MMTV superantigens coerce an unconventional topology between the TCR and MHC class II.
Mouse mammary tumor virus superantigens (vSAGs) are notorious for defying structural characterization, and a consensus has yet to be reached regarding their ability to bridge the TCR to MHC class II (MHCII). In this study, we determined the topology of the T cell signaling complex by examining the respective relation of vSAG7 with the MHCII molecule, MHCII-associated peptide, and TCR. We used covalently linked peptide/MHCII complexes to demonstrate that vSAG presentation is tolerant to variation in the protruding side chains of the peptide, but can be sensitive to the nature of the protruding N-terminal extension. An original approach in which vSAG was covalently linked to either MHCII chain confirmed that vSAG binds outside the peptide binding groove. Also, whereas the C-terminal vSAG segment binds to the MHCII α-chain in a conformation-sensitive manner, the membrane-proximal N-terminal domain binds the β-chain. Because both moieties of the mature vSAG remain noncovalently associated after processing, our results suggest that vSAG crosslinks MHCII molecules. Comparing different T cell hybridomas, we identified key residues on the MHCII α-chain that are differentially recognized by the CDR3β when engaged by vSAG. Finally, we show that the highly conserved tyrosine residue found in the vSAg TGXY motif is required for T cell activation. Our results reveal a novel SAG/MHCII/TCR architecture in which vSAGs coerce a near-canonical docking between MHCII and TCR that allows eschewing of traditional CDR3 binding with the associated peptide in favor of MHCII α-chain binding. Our findings highlight the plasticity of the TCR CDRs.


Seroprevalence of human granulocytic ehrlichiosis in high-risk groups in Denmark.
The aim of this study was to evaluate the seroprevalence of human granulocytic ehrlichiosis (HGE) among 300 residents in the county of Funen, Denmark. All of these people had either suspected or confirmed borreliosis. Two hundred control sera were included in the study. Samples were submitted by general practitioners and by hospital departments. An indirect immunofluorescence assay was used to identify sera reactive to HGE and an enzyme-linked immunosorbent assay was used to analyse Borrelia burgdorferi antibodies. There were 63 (21%) HGE-positive sera, 53 of which came from Borrelia-seropositive patients. Among patients with negative Borrelia serology, but with clinical suspicion of borreliosis, 14.3% were HGE-positive (n = 70). Of the 200 control sera, 3.5% were HGE-positive and 10.5% were Borrelia-positive. No HGE-positive samples were found among subjects < 20 y of age, wheras 20.4% of Borrelia seropositive samples where from subjects < 20 y of age. No mortality was observed in the HGE-positive group and the percentage of serum samples positive for both Borrelia and HGE did not differ significantly between hospitalized and non-hospitalized patients. Our study indicates that HGE infection with or without concomitant or previous Borrelia burgdorferi infection is common in tick-exposed individuals > 20 y old in the county of Funen, Denmark.


Standardized Outcomes in Nephrology-Peritoneal Dialysis (SONG-PD): Study Protocol for Establishing a Core Outcome Set in PD.
Worldwide, approximately 11% of patients on dialysis receive peritoneal dialysis (PD). Whilst PD may offer more autonomy to patients compared with hemodialysis, patient and caregiver burnout, technique failure, and peritonitis remain major challenges to the success of PD. Improvements in care and outcomes are likely to be mediated by randomized trials of innovative therapies, but will be limited if the outcomes measured and reported are not important for patients and clinicians. The aim of the Standardised Outcomes in Nephrology-Peritoneal Dialysis (SONG-PD) study is to establish a set of core outcomes for trials in patients on PD based on the shared priorities of all stakeholders, so that outcomes of most relevance for decision-making can be evaluated, and that interventions can be compared reliably. The 5 phases in the SONG-PD project are: a systematic review to identify outcomes and outcome measures that have been reported in randomized trials involving patients on PD; focus groups using nominal group technique with patients and caregivers to identify, rank, and describe reasons for their choice of outcomes; semi-structured key informant interviews with health professionals; a 3-round international Delphi survey involving a multi-stakeholder panel; and a consensus workshop to review and endorse the proposed set of core outcome domains for PD trials. The establishment of 3 to 5 high-priority core outcomes, to be measured and reported consistently in all trials in PD, will enable patients and clinicians to make informed decisions about the relative effectiveness of interventions, based upon outcomes of common importance.


A case of Varicella zoster virus polyneuropathy: involvement of the glossopharyngeal and vagus nerves mimicking a tumor.
A 36-year-old woman presented with glossopharyngeal and vagus nerve palsy, which proved to be herpes zoster based on the high titers of Varicella zoster virus antibody in her serum. Thin-section T1-weighted images with contrast media demonstrated swelling and distinct contrast enhancement of the glossopharyngeal and vagus complex, mimicking a tumor. Following MR imaging, the size of the nerve complex returned to normal; however, the contrast enhancement remained longer than the symptoms.


A psychopharmacological treatment algorithm for generalised anxiety disorder (GAD).
Generalised anxiety disorder (GAD) is defined as excessive and uncontrollable worry and anxiety about everyday life situations. It is a chronic disorder, and is associated with substantial somatisation, high rates of comorbid depression and other anxiety disorders, and significant disability. The evidence base for pharmacotherapy and psychotherapy has continued to grow, and a wide range of drug choices for GAD now exists. Current guidelines for GAD generally restrict themselves to presentation of the evidence for various treatments, which, as a result, generally do not offer detailed discussion or recommendation of strategies beyond the first level of treatment, or take into account the individual circumstances of the patient. Thus, there is a lack of algorithm-based treatment guidelines for GAD. Our aim is, therefore, to present an algorithm for the psychopharmacologic management of GAD, intended for all clinicians who treat patients with GAD, where issues of pharmacotherapy are under consideration. We also hope that these GAD algorithms and other guidelines can help to identify high-priority areas that need further study. In this algorithm, we provide a sequenced approach to the pharmacotherapy of GAD, taking into account salient symptomatology and comorbidity, levels of evidence and extent of response. Special issues, including comorbidity, insomnia, suicidality, substance abuse, treatment adherence, pregnancy and lactation, cross-cultural issues, use of medication in the elderly, psychosocial treatment and dosing issues are also addressed.


20-State Molecular Switch in a Li@C60 Complex.
A substantial potential advantage of industrial electric and thermoelectric devices utilizing endohedral metallofullerenes (EMFs) is their ability to accommodate metallic moieties inside their empty cavities. Experimental and theoretical studies have elucidated the merit of this extraordinary feature with respect to developing electrical conductance and thermopower. Published research studies have demonstrated multiple state molecular switches initiated with 4, 6, and 14 distinguished switching states. Through comprehensive theoretical investigations involving electronic structure and electric transport, we report 20 molecular switching states that can be statistically recognized employing the endohedral fullerene Li@C60 complex. We propose a switching technique that counts on the location of the alkali metal that encapsulates inside a fullerene cage. The 20 switching states correspond to the 20 hexagonal rings that the Li cation energetically prefers to reside close to. We demonstrate that the multiswitching feature of such molecular complexes can be controlled by taking advantage of the off-center displacement and charge transfer from the alkali metal to the C60 cage. The most energetically favorable optimization suggests 1.2-1.4 Å off-center displacement, and Mulliken, Hirshfeld, and Voronoi simulations articulate that the charge migrates from the Li cation to C60 fullerene; however, the amount of the charge transferred depends on the nature and location of the cation within the complex. We believe that the proposed work suggests a relevant step toward the practical application of molecular switches in organic materials.


A round Bouba is easier to remember than a curved Kiki: Sound-symbolism can support associative memory.
Past research has shown that prior knowledge can support our episodic memory for recently encountered associations. According to the model proposed by Cox and Criss (Proceedings of the 40th Annual Conference of the Cognitive Science Society, pp. 250-255, Madison, MI: Cognitive Science Society, 2018) and Cox and Shiffrin (Cognitive Psychology, 97, 31-61, 2017), any features shared by associated items should facilitate encoding and retrieval. We implemented a strict test of this prediction by taking advantage of sound-symbolism associations; here, the latter refer to relationships between phonemes and object characteristics - relationships that participants readily find natural - even if they have never encountered the items before. For instance, the non-word "maluma" is much more readily seen to refer to a random shape with rounded contours than to a shape that has sharp angles. In our study, 70 participants completed paired-associate memory tests after studying lists of three pairs, each composed of a random shape and a non-word. As predicted, there was better associative memory performance for sound-shape pairs that could rely on sound-symbolism links.


The lymphocyte activity of adenosine deaminase and enzymes of AMP metabolism in mammary carcinogenesis: the effect of thymostimulin.
Studies were undertaken on the activity of adenosine deaminase (ADA) and metabolic enzymes of AMP in the spleen and thymus cells of rats with DMBA-induced carcinogenesis of the mammary glands and on the effect of thymostimulin (TS) on this activity. In addition, we investigated the activity of ADA in the peripheral blood lymphocytes of the patients with premalignant diseases and cancer of the mammary glands (Stages I-IV) as well as the possibility of its enhancement by TS. The disturbances of these enzyme activities characterized by a decrease in ADA activity and an increase in activity of the AMP metabolism enzymes, predisposed for adenosine accumulation in lymphoid cells. Injection of TS normalized the ADA activity and decreased the activity of the AMP metabolic enzymes. Analogous results were obtained in the studies of ADA activity in the human materials. With the human tumor growth the in vitro effect of TS on the lymphocyte ADA activity decreased. We may suggest that measurement of the ADA activity in the lymphocytes may serve as a reliable tool to control the state of immune system and the effectiveness of immunotherapy with the thymic humoral factors.


