display high Raman intensities, due to their resonance properties, we observed that non-resonant SERS NP concentrations can be quantitated by the intensity of their unique emissions with higher accuracy. Spectral unmixing of five-plex mixtures revealed that the studied non-resonant SERS NPs maintain their detection limits more robustly as compared to the NIR resonant SERRS NP flavors when introducing more components into a mixture.


Reaching toward underexplored targets in antibacterial drug design.
The increase of antimicrobial resistance necessitates the renewal and strong research involvement in antibacterial drug design. In the following work, we comment on the key approaches used in development of new antibacterials, focusing on intracellular therapeutic targets that have been so far mostly underexplored: the enzymes of the Mur pathway MurA to MurF. We identify common obstacles observed during research on MurA, MurB, and Mur ligases inhibitors and their development into potential antibacterial compounds, and discern several approaches and solutions to tackle the whole-cell activity of designed compounds. Furthermore, we consolidate recent literature reports and encourage the further research on Mur enzymes.


Ovarian leukocyte distribution and cytokine/chemokine mRNA expression in follicular fluid cells in women with polycystic ovary syndrome.
Polycystic ovary syndrome (PCOS) affects 5-10% of reproductive-aged women and is commonly associated with anovulatory infertility. Leukocytes, together with granulosa cells, may contribute to the pathogenesis of PCOS via their ability to secrete an array of cytokines implicated in follicle growth. The aim of this study was to examine leukocyte subtypes in follicular phase ovaries and to quantify cytokine and chemokine mRNA expression in follicular fluid cells obtained at the time of oocyte retrieval before IVF in women with and without PCOS. Ovaries were immunostained for various leukocyte markers [CD3, CD4, CD14, CD15, CD45, CD45RA, CD45RO, CD57 and major histocompatibility complex (MHC) class II]. In addition, follicular fluid cells were subjected to quantitative RT-PCR to evaluate colony-stimulating factor-1 (CSF-1), granulocyte-macrophage (GM)-CSF, interleukins (IL-1beta, IL-6, IL-8 and IL-10), monocyte chemotactic protein (MCP-1) and tumour necrosis factor (TNFalpha) mRNA expression relative to beta-actin. CD45RO+ cells (activated/memory T lymphocytes) were reduced by 60% in the theca layer of follicles from PCOS women. The relative abundance of macrophages and neutrophils was unchanged. Cytokine and chemokine mRNA transcripts examined were not affected by PCOS status. There was an association between high BMI and high TNFalpha and low IL-6 mRNA expression in follicular cells. IL-6 expression was higher in women who subsequently achieved pregnancy. T lymphocytes potentially play a role in the local pathological mechanisms of PCOS. Further studies are required to identify their contribution to the aetiology of this common condition.


China's rural public health system performance: a cross-sectional study.
In the past three years, the Government of China initiated health reform with rural public health system construction to achieve equal access to public health services for rural residents. The study assessed trends of public health services accessibility in rural China from 2008 to 2010, as well as the current situation about the China's rural public health system performance. The data were collected from a cross-sectional survey conducted in 2011, which used a multistage stratified random sampling method to select 12 counties and 118 villages from China. Three sets of indicators were chosen to measure the trends in access to coverage, equality and effectiveness of rural public health services. Data were disaggregated by provinces and by participants: hypertension patients, children, elderly and women. We examined the changes in equality across and within region. China's rural public health system did well in safe drinking water, children vaccinations and women hospital delivery. But more hypertension patients with low income could not receive regular healthcare from primary health institutions than those with middle and high income. In 2010, hypertension treatment rate of Qinghai in Western China was just 53.22% which was much lower than that of Zhejiang in Eastern China (97.27%). Meanwhile, low performance was showed in effectiveness of rural public health services. The rate of effective treatment for controlling their blood pressure within normal range was just 39.7%. The implementation of health reform since 2009 has led the public health development towards the right direction. Physical access to public health services had increased from 2008 to 2010. But, inter- and intra-regional inequalities in public health system coverage still exist. Strategies to improve the quality and equality of public health services in rural China need to be considered.