A systematic review of interactive multimedia interventions to promote children's communication with health professionals: implications for communicating with overweight children.
Interactive multimedia is an emerging technology that is being used to facilitate interactions between patients and health professionals. The purpose of this review was to identify and evaluate the impact of multimedia interventions (MIs), delivered in the context of paediatric healthcare, in order to inform the development of a MI to promote the communication of dietetic messages with overweight preadolescent children. Of particular interest were the effects of these MIs on child engagement and participation in treatment, and the subsequent effect on health-related treatment outcomes. An extensive search of 12 bibliographic databases was conducted in April 2012. Studies were included if: one or more child-participant was 7 to 11-years-of-age; a MI was used to improve health-related behaviour; child-participants were diagnosed with a health condition and were receiving treatment for that condition at the time of the study. Data describing study characteristics and intervention effects on communication, satisfaction, knowledge acquisition, changes in self-efficacy, healthcare utilisation, and health outcomes were extracted and summarised using qualitative and quantitative methods. A total of 14 controlled trials, published between 1997 and 2006 met the selection criteria. Several MIs had the capacity to facilitate engagement between the child and a clinician, but only one sought to utilise the MI to improve communication between the child and health professional. In spite of concerns over the quality of some studies and small study populations, MIs were found useful in educating children about their health, and they demonstrated potential to improve children's health-related self-efficacy, which could make them more able partners in face-to-face communications with health professionals. The findings of this review suggest that MIs have the capacity to support preadolescent child-clinician communication, but further research in this field is needed. Particular attention should be given to designing appropriate MIs that are clinically relevant.


A photo-induced electron transfer study of an organic dye anchored on the surfaces of TiO2 nanotubes and nanoparticles.
We report on femtosecond-nanosecond (fs-ns) studies of the triphenylamine organic dye (TPC1) interacting with titania nanoparticles of different sizes, nanotubes and nanorods. We used time-resolved emission and absorption spectroscopy to measure the photoinduced dynamics of forward and back electron transfer processes taking place in TPC1-titania complexes in acetonitrile (ACN) and dichloromethane (DCM) solutions. We observed that the electron injection from the dye to titania occurs in a multi-exponential way with the main contribution of 100 fs from the hot excited charge-transfer state of anchored TPC1. This process competes with the relaxation of the excited state, mainly governed by solvation, that takes place with average time constants of 400 fs in ACN and 1.3 ps in DCM solutions. A minor contribution to the electron injection process takes place with longer time constants of about 1-10 ps from the relaxed excited state of TPC1. The latter times and their contribution do not depend on the size of the nanoparticles, but are substantially smaller in the case of nanotubes (1-3 ps), probably due to the caging effect. The contribution is also smaller in DCM than in ACN. The efficient back recombination takes place also in a multi-exponential way with times of 1 ps, 15 ps and 1 ns, and only 20-30% of the initial injected electrons in the conduction band are left within the first 1 ns after excitation. The faster recombination rates are suggested due to those originating from the free electrons in the conduction band of titania or the electrons in the shallow trap states, while the slower recombination is due to the electrons in the deep trap states. The results reported here should be relevant to a better understanding of the photobehaviour of an organic dye with promising potential for use in solar cells. They should also help to determine the important factors that limit the efficiency of solar cells based on the triphenylamine-based dyes for solar energy conversion.


Cloning and expression of IDDM-specific human autoantigens.
A DNA cloning approach was taken to identify islet cell protein antigens that are recognized specifically by insulin-dependent diabetes mellitus (IDDM) sera. A human islet cDNA library was generated and screened with diabetic sera. In this article, identification of two clones is described. Proteins expressed by these lambda phages appeared to react specifically with newly diagnosed diabetic sera. Islet cell antibody 12 (ICA12) was tested by Western blotting. ICA512 was not reactive with sera in the Western format but was specifically immunoprecipitated by diabetic sera from an Escherichia coli extract.


Changes in Lower Gastrointestinal Bleeding Presentation, Management, and Outcomes Over a 10-Year Span.
There are only limited data available on changes in the etiology, management, and clinical outcomes in patients with lower gastrointestinal bleeding over the past decade. We compared 2 groups of consecutive patients hospitalized with lower gastrointestinal bleeding during 2 time periods: 2005 to 2007 (301 patients) and 2015 to 2017 (249 patients). Compared with the 2005 to 2007 group, the mean Charlson comorbidity index in the 2015 to 2017 group was higher (5.0±2.6 vs. 6.0±3.0, P=0.028), whereas the use of computerized tomographic angiography and small bowel capsule endoscopy was more common (12.9% vs. 58.1%, P<0.001, and 28.8% vs. 69.0%, P=0.031, respectively). In 2005 to 2007, ischemic colitis (12.0%) was the most common confirmed etiology of bleeding and diverticular bleeding the second most common (8.6%), whereas in 2015 to 2017, diverticular bleeding was the most common etiology (10.4%), followed by angiodysplasia (8.4%). Small bowel bleeding sources were confirmed more often in the 2015 to 2017 group (P=0.017). Endoscopic treatment was attempted in 16.6% of patients in 2005 to 2007 versus 25.3% in 2015 to 2017 (P=0.015). Higher rebleeding rates, longer hospitalization durations (4.6±4.3 vs. 5.5±3.4 d, P=0.019), and a higher proportion of patients needing a transfusion (62.0% vs. 78.4%, P=0.016) were noted in 2015 to 2017. Over a 10-year span, there were several notable changes: (1) more comorbidities in patients hospitalized for lower gastrointestinal bleeding; (2) marked increase in the use of computerized tomographic angiography and capsule endoscopy for diagnostic evaluation; and (3) longer hospitalization durations and greater need for blood transfusion, possibly reflecting the selection of sicker patients for in-patient management in 2015 to 2017.


Inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopenicillanic acid: kinetics.
The kinetics of the inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopenicillanic acid are described. Loss of beta-lactamase activity is accompanied by a decrease in protein fluorescence, by the appearance of a protein-bound chromophore at 326 nm, and by loss of tritium from 6 alpha-[3H]-6 beta-bromopenicillanic acid. It is shown that all of the above changes probably have the same rate-determining step. The inactivation reaction is competitively inhibited by cephalosporin C, a competitive inhibitor of this enzyme, and by covalently bound clavulanic acid, suggesting that 6 beta-bromopenicillanic acid reacts directly with the beta-lactamase active site. It is proposed that this inhibitor reacts initially as a normal substrate and that the rate-determining step of the inactivation is acylation of the enzyme. A rapid irreversible inactivation reaction rather than normal hydrolysis of the acyl-enzyme then follows acylation; 6 beta-bromopenicillanic acid is thus a suicide substrate.


Privacy-Preserving Patient-Centric Clinical Decision Support System on Naïve Bayesian Classification.
Clinical decision support system, which uses advanced data mining techniques to help clinician make proper decisions, has received considerable attention recently. The advantages of clinical decision support system include not only improving diagnosis accuracy but also reducing diagnosis time. Specifically, with large amounts of clinical data generated everyday, naïve Bayesian classification can be utilized to excavate valuable information to improve a clinical decision support system. Although the clinical decision support system is quite promising, the flourish of the system still faces many challenges including information security and privacy concerns. In this paper, we propose a new privacy-preserving patient-centric clinical decision support system, which helps clinician complementary to diagnose the risk of patients' disease in a privacy-preserving way. In the proposed system, the past patients' historical data are stored in cloud and can be used to train the naïve Bayesian classifier without leaking any individual patient medical data, and then the trained classifier can be applied to compute the disease risk for new coming patients and also allow these patients to retrieve the top- k disease names according to their own preferences. Specifically, to protect the privacy of past patients' historical data, a new cryptographic tool called additive homomorphic proxy aggregation scheme is designed. Moreover, to leverage the leakage of naïve Bayesian classifier, we introduce a privacy-preserving top- k disease names retrieval protocol in our system. Detailed privacy analysis ensures that patient's information is private and will not be leaked out during the disease diagnosis phase. In addition, performance evaluation via extensive simulations also demonstrates that our system can efficiently calculate patient's disease risk with high accuracy in a privacy-preserving way.


Treatment of blastomycosis with itraconazole in 112 dogs.
One hundred twelve client-owned dogs with blastomycosis were treated with itraconazole, 5 or 10 mg/kg/d. The first group of 70 dogs treated in 1987 and 1988 received 10 mg/kg/d (group 1), and the second group of 42 dogs treated after October 1988 received 5 mg/kg/d (group 2). Even though the groups were treated at different times, the dogs were similar in age and gender distribution, number of sites involved, and percent and severity of pulmonary involvement. The proportion of dogs cured with a 60-day course of itraconazole was similar for both groups (53.6% versus 54.3%) and for a second historical control group treated with amphotericin B (57%); the recurrence rate was also similar, 20%, 21.4%, and 20%, respectively. Dogs treated with itraconazole had similar mortality rates (25.7% at 5 mg/kg/d; 25% at 10 mg/kg/day) to those treated with amphotericin B (23%). Seventeen of the 23 dogs that died (74%), did so during the first week of treatment; these early deaths were usually attributed to respiratory failure. The only site of infection that was significantly associated with failure (death or recurrence) was the brain. There was a marked difference in survival times between dogs without lung disease or with mild lung disease compared with dogs with moderate or severe lung disease. Serum itraconazole concentrations reached steady state by 14 days of treatment. Dogs receiving 5 mg/kg/d of itraconazole (group 2) had mean serum concentrations of 3.55 +/- 2.81 mg/mL (range, 0.67 to 10.8 micrograms/mL), whereas dogs receiving 10 micrograms/kg/d (group 1) had mean concentrations of 13.46 +/- 8.49 micrograms/mL (range, 1.8 to 28 micrograms/mL) (P < or = .001). There was no association between cure and serum itraconazole concentrations. Dogs in group 1 had significantly more adverse effects than dogs in group 2 (P = .046). Anorexia was the most common adverse effect, occurring in 14.9% of dogs in group 1. Only 8% of dogs in group 2 had adverse effects. Serum concentrations of itraconazole were positively correlated with serum alkaline phosphatase and alanine aminotransferase activities. Our findings indicate that itraconazole administered at a dose of 5 mg/kg/d is the drug of choice for blastomycosis in dogs.


High efficiency of energy flux controls within mitochondrial interactosome in cardiac intracellular energetic units.
The aim of our study was to analyze a distribution of metabolic flux controls of all mitochondrial complexes of ATP-Synthasome and mitochondrial creatine kinase (MtCK) in situ in permeabilized cardiac cells. For this we used their specific inhibitors to measure flux control coefficients (C(vi)(JATP)) in two different systems: A) direct stimulation of respiration by ADP and B) activation of respiration by coupled MtCK reaction in the presence of MgATP and creatine. In isolated mitochondria the C(vi)(JATP) were for system A: Complex I - 0.19, Complex III - 0.06, Complex IV 0.18, adenine nucleotide translocase (ANT) - 0.11, ATP synthase - 0.01, Pi carrier - 0.20, and the sum of C(vi)(JATP) was 0.75. In the presence of 10mM creatine (system B) the C(vi)(JATP) were 0.38 for ANT and 0.80 for MtCK. In the permeabilized cardiomyocytes inhibitors had to be added in much higher final concentration, and the following values of C(vi)(JATP) were determined for condition A and B, respectively: Complex I - 0.20 and 0.64, Complex III - 0.41 and 0.40, Complex IV - 0.40 and 0.49, ANT - 0.20 and 0.92, ATP synthase - 0.065 and 0.38, Pi carrier - 0.06 and 0.06, MtCK 0.95. The sum of C(vi)(JATP) was 1.33 and 3.84, respectively. Thus, C(vi)(JATP) were specifically increased under conditions B only for steps involved in ADP turnover and for Complex I in permeabilized cardiomyocytes within Mitochondrial Interactosome, a supercomplex consisting of MtCK, ATP-Synthasome, voltage dependent anion channel associated with tubulin βII which restricts permeability of the mitochondrial outer membrane.


The open reading frame of bamboo mosaic potexvirus satellite RNA is not essential for its replication and can be replaced with a bacterial gene.
A satellite RNA of 836 nt depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsulation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein. A full-length infectious cDNA clone has been generated downstream of the T7 RNA polymerase promoter. To investigate the role of the 20-kDa protein encoded by satBaMV, satBaMV transcripts containing mutations in the open reading frame were tested for their ability to replicate in barley protoplasts and in Chenopodium quinoa using BaMV RNA as a helper genome. Unlike other large satellite RNAs, mutants in the open reading frame did not block their replication, suggesting that the 20-kDa protein is not essential for satBaMV replication. Precise replacement of the open reading frame with sequences encoding chloramphenicol acetyltransferase resulted in high level expression of chloramphenicol acetyltransferase in infected C. quinoa, indicating that satBaMV is potentially useful as a satellite-based expression vector.


Pharmacotherapeutic options for the treatment of preeclampsia.
Pharmacotherapeutic options for the treatment of preeclampsia are reviewed. Risk factors for the development of preeclampsia include microvascular diseases, such as diabetes mellitus; vascular and connective tissue disorders; hypertension; antiphospholipid antibody syndrome; and nephropathy. Several pathophysiological factors contribute to the development of the preeclamptic state, including vasospasm onset, coagulation system activation, increased inflammatory response, and ischemia. The specific agents used for the treatment of preeclampsia are dependent on a number of factors including symptom severity, maternal or fetal compromise, the progression to eclampsia, gestational period, and cervical status. The diagnosis of preeclampsia beyond the gestation period of 38 weeks requires delivery. The presence of maternal compromise or eclampsia at gestation greater than 20 weeks also necessitates delivery. In cases of chronic or mild hypertension, oral methyldopa may be administered on an outpatient basis. Intravenous hydralazine is a commonly administered arteriolar vasodilator that is effective for hypertensive emergencies associated with pregnancies. The most common adverse effect of hydralazine administration is unpredictable hypotension. Labetalol decreases heart rate and may be preferred because of a lack of reflex tachycardia, hypotension, or increased intracranial pressure. However, the drug of choice for the prevention and control of maternal seizures in patients with severe preeclampsia or eclampsia during the peripartum period is i.v. magnesium sulfate. Therapeutic serum magnesium levels cause cerebral vasodilation, thereby reversing the ischemia produced by cerebral vasospasm during an eclamptic episode. The results of one study indicated that women receiving magnesium sulfate therapy had a 58% lower risk of eclampsia than placebo. Magnesium sulfate remains the drug of choice for the prevention and treatment of preeclampsia. Alternative antihypertensive agents may provide additional benefit in the management of hypertension for preeclamptic patients.


Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale.
Adeno-associated viral (AAV) manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. Although scalable systems based on AAV-adenovirus, AAV-herpesvirus, and AAV-baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate-scale preclinical studies in large animals, in which several combinations of serotype and genome may need to be tested. We observed that during production of many AAV serotypes, large amounts of vector are found in the culture supernatant, a relatively pure source of vector in comparison with cell-derived material. Here we describe a high-yielding, recombinant AAV production process based on polyethylenimine (PEI)-mediated transfection of HEK293 cells and iodixanol gradient centrifugation of concentrated culture supernatant. The entire process can be completed in 1 week and the steps involved are universal for a number of different AAV serotypes. Process conditions have been optimized such that final purified yields are routinely greater than 1 x 10(14) genome copies per run, with capsid protein purity exceeding 90%. Initial experiments with vectors produced by the new process demonstrate equivalent or better transduction both in vitro and in vivo when compared with small-scale, CsCl gradient-purified vectors. In addition, the iodixanol gradient purification process described effectively separates infectious particles from empty capsids, a desirable property for reducing toxicity and unwanted immune responses during preclinical studies.


Forest composition change and biophysical climate feedbacks across boreal North America.
Deciduous tree cover is expected to increase in North American boreal forests with climate warming and wildfire. This shift in composition has the potential to generate biophysical cooling via increased land surface albedo. Here we use Landsat-derived maps of continuous tree canopy cover and deciduous fractional composition to assess albedo change over recent decades. We find, on average, a small net decrease in deciduous fraction from 2000 to 2015 across boreal North America and from 1992 to 2015 across Canada, despite extensive fire disturbance that locally increased deciduous vegetation. We further find near-neutral net biophysical change in radiative forcing associated with albedo when aggregated across the domain. Thus, while there have been widespread changes in forest composition over the past several decades, the net changes in composition and associated post-fire radiative forcing have not induced systematic negative feedbacks to climate warming over the spatial and temporal scope of our study.


Large-scale carbon stock assessment of woody vegetation in tropical dry deciduous forest of Sathanur reserve forest, Eastern Ghats, India.
Tropical dry forests are one of the most widely distributed ecosystems in tropics, which remain neglected in research, especially in the Eastern Ghats. Therefore, the present study was aimed to quantify the carbon storage in woody vegetation (trees and lianas) on large scale (30, 1 ha plots) in the dry deciduous forest of Sathanur reserve forest of Eastern Ghats. Biomass of adult (≥10 cm DBH) trees was estimated by species-specific allometric equations using diameter and wood density of species whereas in juvenile tree population and lianas, their respective general allometric equations were used to estimate the biomass. The fractional value 0.4453 was used to convert dry biomass into carbon in woody vegetation of tropical dry forest. The mean aboveground biomass value of juvenile tree population was 1.86 Mg/ha. The aboveground biomass of adult trees ranged from 64.81 to 624.96 Mg/ha with a mean of 245.90 Mg/ha. The mean aboveground biomass value of lianas was 7.98 Mg/ha. The total biomass of woody vegetation (adult trees + juvenile population of trees + lianas) ranged from 85.02 to 723.46 Mg/ha, with a mean value of 295.04 Mg/ha. Total carbon accumulated in woody vegetation in tropical dry deciduous forest ranged from 37.86 to 322.16 Mg/ha with a mean value of 131.38 Mg/ha. Adult trees accumulated 94.81% of woody biomass carbon followed by lianas (3.99%) and juvenile population of trees (1.20%). Albizia amara has the greatest biomass and carbon stock (58.31%) among trees except for two plots (24 and 25) where Chloroxylon swietenia contributed more to biomass and carbon stock. Similarly, Albizia amara (52.4%) showed greater carbon storage in juvenile population of trees followed by Chloroxylon swietenia (21.9%). Pterolobium hexapetalum (38.86%) showed a greater accumulation of carbon in liana species followed by Combretum albidum (33.04%). Even though, all the study plots are located within 10 km radius, they show a significant spatial variation among them in terms of biomass and carbon stocks which could be attributed to variation in anthropogenic pressures among the plots as well as to changes in tree density across landscapes. Total basal area of woody vegetation showed a significant positive (R 2 = 0.978; P = 0.000) relationship with carbon storage while juvenile tree basal area showed the negative relationship (R 2 = 0.4804; P = 0.000) with woody carbon storage. The present study generates a large-scale baseline data of dry deciduous forest carbon stock, which would facilitate carbon stock assessment at a national level as well as to understand its contribution on a global scale.