Clinical Potential of Mesenchymal Stem Cell-Derived Exosomes in Bone Regeneration.
Bone metabolism is regulated by osteoblasts, osteoclasts, osteocytes, and stem cells. Pathologies such as osteoporosis, osteoarthritis, osteonecrosis, and traumatic fractures require effective treatments that favor bone formation and regeneration. Among these, cell therapy based on mesenchymal stem cells (MSC) has been proposed. MSC are osteoprogenitors, but their regenerative activity depends in part on their paracrine properties. These are mainly mediated by extracellular vesicle (EV) secretion. EV modulates regenerative processes such as inflammation, angiogenesis, cell proliferation, migration, and differentiation. Thus, MSC-EV are currently an important tool for the development of cell-free therapies in regenerative medicine. This review describes the current knowledge of the effects of MSC-EV in the different phases of bone regeneration. MSC-EV has been used by intravenous injection, directly or in combination with different types of biomaterials, in preclinical models of bone diseases. They have shown great clinical potential in regenerative medicine applied to bone. These findings should be confirmed through standardization of protocols, a better understanding of the mechanisms of action, and appropriate clinical trials. All that will allow the translation of such cell-free therapy to human clinic applications.


Expression profiles of matrix metalloproteinases and their inhibitors in colonic inflammation related to pediatric inflammatory bowel disease.
The differential diagnosis of chronic colitis in inflammatory bowel disease (IBD) is challenging and a distinction between Crohn's disease (CD) and ulcerative colitis (UC) is not always possible. Matrix metalloproteinases (MMPs) cleave components of the extracellular matrix and their dysregulation leads to damage to the mucosa. They are involved in inflammation in IBD, as well as in eventual tissue repair. We aimed to examine putative differences in the profiles of MMPs and their tissue inhibitors [tissue inhibitors of metalloproteinase (TIMPs)] in pediatric IBD to find better tools for differential diagnosis of various IBD subgroups at the tissue level in the colon. Expression of MMPs -1, -7, -8, -9, -10, -12 and -26 and TIMPs -1 and -3 was studied by immunohistochemistry in colonic tissue samples of 32 pediatric patients with IBD and 11 non-IBD cases. In the colon, expression of MMP-7 in epithelium was greater in CD samples compared to UC samples (1.09 versus 0.33; P = 0.010). Furthermore, epithelial MMP-10 expression was elevated in CD and UC samples compared to non-IBD samples (1.55 versus 1.00; P = 0.041 and 1.58 versus 1.00; P = 0.025, respectively). TIMP-3 expression in the stroma was higher in both the CD and UC groups when compared to non-IBD samples (2.18 versus 1.36; P = 0.026 and 2.50 versus 1.36; P = 0.002, respectively), but differences between UC and CD could not be observed. Increased expression of epithelial MMP-10 and stromal TIMP-3 could serve as histological indicators of IBD etiology. Epithelial MMP-7 expression, on the other hand, could help to differentiate between CD-related colitis and UC.


Gabapentinoid Dosing and Associated Toxicities in Patients With or Without Concomitant Opioids During Hospitalization.
Gabapentinoid drugs (gabapentin and pregabalin) are increasingly used for pain as both patients and physicians seek opioid-sparing or opioid-reducing strategies. Such widespread use for off-label pain indications is not supported by robust evidence, risking potential unintended consequences relating to adverse events. This study evaluated adult patients who were administered at least one dose of a gabapentinoid during a hospital admission between January 1, 2018 and December 31, 2018. The primary objective was to compare the difference in administered gabapentinoid total daily dose in patients with or without concomitant opioid. Secondary objectives included comparing the difference in sedation documentation, renal function, fall risk scores using the Hester Davis Fall Risk Scale, and central nervous system (CNS) related side effects between the 2 study groups. Four hundred and ninety nine patients (pregabalin n = 32 and gabapentin n = 467) were included. Due to small sample size for pregabalin, outcome results were presented for gabapentin only. The mean gabapentin total daily dose was 706 mg (SD = 614 mg) in the non-opioid group versus 860 mg (SD = 553 mg) in the opioid group (p = 0.007). There was more sedation documentation observed in the opioid group (p < 0.001). Patients in the opioid group used a significantly higher gabapentinoid total daily dose. Sedation documentation was significantly more in the opioid group despite commonly reported CNS related adverse events with gabapentinoids.


A novel proteolytic event controls Hedgehog intracellular sorting and distribution to receptive fields.
The patterning activity of a morphogen depends on secretion and dispersal mechanisms that shape its distribution to the cells of a receptive field. In the case of the protein Hedgehog (Hh), these mechanisms of secretion and transmission remain unclear. In the developing Drosophila visual system, Hh is partitioned for release at opposite poles of photoreceptor neurons. Release into the retina regulates the progression of eye development; axon transport and release at axon termini trigger the development of postsynaptic neurons in the brain. Here we show that this binary targeting decision is controlled by a C-terminal proteolysis. Hh with an intact C-terminus undergoes axonal transport, whereas a C-terminal proteolysis enables Hh to remain in the retina, creating a balance between eye and brain development. Thus, we define a novel mechanism for the apical/basal targeting of this developmentally important protein and posit that similar post-translational regulation could underlie the polarity of related ligands.


Bipolar manic-depressives and unipolar depressives distinguished by tests of lateral asymmetry.
Tests of lateral asymmetry in hand preference and superiority in thumb opposition rotation (opposing the thumb's pulp surface to that of the little finger) have been applied to bipolar and unipolar affective patients in an attempt to evaluate the effectiveness of these tests in differentiating the bipolar and unipolar patient populations. Two comparison samples were also tested: nonpsychotic central nervous system (CNS) disease patients, and normal controls. The normals divided almost evenly into pure dominance (e.g., right-handed, and superior right thumb opposition) and cross-dominance (e.g., right-handed, but superior left thumb opposition). All but one of the CNS disease patients were cross-dominant; the bipolars were predominantly pure dominant; while the unipolars, in contrast to the pure dominant bipolars, were in the majority cross-dominant. This result is consistent with the view that there are two types of affective disorder, bipolar manic-depressive and unipolar depressive illness.


Alcohol screening and brief intervention among military service members and veterans: rural-urban disparities.
Access to screening, brief intervention and referral to treatment programmes for alcohol use have been shown to be effective; however, little is known about access to these services among service members and veterans. We examined the association of service member or veteran rural-dwelling area and the following outcomes: recent general health check-up, alcohol screening and alcohol brief intervention. Data on 5080 military service members and veterans were obtained from the 2017 Behavioural Risk Factor Surveillance System of the USA. We estimated rural-urban disparities in the receipt of a recent voluntary general health check-up, as well as the receipt of alcohol screening and brief intervention, using a mixed logit model. Of the 5080 participants in the study, a total of 4666 (90.49%, 95% CI 89.39% to 91.48%) reported a general health check-up in the last 2 years. Results showed 7.48% of the sample (95% CI 6.64% to 8.41%) exhibited heavy alcohol consumption patterns. Of the 414 participants who did not undergo a general health check-up, 13.80% (95% CI 9.63% to 19.41%) exhibited a pattern of heavy alcohol consumption. Rural individuals were less likely to report a recent health check-up (adjusted OR=0.82, 95% CI 0.79 to 0.87). Rurality was also independently associated with decreased likelihood of receiving an alcohol screening and brief intervention. Greater access to telehealth or other geographically flexible screening and brief intervention programmes is needed in rural areas for service members and veterans.


From genome scissors to molecular scalpel: evolution of CRISPR systems.
From bizarre palindromic repeats to a bacterial defense mechanism, to genome editing tool, and more, Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR has significantly impacted the way we study genome modification in less than a decade. In this review, we would like to highlight some key players over 30 years of research and explain this biotechnological tool's basic mechanisms. We also refer to the evolution of the CRISPR variants and some of the applications derived from them. The understanding and upgrading of this system will be a valuable tool in the years to come to solve some of the challenges in diverse fields from pharmaceuticals to therapeutics, from basic plant genetics to crop improvement, from metabolic engineering to waste management and industrial processing.