The radiation field characteristics of piezoelectric polymer membrane transducers when operating into air.
This paper describes the evaluation of the radiation field characteristics of PVDF membrane transducers, whereby the active area is defined by an appropriate electrode pattern when operating into air. Measurement, including conventional scanning of the fields and laser vibrometry, is supplemented by finite element modeling over the frequency range 500 kHz to 1.1 MHz. Contrary to expectation, the surface displacements are nonuniform, giving rise to premature axial focusing of the field. This is shown to be caused by generation of the zero order symmetrical Lamb wave, arising from the electric field discontinuity at the electrode boundary of the transducer.


Role of prostatic fluid in cooled canine epididymal sperm.
In this study, sperms collected from the right and left cauda epididymis were grouped into having canine prostatic fluid (PF) sensitization or not diluted with egg yolk Tris-fructose citrate extender, and stored at 4°C. The necessity of canine PF in cooled preservation was determined by elucidating the sperm quality after the storage. As a result, while there was no difference among all groups up to 48 hr of storage, after storage for 96 hr and more, a significantly lower sperm motility was observed in the group without being sensitized to PF than the groups with being sensitized to PF (p < .05, p < .01). Although sperm abnormality increased in all groups with increased storage time, the group without being sensitized to PF showed significantly higher sperm abnormality than did the groups with being sensitized to PF after storage for 24 hr and more (p < .01). From these findings, we concluded that PF was necessary for the cooled preservation of the canine sperm because these sperms were protected from any effects of low temperatures by being sensitized to PF.


Protective immunity against avian influenza induced by a fowlpox virus recombinant.
Fowlpox virus, the prototypic virus of the genus Avipoxvirus has a natural host range limited to avian species. As such, fowlpox virus provides a suitable candidate for the development of a species-specific recombinant viral vector. This paper reports the development of a fowlpox virus recombinant expressing the haemagglutinin molecule from a highly virulent avian influenza virus. On immunization of chickens and turkeys with the recombinant, protection is afforded against a lethal challenge with either the homologous or a heterologous influenza virus strain.


Comparison of Drugs Used for Adjuvant and Metastatic Therapy of Colon, Breast, and Non-Small Cell Lung Cancers.
Drugs are used in the adjuvant, metastatic, or both settings in cancer, but the rate, direction, and speed with which drugs are tested and indicated in each setting are unknown. To identify the number of unique agents that are currently category 1 or 2A per National Comprehensive Cancer Network (NCCN) guidelines in metastatic and adjuvant settings of non-small cell lung cancer (NSCLC), breast cancer, and colon cancer, as well as the mean delay between use in these 2 settings and the quality of supporting evidence. This cross-sectional study used NCCN treatment guidelines current as of May 15, 2019, and the clinical trials cited either by these guidelines or within corresponding drug labels. Trials published between 1970 and 2019 were evaluated. The analysis included published clinical trials of systemic therapy options deemed by the NCCN as category 1 or 2A. Participants included patients with early or metastatic NSCLC, breast cancer, or colon cancer who were included in clinical trials evaluating current NCCN-recommended systemic therapy options. Data analysis was performed from March 2019 to May 2019. Systemic therapy regimens used as either adjuvant treatment or as therapy for metastatic disease in the 3 cancer types. Number of agents recommended for use in adjuvant and metastatic settings of NSCLC, colon cancer, and breast cancer, the mean delay between use in these 2 settings, and the percentage of agents supported by trials with substantial improvement in either progression-free survival (disease-free survival for adjuvant agents) or overall survival. This study identified 69 agents recommended for use in metastatic disease compared with 25 agents recommended for adjuvant use. For agents used in both settings, the mean (SD) delay between use in metastatic disease and as adjuvant therapy was 10.0 (7.5) years. On the basis of trials with positive outcomes, 39 of 69 agents (56.5%) were approved or recommended in the metastatic setting, compared with 23 of 25 agents (92.0%) approved for use as adjuvant therapy. There is a substantial difference in the number of agents available for use, as well as the timing of supporting evidence, in the metastatic and adjuvant settings for NSCLC, breast cancer, and colon cancer. Given the potential benefit of adjuvant therapy in these cancer types, further investigation into additional adjuvant systemic therapy options is warranted.


Prostate-Specific Membrane Antigen Targeted Therapy of Prostate Cancer Using a DUPA-Paclitaxel Conjugate.
Prostate cancer (PCa) is the most prevalent cancer among men in the United States and remains the second-leading cause of cancer mortality in men. Paclitaxel (PTX) is the first line chemotherapy for PCa treatment, but its therapeutic efficacy is greatly restricted by the nonspecific distribution in vivo. Prostate-specific membrane antigen (PSMA) is overexpressed on the surface of most PCa cells, and its expression level increases with cancer aggressiveness, while being present at low levels in normal cells. The high expression level of PSMA in PCa cells offers an opportunity for target delivery of nonspecific cytotoxic drugs to PCa cells, thus improving therapeutic efficacy and reducing toxicity. PSMA has high affinity for DUPA, a glutamate urea ligand. Herein, a novel DUPA-PTX conjugate is developed using DUPA as the targeting ligand to deliver PTX specifically for treatment of PSMA expressing PCa. The targeting ligand DUPA enhances the transport capability and selectivity of PTX to tumor cells via PSMA mediated endocytosis. Besides, DUPA is conjugated with PTX via a disulfide bond, which facilitates the rapid and differential drug release in tumor cells. The DUPA-PTX conjugate exhibits potent cytotoxicity in PSMA expressing cell lines and induces a complete cessation of tumor growth with no obvious toxicity. Our findings give new insight into the PSMA-targeted delivery of chemotherapeutics and provide an opportunity for the development of novel active targeting drug delivery systems for PCa therapy.


Diagnostic Accuracy of Magnetic Resonance Imaging Measures of Brain Atrophy Across the Spectrum of Progressive Supranuclear Palsy and Corticobasal Degeneration.
The accurate diagnosis of progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) is hampered by imperfect clinical-pathological correlations. To assess and compare the diagnostic value of the magnetic resonance parkinsonism index (MRPI) and other magnetic resonance imaging-based measures of cerebral atrophy to differentiate between PSP, CBD, and other neurodegenerative diseases. This prospective diagnostic study included participants with 4-repeat tauopathies (4RT), PSP, CBD, other neurodegenerative diseases and available MRI who appeared in the University of California, San Francisco, Memory and Aging Center database. Data were collected from October 27, 1994, to September 29, 2019. Data were analyzed from March 1 to September 14, 2021. The main outcome of this study was the neuropathological diagnosis of PSP or CBD. The clinical diagnosis at the time of the MRI acquisition was noted. The imaging measures included the MRPI, cortical thickness, subcortical volumes, including the midbrain, pons, and superior cerebellar peduncle volumes. Multinomial logistic regression models (MLRM) combining different cortical and subcortical regions were defined to discriminate between PSP, CBD, and other pathologies. The areas under the receiver operating characteristic curves (AUROC) and cutoffs were calculated to differentiate between PSP, CBD, and other diseases. Of the 326 included participants, 176 (54%) were male, and the mean (SD) age at MRI was 64.1 (8.0) years. The MRPI showed good diagnostic accuracy for the differentiation between PSP and all other pathologies (accuracy, 87%; AUROC, 0.90; 95% CI, 0.86-0.95) and between 4RT and other pathologies (accuracy, 80%; AUROC, 0.82; 95% CI, 0.76-0.87), but did not allow the discrimination of participants with CBD. Its diagnostic accuracy was lower in the subgroup of patients without the canonical PSP-Richardson syndrome (PSP-RS) or probable corticobasal syndrome (CBS) at MRI. MLRM combining cortical and subcortical measurements showed the highest accuracy for the differentiation between PSP and other pathologies (accuracy, 95%; AUROC, 0.98; 95% CI, 0.97-0.99), CBD and other pathologies (accuracy, 83%; AUROC, 0.86; 95% CI, 0.81-0.91), 4RT and other pathologies (accuracy, 89%; AUROC, 0.94; 95% CI, 0.92-0.97), and PSP and CBD (accuracy, 91%; AUROC, 0.95; 95% CI, 0.91-0.99), even in participants without PSP-RS or CBS at MRI. In this study, the combination of widely available cortical and subcortical measures of atrophy on MRI discriminated between PSP, CBD, and other pathologies and could be used to support the diagnosis of 4RT in clinical practice.


Drug Delivery Strategies and Nanozyme Technologies to Overcome Limitations for Targeting Oxidative Stress in Osteoarthritis.
Oxidative stress is an important, but elusive, therapeutic target for osteoarthritis (OA). Antioxidant strategies that target oxidative stress through the elimination of reactive oxygen species (ROS) have been widely evaluated for OA but are limited by the physiological characteristics of the joint. Current hallmarks in antioxidant treatment strategies include poor bioavailability, poor stability, and poor retention in the joint. For example, oral intake of exogenous antioxidants has limited access to the joint space, and intra-articular injections require frequent dosing to provide therapeutic effects. Advancements in ROS-scavenging nanomaterials, also known as nanozymes, leverage bioactive material properties to improve delivery and retention. Material properties of nanozymes can be tuned to overcome physiological barriers in the knee. However, the clinical application of these nanozymes is still limited, and studies to understand their utility in treating OA are still in their infancy. The objective of this review is to evaluate current antioxidant treatment strategies and the development of nanozymes as a potential alternative to conventional small molecules and enzymes.


Cost and Cost Driver Analysis of Anterior Cruciate Ligament Reconstruction Using Time-Driven Activity-Based Costing: Bone-Tendon-Bone Autograft Versus Hamstring Autograft.
As health care transitions toward value-based care, orthopaedics has started to implement time-driven activity-based costing (TDABC) to understand costs and cost drivers. TDABC has not previously been used to study cost drivers in anterior cruciate ligament reconstruction (ACLR). The purpose of this study was to use TDABC to (1) calculate bone-tendon-bone (BTB) and hamstring ACLR total costs of care and (2) evaluate the impact of graft choice and other factors on ACLR costs. Data were collected from electronic medical records for primary ACLR from the institutional patient-reported outcome registry between 2009 and 2016 in 1 ambulatory surgery center. Patients receiving allograft, revision ACLR, or concomitant meniscal repair or ligament reconstruction were excluded. The total cost of care was determined using TDABC. Multivariate regression analysis was conducted between ACLR cost and group characteristics. A total of 328 patients were included; 211 (64.3%) received BTB autograft and 117 (35.7%) received hamstring autograft. The mean cost was $2,865.01 ± $263.45 (95% confidence interval: $2,829.26, $2,900.77) for BTB ACLR versus $3,377.44 ± $320.12 ($3,318.82, $3,436.05) for hamstring ACLR (p < 0.001). Operative time was 103.1 ± 25.1 (99.7, 106.5) minutes for BTB ACLR versus 113.1 ± 27.9 (108.0, 118.2) minutes for hamstring ACLR (p = 0.001). The total implant cost was $270.32 ± $97.08 ($257.15, $283.50) for BTB ACLR versus $587.36 ± $108.78 ($567.44, $607.28) for hamstring ACLR (p < 0.001). Hamstring graft (p = 0.006) and suspensory fixation on the femoral side (p = 0.011) were associated with increased costs. The mean cost of care and operative time for BTB autograft ACLR are less than those for hamstring autograft ACLR. Operative time, implant choice, and graft choice were identified as modifiable cost drivers that can empower surgeons to manage primary ACLR costs while maximizing the value of the procedure. Economic and Decision Analysis Level IV. See Instructions for Authors for a complete description of levels of evidence.


Level of Palliative Care Complexity in Advanced Cancer Patients: A Multinomial Logistic Analysis.
The current treatment approach for patients in palliative care (PC) requires a health model based on shared and individualised care, according to the degree of complexity encountered. The aims of this study were to describe the levels of complexity that may be present, to determine their most prevalent elements and to identify factors that may be related to palliative complexity in advanced-stage cancer patients. An observational retrospective study was performed of patients attended to at the Cudeca Hospice. Socio-demographic and clinical data were compiled, together with information on the patients' functional and performance status (according to the Palliative Performance Scale (PPS)). The level of complexity was determined by the Diagnostic Instrument of Complexity in Palliative Care (IDC-Pal©) and classified as highly complex, complex or non-complex. The impact of the independent variables on PC complexity was assessed by multinomial logistic regression analysis. Of the 501 patients studied, 44.8% presented a situation classed as highly complex and another 44% were considered complex. The highly complex items most frequently observed were the absence or insufficiency of family support and/or caregivers (24.3%) and the presence of difficult-to-control symptoms (17.3%). The complex item most frequently observed was an abrupt change in the level of functional autonomy (47.6%). The main factor related to the presence of high vs. non-complexity was that of performance status (odds ratio (OR) = 10.68, 95% confidence interval (CI) = 2.81-40.52, for PPS values < 40%). However, age was inversely related to high complexity. This study confirms the high level of complexity present in patients referred to a PC centre. Determining the factors related to this complexity could help physicians identify situations calling for timely referral for specialised PC, such as a low PPS score.


Acute MI in the elderly: choosing the best revascularization method. Risks and benefits of thrombolysis, angioplasty, and bypass surgery.
MI is often recognized less promptly in elderly patients than in younger patients; thus, the best opportunity for reperfusion is often missed. If infarction is diagnosed in less than 12 hours and there are no strong contraindications, thrombolytic therapy is appropriate for the elderly. Coronary angioplasty is a suitable alternative if performed promptly, especially because elderly patients are more likely to have contraindications to, or higher mortality from, thrombolysis. Predictors of unfavorable outcome following angioplasty for acute MI in the elderly include multivessel disease, occlusion of the infarcted artery, and cardiogenic shock. CABG surgery (performed during infarction or in the peri-infarct setting) is also an option for those elderly patients who are hemodynamically stable.


Cardiac structural changes after transcatheter aortic valve replacement: systematic review and meta-analysis of cardiovascular magnetic resonance studies.
Transcatheter aortic valve replacement (TAVR) is increasingly used to treat patients with severe aortic stenosis (AS). Cardiovascular magnetic resonance imaging (CMR) provides reliable and reproducible estimates for assessment of cardiac structure and function after TAVR. The goal of this study was to conduct a systematic review and meta-analysis of the literature to assess left ventricular (LV) volumes, mass and function by CMR after TAVR. Using Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines, we searched PubMed and Embase for studies reporting CMR findings before and at least 1 month after TAVR. Main factors of interest were LV end-diastolic volume index (LVEDVi), LV end-systolic volume index (LVESVi), LV mass index (LVMi), and left ventricular ejection fraction (LVEF). Standardized mean differences (SMD) were pooled by random effects meta-analytic techniques. Of 453 screened publications, 10 studies (published between 2012 and 2018) were included. A total of 305 patients completed pre- and post-TAVR follow-up CMR (mean age range 78.6-85.0 years, follow-up range 6-15 months). Random effects analysis showed TAVR resulted in reduced LVEDVi (SMD: -0.25, 95% CI: - 0.43 to - 0.07, P = 0.006), LVESVi (SMD: -0.24, 95% CI: - 0.44 to - 0.05, P = 0.01), LVMi (SMD: -0.82, 95% CI: - 1.0 to - 0.63, P < 0.001) and increased LVEF (SMD: 22, 95% CI: 6 to 38%, P = 0.006). Heterogeneity across studies was low (I2: 0%, Pheterogeneity > 0.05 for all). The median reduction was 4 ml/m2 (IQR: 3.1 to 8.2) for LVEDVi, 5 ml/m2 (IQR: 3.0 to 6.0) for LVESVi, and 15.1 g/m2 (IQR: 11.8 to 18.3) for LVMi. The median increase for LVEF was 3.4% (IQR 1.0 to 4.6%). CMR demonstrates reverse LV remodeling occurrs within 6-15 months after TAVR, with reductions in LVEDVi, LVESVi and LVMi, and increased LVEF.


Inheritance of female mating preference in a sympatric sibling species pair of Lake Victoria cichlids: implications for speciation.
Female mate choice has often been proposed to play an important role in cases of rapid speciation, in particular in the explosively evolved haplochromine cichlid species flocks of the Great Lakes of East Africa. Little, if anything, is known in cichlid radiations about the heritability of female mating preferences. Entirely sympatric distribution, large ecological overlap and conspicuous differences in male nuptial coloration, and female preferences for these, make the sister species Pundamilia pundamilia and P. nyererei from Lake Victoria an ideally suited species pair to test assumptions on the genetics of mating preferences made in models of sympatric speciation. Female mate choice is necessary and sufficient to maintain reproductive isolation between these species, and it is perhaps not unlikely therefore, that female mate choice has been important during speciation. A prerequisite for this, which had remained untested in African cichlid fish, is that variation in female mating preferences is heritable. We investigated mating preferences of females of these sister species and their hybrids to test this assumption of most sympatric speciation models, and to further test the assumption of some models of sympatric speciation by sexual selection that female preference is a single-gene trait. We find that the differences in female mating preferences between the sister species are heritable, possibly with quite high heritabilities, and that few but probably more than one genetic loci contribute to this behavioural speciation trait with no apparent dominance. We discuss these results in the light of speciation models and the debate about the explosive radiation of cichlid fishes in Lake Victoria.


Looking for age-related growth decline in natural forests: unexpected biomass patterns from tree rings and simulated mortality.
Forest biomass growth is almost universally assumed to peak early in stand development, near canopy closure, after which it will plateau or decline. The chronosequence and plot remeasurement approaches used to establish the decline pattern suffer from limitations and coarse temporal detail. We combined annual tree ring measurements and mortality models to address two questions: first, how do assumptions about tree growth and mortality influence reconstructions of biomass growth? Second, under what circumstances does biomass production follow the model that peaks early, then declines? We integrated three stochastic mortality models with a census tree-ring data set from eight temperate forest types to reconstruct stand-level biomass increments (in Minnesota, USA). We compared growth patterns among mortality models, forest types and stands. Timing of peak biomass growth varied significantly among mortality models, peaking 20-30 years earlier when mortality was random with respect to tree growth and size, than when mortality favored slow-growing individuals. Random or u-shaped mortality (highest in small or large trees) produced peak growth 25-30% higher than the surviving tree sample alone. Growth trends for even-aged, monospecific Pinus banksiana or Acer saccharum forests were similar to the early peak and decline expectation. However, we observed continually increasing biomass growth in older, low-productivity forests of Quercus rubra, Fraxinus nigra, and Thuja occidentalis. Tree-ring reconstructions estimated annual changes in live biomass growth and identified more diverse development patterns than previous methods. These detailed, long-term patterns of biomass development are crucial for detecting recent growth responses to global change and modeling future forest dynamics.