Novel mutations in COL4A3, COL4A4, and COL4A5 in Chinese patients with Alport Syndrome.
Alport syndrome (AS) is a clinically and genetically heterogeneous, progressive nephropathy caused by mutations in COL4A3, COL4A4, and COL4A5, which encode type IV collagen. The large sizes of these genes and the absence of mutation hot spots have complicated mutational analysis by routine polymerase chain reaction (PCR)-based approaches. Here, in order to design a rapid and effective method for the genetic diagnosis of AS, we developed a strategy by utilizing targeted capture associated with next-generation sequencing (NGS) to analyze COL4A3, COL4A4, and COL4A5 simultaneously in 20 AS patients. All the coding exons and flanking sequences of COL4A3, COL4A4, and COL4A5 from the probands were captured followed by HiSeq 2500 sequencing. Candidate mutations were validated by classic Sanger sequencing and quantitative (q)PCR. Sixteen patients (16/20, 75%) showed X-linked inheritance, and four patients (4/20, 20%) showed autosomal recessive inheritance. None of the individuals had autosomal-dominant AS. Fifteen novel mutations, 6 known mutations, and 2 novel fragment deletions were detected by targeted capture and NGS. Of these novel mutations, 12, 3, and 2 mutations were detected in COL4A5, COL4A4, and COL4A3, respectively. A comparison of the clinical manifestations caused by different types of mutations in COL4A5 suggested that nonsense mutations and glycine substitution by an acidic amino acid are more severe than the other missense mutations. Pathogenic mutations were detected in 20 patients. These novel mutations can expand the genotypic spectrum of AS. Our results demonstrated that targeted capture and NGS technology are effective in the genetic diagnosis of AS.


Emulsifying Properties of Oxidatively Stressed Myofibrillar Protein Emulsion Gels Prepared with (-)-Epigallocatechin-3-gallate and NaCl.
The dose-dependent effects of (-)-epigallocatechin-3-gallate (EGCG; 0, 100, or 1000 ppm) on the textural properties and stability of a myofibrillar protein (MP) emulsion gel were investigated. Addition of EGCG significantly inhibited formation of carbonyl but promoted the loss of both thiol and free amine groups. Addition of EGCG, particularly at 1000 ppm, initiated irreversible protein modifications, as evidenced by surface hydrophobicity changes, patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and differential scanning calorimetry. These results indicated that MP was modified by additive reactions between the quinone of EGCG and thiols and free amines of proteins. These adducts increased cooking loss and destabilized the texture, especially with a large EGCG dose. Confocal laser scanning microscopy and scanning electron microscopy images clearly indicated the damage to the emulsifying properties and the collapse of the internal structure when the MP emulsion gel was treated with a large EGCG dose. A high concentration of NaCl (0.6 M) improved modification of MP and increased the rate of deterioration of the internal structure, especially with the large EGCG dose (1000 ppm), resulting in an MP emulsion gel with extremely unstable emulsifying properties.


[Purification of Bacillus mesentericus proteolytic enzymes by affinity chromatography].
By means of affinity chromatography on Ovomucoid-Sepharose two proteinases hydrolyzing casein and elastin were isolated from the supernatant of the Bacillus mesentericus culture medium. The activity yield of proteinases was 100%. The characteristics of the purified enzymes were studied. It is demonstrated that B. mesentericus possesses several proteinases.


A novel protein kinase C (PKCepsilon) is required for fMet-Leu-Phe-induced activation of NF-kappaB in human peripheral blood monocytes.
We have reported that the chemoattractant, fMet-Leu-Phe (fMLP), induces the activation of NF-kappaB in human peripheral blood monocytes and that this requires the activity of small GTPase, RhoA (Huang, S., Chen, L.-Y., Zuraw, B. L., Ye, R. D., and Pan, Z. K. (2001) J. Biol. Chem. 276, 40977-40981). Here we showed that the novel protein kinase C isozyme, PKCepsilon, associates functionally with RhoA in fMLP-stimulated monocytes and that PKCepsilon acted as a signaling component downstream of the GTPase RhoA during fMLP-induced activation of NF-kappaB. Stimulation of monocytes with fMLP resulted in activation of both PKCepsilon and NF-kappaB. This latter activation was largely blocked by specific inhibitors of PKCepsilon by transient expression of a dominant-negative form of PKCepsilon and by PKCepsilon-specific short interfering RNA. These findings demonstrate, for the first time, that fMLP-induced activation of NF-kappaB utilizes a signaling pathway, which requires activity of PKCepsilon, and that PKCepsilon acts as a signaling component downstream of RhoA in cytokine gene transcription stimulated by a chemoattractant. The specificity of this response suggests an important role for the Rho GTPase-PKCepsilon-NF-kappaB pathway in host defense and represents a novel and potentially important mechanism through which fMLP not only attracts leukocytes but may also contribute directly to inflammation.