2016 Laboratory guidelines for postvasectomy semen analysis: Association of Biomedical Andrologists, the British Andrology Society and the British Association of Urological Surgeons.
Post-vasectomy semen analysis (PVSA) is the procedure used to establish whether sperm are present in the semen following a vasectomy. PVSA is presently carried out by a wide variety of individuals, ranging from doctors and nurses in general practitioner (GP) surgeries to specialist scientists in andrology laboratories, with highly variable results.Key recommendations are that: (1) PVSA should take place a minimum of 12 weeks after surgery and after a minimum of 20 ejaculations. (2) Laboratories should routinely examine samples within 4 h of production if assessing for the presence of sperm. If non-motile sperm are observed, further samples must be examined within 1 h of production. (3) Assessment of a single sample is acceptable to confirm vasectomy success if all recommendations and laboratory methodology are met and no sperm are observed. Clearance can then be given. (4) The level for special clearance should be <100 000/mL non-motile sperm. Special clearance cannot be provided if any motile sperm are observed and should only be given after assessment of two samples in full accordance with the methods contained within these guidelines. Surgeons are responsible both preoperatively and postoperatively for the counselling of patients and their partners regarding complications and the possibility of late recanalisation after clearance. These 2016 guidelines replace the 2002 British Andrology Society (BAS) laboratory guidelines and should be regarded as definitive for the UK in the provision of a quality PVSA service, accredited to ISO 15189:2012, as overseen by the United Kingdom Accreditation Service (UKAS).


Human Coronavirus Infections in Israel: Epidemiology, Clinical Symptoms and Summer Seasonality of HCoV-HKU1.
Human coronaviruses (HCoVs) cause mild to severe respiratory diseases. Six types of HCoVs have been discovered, the most recent one termed the Middle East respiratory syndrome coronavirus (MERS-CoV). The aim of this study is to monitor the circulation of HCoV types in the population during 2015⁻2016 in Israel. HCoVs were detected by real-time PCR analysis in 1910 respiratory samples, collected from influenza-like illness (ILI) patients during the winter sentinel influenza survey across Israel. Moreover, 195 HCoV-positive samples from hospitalized patients were detected during one year at Soroka University Medical Center. While no MERS-CoV infections were detected, 10.36% of patients in the survey were infected with HCoV-OC43 (43.43%), HCoV-NL63 (44.95%), and HCoV-229E (11.62%) viruses. The HCoVs were shown to co-circulate with respiratory syncytial virus (RSV) and to appear prior to influenza virus infections. HCoV clinical symptoms were more severe than those of RSV infections but milder than influenza symptoms. Hospitalized patients had similar HCoV types percentages. However, while it was absent from the public winter survey, 22.6% of the patients were HCoV-HKU1 positives, mainly during the spring-summer period.


Influence of frequent infectious exposures on general and varicella-zoster virus-specific immune responses in pediatricians.
Reexposure to viruses is assumed to strengthen humoral and cellular immunity via the secondary immune response. We studied the effects of frequent exposure to viral infectious challenges on immunity. Furthermore, we assessed whether repetitive exposures to varicella-zoster virus (VZV) elicited persistently high immune responses. Blood samples from 11 pediatricians and matched controls were assessed at 3 time points and 1 time point, respectively. Besides the assessment of general immunity by means of measuring T-cell subset percentages, antibody titers and gamma interferon (IFN-γ)/interleukin 2 (IL-2)-producing T-cell percentages against adenovirus type 5 (AdV-5), cytomegalovirus (CMV), tetanus toxin (TT), and VZV were determined. Pediatricians had lower levels of circulating CD4(+)-naive T cells and showed boosting of CD8(+) effector memory T cells. Although no effect on humoral immunity was seen, repetitive exposures to VZV induced persistently higher percentages of IFN-γ-positive T cells against all VZV antigens tested (VZV glycoprotein E [gE], VZV intermediate-early protein 62 [IE62], and VZV IE63) than in controls. T cells directed against latency-associated VZV IE63 benefitted the most from natural exogenous boosting. Although no differences in cellular or humoral immunity were found between the pediatricians and controls for AdV-5 or TT, we did find larger immune responses against CMV antigens in pediatricians. Despite the high infectious burden, we detected a robust and diverse immune system in pediatricians. Repetitive exposures to VZV have been shown to induce a stable increased level of VZV-specific cellular but not humoral immunity. Based on our observations, VZV IE63 can be considered a candidate for a zoster vaccine.


Impact of invalidation and trust in physicians on health outcomes in fibromyalgia patients.
Patients with fibromyalgia have reported experiencing discouragement, rejection, suspicion, and stigma during their encounters with health care professionals. The impact of these experiences on health outcomes has not been extensively examined. The aim of this study was to assess fibromyalgia patients' self-reported quality of life (QoL) and pain based on the following: perceptions of physician attitudes, trust in physicians, perceptions of medical professionals, type of treatment, and various demographic variables. An online survey was advertised in the electronic newsletter of the National Fibromyalgia and Chronic Pain Association and data were collected in February 2013. A new scale was developed to measure patient perceptions of physician attitudes. Patients' trust in physicians, patients' perceptions of medical professionals, and QoL were measured using the following standardized scales: Trust in Physician Scale, Illness Invalidation Inventory (3*I), and Quality of Life Scale-16 (QOLS-16). The survey resulted in 670 usable responses. The Patient Perceptions of Physician Attitudes Scale showed high internal consistency and convergent validity (Cronbach α = 0.91). Factor analysis of the Trust in Physician scale, 3*I, and QOLS-16 showed a 1-dimensional structure. Invalidation, use of complementary and alternative medicine, income, age, and marital status were significant predictors of QoL (P < .001). Trust in physician, income, education, and number of referrals to health care providers were significant predictors of pain (P < .001). Invalidation, trust in physician, and use of complementary medicine can have significant impact on QoL and pain in fibromyalgia. Further research in more representative fibromyalgia samples may help confirm findings.


A model of perinatal stress and childhood wheezing: ELSPAC-CZ cohort.
Prenatal origins of wheezing are not fully understood. This study develops a model of mechanisms linking perinatal stress exposure to wheeze phenotypes in children. Data were obtained from 1880 mother-child dyads participating in ELSPAC-CZ birth cohort. Wheeze phenotypes assessed between birth and age 7 years included "never wheeze," "early-onset transient (EOT) wheeze," "early-onset persistent (EOP) wheeze," and "late-onset (LO) wheeze." Prenatal and postnatal stress exposures were assessed in mid-pregnancy and 6 months after delivery, respectively, using an inventory of 42 life events. In adjusted models, children in the highest tercile (high) versus lowest tercile (low) for prenatal life events had a 38% higher risk of EOT wheeze (relative risk ratio [RRR] = 1.38; 95% confidence interval [CI] = 1.01-1.88; p = .041) and 50% higher risk of LO wheeze (RRR = 1.50; 95% CI = 1.00-2.25; p = .047). High versus low exposure to postnatal life events predicted a 60% increase in relative risk of EOT wheeze (RRR = 1.60; 95% CI = 1.17-2.19; p = .003) and medium versus low exposure was related to an 85% increase in relative risk of EOP wheeze (RRR = 1.85; 95% CI = 1.16-2.95; p = .010). Lower respiratory tract infections and postpartum depression partially mediated between postnatal life events and any wheeze (indirect effects 1.06, 95% CI = 1.02-1.09, p = .003 and odds ratio [OR] = 1.08, 95% CI = 1.02-1.15, p = .012, respectively), while postnatal events mediate for prenatal events (indirect effect OR = 1.11; 95% CI = 1.03-1.18; p = .005). Exposures to prenatal and postnatal life events are risk factors for the development of wheezing. Prenatal stress contributes to wheeze directly and also through postnatal life events, respiratory infections, and maternal depression.


Chapter 6: Nonallergic rhinitis.
Nonallergic rhinitis represents a non-IgE-mediated group of disorders that share the symptoms of nasal congestion, rhinorrhea, sneezing, and/or postnasal discharge but not pruritus that characterizes allergic rhinitis. Nonallergic rhinitis may be divided into two broad categories, inflammatory and noninflammatory etiologies. The inflammatory causes include postinfectious (viral and bacterial), rhinitis associated with nasal polyps, and nonallergic rhinitis with eosinophilia, where eosinophils are present in nasal smears but skin testing for aeroallergens is negative. The noninflammatory causes include idiopathic nonallergic rhinitis (formerly referred to as vasomotor rhinitis or colloquially as an "overreactive nose"); rhinitis medicamentosa, which is medication-induced rhinitis; hormone related (pregnancy); systemic disease related (severe hypothyroidism); and structural defect related (deviated septum, head trauma causing cerebrospinal fluid rhinorrhea). The classic symptoms of idiopathic nonallergic rhinitis are nasal congestion, postnasal drip, and sneezing triggered by irritant odors, perfumes, wine, and weather changes. The diagnosis of rhinitis begins with a directed history and physical exam. Examination of the nasal cavity with attention to appearance of the septum and inferior turbinates is recommended. Skin testing for seasonal and perennial aeroallergens is helpful in establishing the presence or absence of IgE antibodies and to help differentiate nonallergic from allergic rhinitis. Topical H(1)-receptor antagonist (antihistamine) nasal sprays, intranasal steroids, intranasal anticholinergics, and oral decongestants are options for pharmacotherapy. It is important to inquire about hypertension, arrhythmias, insomnia, prostate hypertrophy, or glaucoma to prevent undesirable side effects associated with the oral decongestant pseudoephedrine.


Improving women's access to emergency contraception: innovative information and service delivery strategies.
Barriers to widespread use of emergency contraception (EC) include lack of knowledge on the part of women and providers, lack of support for the method from providers, and lack of a dedicated product in many countries. This article reviews strategies to improve women's access to EC launched by national or regional health authorities, clinicians, grass-roots health organizations, and women's groups. Information campaigns have targeted women to improve their knowledge of EC and providers to improve their comfort with it. Local groups and individual providers have also provided leaflets or designed innovative service strategies in order to improve women's access to the method. Expanding the scope or number of these programs and introducing them in areas where women do not currently have adequate knowledge of or access to EC will insure that more women will be able to use this method.