Mechanisms underlying the modulation of arrhythmogenic events by components of ischemia in guinea pig cardiac myocytes.
The effects of some components of ischemia on the oscillatory (Vos) and nonoscillatory (V(ex)) potentials and respective currents (Ios and Iex), as well as their mechanisms, were studied in guinea pig isolated ventricular myocytes by means of a single-microelectrode, discontinuous voltage clamp method. Repetitive activations induced not only Vos and Ios, but also V(ex) and Iex. A small decrease in resting potential caused an immediate increase in Vos followed by a gradual increase due to the longer action potential. Immediate and gradual increases in Ios also occurred during voltage clamp steps. A small depolarization increased Vos and V(ex), and facilitated the induction of spontaneous discharge by fast drive. At Vh where INa is inactivated, depolarizing steps induced larger Ios and Iex, indicating the importance of the Na-independent Ca loading. High [K]o decreased the resting potential, but also Vos, V(ex), Ios, Iex, and ICa. In high [K]o, depolarization still increased Vos and V(ex). Norepinephrine (NE) enhanced Vos and V(ex), and also Ios and Iex, during voltage clamp steps. High [K]o antagonized NE effects, and NE those of high [K]o. In conclusion, on depolarization, Vos and Ios immediately increase through a voltage-dependent mechanism; and then Vos and Ios gradually increase, apparently through an increased Ca load related to the longer action potentials and the Na-Ca exchange. The depolarization induced by V(ex) may contribute to increase Vos size. Vos and V(ex) are similarly influenced by different procedures that modify Ca load. The arrhythmogenic events are enhanced by the simultaneous presence of depolarization, faster rate, or NE. Instead, high [K]o decreases Vos and V(ex) by decreasing ICa and opposes the effects of NE. The voltage clamp results show that potentiation and antagonism between different components of ischemia are due primarily to changes in Ca loading and not to changes in action potential configuration.


Compassion fatigue resiliency training: the experience of facilitators.
This qualitative evaluation examined compassion fatigue facilitators' perceptions of the effects of a compassion fatigue resiliency training program in an urban medical center in the midwestern United States. Nine months after completing a compassion fatigue resiliency facilitator training program, 15 participants wrote short narratives describing how the program affected them. Participants described how the training program benefited them both personally and professionally. Two main themes were identified from the narrative analysis: self-improvement and application of resiliency. All of the participants described one or more self-improvements as a result of the program, particularly in regard to emotional health. All of the participants also described how they regularly applied one or more of the resiliency skills taught in the class to improve their ability to manage stress and prevent compassion fatigue. This program shows promise in ameliorating compassion fatigue and burnout in health care providers.


Structural and Optical Properties of Sputtered Cadmium Telluride Thin Films Deposited on Flexible Substrates for Photovoltaic Applications.
Cadmium telluride (CdTe) is a photovoltaic technology based on the use of thin films of CdTe to absorb and convert sunlight into electricity. In this paper, polycrystalline CdTe thin films were deposited using radio frequency magnetron sputtering onto flexible substrates including polyimide and molybdenum foil. The structural and optical properties of the films grown at various sputtering pressures were investigated using X-ray diffraction (XRD), field-emission scanning electron microscope (FE-SEM), and UV/Nis/NIR spectrophotometry. The sputtering pressure was found to have significant effects on the structural properties, including crystallinity, preferential orientation, and microstructure. Deterioration of the optical properties of CdTe thin films were observed at high sputtering pressure.