Architecture of the replication fork stalled at the 3' end of yeast ribosomal genes.
Every unit of the rRNA gene cluster of Saccharomyces cerevisiae contains a unique site, termed the replication fork barrier (RFB), where progressing replication forks are stalled in a polar manner. In this work, we determined the positions of the nascent strands at the RFB at nucleotide resolution. Within an HpaI-HindIII fragment essential for the RFB, a major and two closely spaced minor arrest sites were found. In the majority of molecules, the stalled lagging strand was completely processed and the discontinuously synthesized nascent lagging strand was extended three bases farther than the continuously synthesized leading strand. A model explaining these findings is presented. Our analysis included for the first time the use of T4 endonuclease VII, an enzyme recognizing branched DNA molecules. This enzyme cleaved predominantly in the newly synthesized homologous arms, thereby specifically releasing the leading arm.


Viviparous-5 encodes phytoene desaturase, an enzyme essential for abscisic acid (ABA) accumulation and seed development in maize.
The role of synthesis in the regulation of abscisic acid accumulation was investigated in the developing maize seed. To do this, expression and regulation of the abscisic acid biosynthetic enzyme phytoene desaturase were examined. Comparison of the gene sequence encoding phytoene desaturase and its transcript in the wild-type and viviparous-5 mutant showed that the mutant gene contains multiple insertions and deletions, resulting in the synthesis of a larger transcript. In addition, the 55-kDa phytoene desaturase protein was not detectable in the viviparous-5 mutant, indicating that this phenotype results from a mutation at the phytoene desaturase locus. Levels of phytoene desaturase transcript and protein were compared to abscisic acid levels during development to determine whether phytoene desaturase might regulate abscisic acid accumulation. In the endosperm, transcript levels were initially high and declined during late maturation and dormancy, while protein levels remained high throughout development. In the embryo, transcript levels were low and constant, while protein levels declined. Both temporal and tissue-specific expression of phytoene desaturase were unrelated to abscisic acid levels. An abscisic acid mutant (viviparous-2) deficient in phytoene desaturation was used to determine whether the wild-type protein encoded by Viviparous-2 regulates phytoene desaturase. Phytoene desaturase transcript and protein levels were compared in wild-type and viviparous-2 mutant embryos and endosperm. Normalized levels of phytoene desaturase were similar in wild-type and mutant tissues, suggesting that the wild-type Viviparous-2 protein does not regulate phytoene desaturase transcript or protein levels.


A deep learning approach for detecting liver cirrhosis from volatolomic analysis of exhaled breath.
Liver disease such as cirrhosis is known to cause changes in the composition of volatile organic compounds (VOC) present in patient breath samples. Previous studies have demonstrated the diagnosis of liver cirrhosis from these breath samples, but studies are limited to a handful of discrete, well-characterized compounds. We utilized VOC profiles from breath samples from 46 individuals, 35 with cirrhosis and 11 healthy controls. A deep-neural network was optimized to discriminate between healthy controls and individuals with cirrhosis. A 1D convolutional neural network (CNN) was accurate in predicting which patients had cirrhosis with an AUC of 0.90 (95% CI: 0.75, 0.99). Shapley Additive Explanations characterized the presence of discrete, observable peaks which were implicated in prediction, and the top peaks (based on the average SHAP profiles on the test dataset) were noted. CNNs demonstrate the ability to predict the presence of cirrhosis based on a full volatolomics profile of patient breath samples. SHAP values indicate the presence of discrete, detectable peaks in the VOC signal.


Retinoic acid inhibits nitric oxide synthase-2 expression through the retinoic acid receptor-alpha.
Retinoids are multipotent modulators of cellular functions and suppress cytokine-induced production of nitric oxide (NO) in several cell types. We have explored the mechanisms by which retinoic acid (RA) regulates NO production in rat aortic smooth muscle cells (VSMC), which express NOS2 in response to proinflammatory cytokines. RA inhibited interleukin-1beta (IL-1beta)-induced NOS2 mRNA expression and NO production. These effects were attenuated by the retinoic acid receptor (RAR) antagonist CD3106, indicating that they were mediated through retinoic acid receptors (RARs). The synthetic retinoid agonists CD336 (which specifically binds RARalpha) and CD367 (which binds all RARs) but not agonists specific for RARbeta, RARgamma, or RXRs reduced IL-1beta-induced NOS2 expression and NO production. When transfecting VSMC with a 1570-bp NOS2 promoter fragment fused to a luciferase reporter gene, the NOS2 promoter activity was inhibited by RA. These results indicate that retinoids modulate NO production in VSMC via RARalpha, which inhibits the transcription of the NOS2 gene.


Turbulence effects on thermal blooming.
Theoretical and experimental studies have been carried out to determine the importance of mechanical turbulence, i.e., velocity fluctuations, on the propagation of high power cw CO(2) laser radiation in the atmosphere. The experimental results were obtained using artificially generated turbulence and show, in agreement with theory based on a diffusion model, that the turbulence tends to replace the asymmetric bending, focusing, and spreading by the mean wind with a symmetric blooming. For sufficiently strong velocity fluctuations, say, greater than two to three times the mean velocity, the turbulence can reduce thermal blooming effects and increase the beam irradiance. Smaller turbulence levels, however, may actually result in decreasing the beam irradiance somewhat. From these results and estimates of the properties of turbulent diffusion in the atmosphere it appears that under typical conditions the mechanical turbulence will not significantly reduce the wind-dominated thermal distortion effects.


An outbreak of gentamicin-resistant Enterobacter cloacae infections in a pediatric intensive care unit.
The intensive care unit at Children's Medical Center in Dallas is a medical-surgical unit that cares for pediatric patients of all ages. In 1978 an outbreak of infections occurred that was caused by a gentamicin-resistant strain of Enterobacter cloacae. Thirty of the 34 patients involved in the outbreak were neonates. Six patients developed bacteremia, five of them neonates. The neonates who became infected were significantly smaller (less 1500 g) and more premature (less than 35 weeks) than control patients. Neonates with bacteremia had a significantly higher incidence of congenital anomalies. In a multi-specialty pediatric intensive care unit newborn infants were the group of patients at high risk for nosocomial infection.


Declarative and nondeclarative sequence learning tasks: closed-head injured patients versus control participants.
Patients who sustained closed-head injury (CHI) have been shown to have impaired memory for temporal order when measured under intentional, but not incidental, retrieval conditions. A group of 26 patients who sustained CHI and a matched control group of 26 individuals were tested on a declarative sequence learning task--"Chain Making" (CM), and a nondeclarative sequence learning task--Tower of Hanoi puzzle (TOHP). The TOHP is a problem solving task that requires planning and a strategic approach. The latter are cognitive processes known to be impaired following frontal lobe damage, as has been frequently documented in CHI patients. The goal of the present study was to test whether CHI patients' nondeclarative learning as measured by the TOHP task is preserved, as seen in amnesic patients, or impaired, as would be predicted following frontal lobe damage. Half of the participants in each group underwent active training, and the other half went through passive training of the tasks. The results demonstrate that the control group outperformed the CHI group (in most measures) in both declarative and nondeclarative sequence learning tasks. The effect of type of training differed for the two tasks: while performance of the control group on the TOHP was better under passive training (CHI patients did not improve on either one of the training modes), performance on the CM task was better under active training for both groups. The results are discussed in light of the role of the frontal lobes in memory generally, and in sequence learning particularly.


Lithological and land-use based assessment of heavy metal pollution in soils surrounding a cement plant in SW Europe.
We study the influence of phasing out a cement plant on the heavy metal (Hg, Pb and Cr) content in the surrounding soils, taking into account factors often neglected, such as contributions due to local lithology or land use. The range of total Hg was 10-144µg kg(-1), reaching up to 41 and 145mgkg(-1) for total contents of Pb and Cr, respectively. Forest soils showed higher concentration of Hg than prairie soils, indicating the importance of land use on the accumulation of volatile heavy metals in soils. In forest soils, total Hg showed a trend to decrease with soil depth, whereas in prairie soils the vertical pattern of heavy metal concentrations was quite homogeneous. In most cases, the distance to the cement plant was not a factor of influence in the soils content of the analyzed heavy metals. Total Pb and Cr contents in soils nearby the cement plant were quite similar to those found in the local lithology, resulting in enrichment factor values (EF's) below 2. This suggests that soil parent material is the main source of these heavy metals in the studied soils, while the contribution of the cement plant to Pb and Cr soil pollution was almost negligible. On the contrary, the soils surrounding the cement plant accumulate a significant amount of Hg, compared to the underlying lithology. This was especially noticeable in forest soils, where Hg EF achieved values up to 36. These results are of relevance, bearing in mind that Hg accumulation in soils may be an issue of environmental concern, particularly in prairie soils, where temporal flooding can favor Hg transformation to highly toxic methyl-Hg. In addition, the concurrence of acid soils and total-Cr concentrations in the range of those considered phytotoxic should be also stressed.


Nanofibers and nanoparticles for orthopaedic surgery applications.
The ability of nanostructures to elicit altered cell behaviors, including cell adhesion, proliferation, orientation, motility, integrin expression, cytoskeletal organization, and modulation of intracellular signaling, has raised heightened interest in these materials for various biomedical applications, including orthopaedic repair and regeneration. Studies have demonstrated that nanofibrous structures can favorably modulate osteoblast, osteoclast, and fibroblast activities toward implant and/or scaffold materials. Nanomaterials based on silver nanoparticles have received significant attention. Apart from their unique wound-healing ability, silver nanoparticles also exhibit high antibacterial properties, making them potential candidates for use in the development of infection-resistant biomaterials.


DMF attenuates cisplatin-induced kidney injury via activating Nrf2 signaling pathway and inhibiting NF-kB signaling pathway.
N,N-dimethylformamide (DMF) exerts anti-inflammatory and anti-oxidant capacities. We aim to explore whether DMF could regulate cisplatin-induced kidney injury in rats via NF-E2-related factor 2 (Nrf2) pathway and nuclear factor-κB (NF-κB) pathway. A total of 30 Sprague Dawley (SD) rats were randomly assigned into sham group, cisplatin treatment group (DDP group) and DMF + cisplatin treatment group (DMF group), with 10 rats in each group. After 10 days of treatment, we collected serum and kidney samples of rats. Serum levels of creatinine (Cr) and urea nitrogen (BUN) were detected using relative commercial kits. Hematoxylin-eosin (HE) staining was performed to observe pathological changes of kidneys. The relevant oxidative stress indicators in the kidney homogenates of each group were detected by commercial kits, including malondialdehyde (MDA), total antioxidant capacity (T-AOC), catalase (CAT), glutathione (GSH), superoxide dismutase (SOD) and monoamine oxidase (MAO). Protein expressions of Nrf2 and NF-κB in kidney tissues were detected by Western blot. Serum levels of Cr and BUN were lower in DMF group than those of DDP group. Higher activities of SOD, GSH, CAT and T-AOC were found in DMF group compared with those of DDP group. However, MAD and ROS contents were remarkably decreased in DMF group than those of DDP group. DMF pretreatment remarkably reduced renal pathological changes. Western blot analysis also indicated that DMF effectively upregulated expression levels of Nrf2, Heme Oxygenase-1 (HO-1) and NAD (P) H, quinine oxidoreductase 1 (NQO-1), while downregulated NF-κB. DMF could inhibit oxidative stress by activating Nrf2 signaling pathway and reduce inflammatory response by attenuating NF-κB signaling pathway, thus protecting cisplatin-induced kidney injury.


Transient hyperphosphatasemia of infancy and early childhood: clinical and biochemical features of 21 cases and literature review.
Clinical and biochemical features of transient hyperphosphatasemia of infancy and early childhood are reviewed in 21 patients we have studied and in a further 93 cases reported in the literature. The diagnosis is suggested by the finding of an increased activity of alkaline phosphatase (EC 18.104.22.168) in plasma, typically more than fivefold the adult upper reference limit, in a child under five years of age, without evidence of liver or bone disease. The condition is confirmed by the presence of a characteristic pattern of alkaline phosphatase isoenzymes and by the normalization of the enzyme's activity in plasma within approximately three months. The etiology of the condition and possible mechanisms of the enzyme increase are discussed.


Stigma and self-esteem: A case of HIV-positive sex-workers.
Stigma associated with sex work and HIV can be easily recognized in public reactions towards the members of discriminated groups. Nevertheless, there are only a few studies examining the impact of discrimination to the self-esteem of individuals who suffer the coexistence of multiple stigmatizing conditions. In our case, the unprecedented stigmatization of sex workers through the media as a menace of public health as well as criminals due to their seropositivity should be examined with respect and scientificity. The sample consisted of the 27 women found to be HIV positive. The small number of subject and the uniqueness of the situation made necessary the use of qualitative research method. Data were collected of through a semi-structured interview during which personal and medical history was taken and Rosenberg self-esteem scale was completed. Information for each domain of interest was systematically collected from multiple interview guide items. Interpretive Phenomenological Analysis was used to analyze data derived from qualitative interview (IPA). Four main categories emerged from the horizontal analysis of the interviews referring to the mechanism used by those women in order to cope with stigma and protect their self-esteem, a description of their felt stigma and feelings about seropositivity, as well as the existence of self-destructive behaviors. The existence of a normal self-esteem on the majority of those women is well explained by the use of certain coping strategies in order to confront the enacted stigma, such as the avoidance of self-blame for their condition (HIV-positive), the disregard of public's discriminating comments and behaviors, the acknowledge of their competence in specific issues they have to deal with in their everyday life, in common with the existence of a strongly supportive network. Despite those women's felt stigma, structured by community's discriminating approach of their families and their feelings of helplessness and incompetence to protect their beloved, their self-esteem is not harmed and the frequency of selfdestructive behaviors remained stable, possibly as a result of those coping mechanisms developed early in their lives. The circle of stigmatization that emerged through the stories of those women is not an isolated social phenomenon related only to prostitution and drug use. This is the reason why educational programs, access to HIV care services and efforts towards de-stigmatization would benefit the society in multiple levels, and would ultimately strengthen the effort of combating the global AIDS epidemic.


Chemical Profile and Bioactivity Evaluation of Salvia Species from Eastern Europe.
The Salvia genus comprises about 1000 species endowed with medicinal, aromatic, cosmetic, and ornamental applications. Even though the genus is one of the most-studied taxa of the Lamiaceae family, data on the chemical composition and biological properties of certain locally used Salvia species are still scarce. The present work aimed to evaluate the phytochemical profile and antimicrobial, antioxidant, and cytotoxic potential of ten Salvia species that grow in Eastern Europe (e.g., the Republic of Moldova). LC-HRMS/MS metabolite profiling allowed for the annotation of 15 phenolic and organic acids, 18 flavonoids, 19 diterpenes, 5 sesterpenes, and 2 triterpenes. Multivariate analysis (e.g., principal component analysis, hierarchical cluster analysis) revealed that S. austriaca, S. nutans, and S. officinalis formed individual clusters, whereas the remaining species had a similar composition. S. officinalis showed the highest activity against Staphylococcus aureus and Streptococcus pneumoniae (MIC = 0.625 mg/mL). As evaluated in DPPH, ABTS, and FRAP assays, S. officinalis was one of the most potent radical scavenging and metal-reducing agents (CE50 values of 25.33, 8.13, and 21.01 μg/mL, respectively), followed by S. verticillata, S. sclarea, S. kopetdaghensis, S. aethiopis, and S. tesquicola. Pearson correlation analysis revealed strong correlations with rosmarinic acid, luteolin-O-glucuronide, and hydroxybenzoic acid. When the cytotoxic activity was evaluated in human breast carcinoma MCF-7 and MDA-MB-231 cells, no significant reduction in cell viability was observed over the concentrations ranging from 25 and 100 μg/mL. The results confirm the potential use of understudied Salvia species as promising sources of antioxidant compounds for developing novel pharmaceutical, nutraceutical, or cosmeceutical products.


Tuberculosis chemotherapy today.
Effective chemotherapy of tuberculosis not only reduces morbidity and mortality, but it is a powerful mechanism to control further spread of infection. Although conventional therapy for 18-24 months is highly effective, a major disadvantage is non-compliance of patients in completing the full course of therapy. With better understanding of bacteriologic action of many antituberculous drugs, the disease can now be cured in six to nine months of bactericidal chemotherapy. Isoniazid and rifampin for nine months, either given daily throughout or initially for one month followed by twice weekly administration for another eight months, is highly effective for cure of the disease. However, this is not recommended for patients in developing countries due to high frequency of initial isoniazid resistance. Bactericidal therapy with four drugs (streptomycin or ethambutol (25 mg/kg), isoniazid, rifampin and pyrazinamide) daily for two months followed by isoniazid and rifampin daily or twice weekly for another four months is very effective. In suspected or proved isoniazid resistance, the therapy has also proven effective. However, therapy may also be changed during the continuation phase to streptomycin, rifampin and pyrazinamide daily or twice weekly for another four to six months. The treatment of extrapulmonary tuberculosis, alone or when associated with other medical disorders, is the same as for pulmonary tuberculosis. There are several short course regimens available for use in developing countries while keeping in consideration the shortage of major drugs and financial constraints. Modern short course chemotherapy has the potential advantage of success in developing countries by increasing the population of patients completing therapy.(ABSTRACT TRUNCATED AT 250 WORDS)


Imaging rRNA Methylation in Bacteria by MR-FISH.
Methylation of RNA is normally monitored in purified cell lysates using next-generation sequencing, gel electrophoresis, or mass spectrometry as readouts. These bulk methods require the RNA from ~104 to 107 cells to be pooled to generate sufficient material for analysis. Here we describe a method-methylation-sensitive RNA in situ hybridization (MR-FISH)-that assays rRNA methylation in bacteria on a cell-by-cell basis, using methylation-sensitive hybridization probes and fluorescence microscopy. We outline step-by-step protocols for designing probes, in situ hybridization, and analysis of data using freely available code.


DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy.
Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of-CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for-CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than-CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.


Impact of Allogeneic Hematopoietic Stem Cell Transplantation on the HIV Reservoir and Immune Response in 3 HIV-Infected Individuals.
Allogeneic hematopoietic stem cell transplantation (HSCT) can lead to significant changes to the HIV reservoir and HIV immune responses, indicating that further characterization of HIV-infected patients undergoing HSCT is warranted. We studied 3 patients who underwent HSCT after either reduced intensity conditioning or myeloablative conditioning regimen. We measured HIV antigens and antibodies (Ag/Ab), HIV-specific CD4 T-cell responses, HIV RNA, and DNA in plasma, peripheral blood mononuclear cells, isolated CD4 T cells from peripheral blood, and lymph node cells. The patients remained on antiretroviral therapy throughout the follow-up period. All patients have been in continued remission for 4-6 years post-HSCT. Analyses of HIV RNA and DNA levels showed substantial reductions in HIV reservoir-related measurements in all 3 patients, changes in immune response varied with pronounced reductions in 2 patients and a less dramatic reduction in 1 patient. One patient experienced unexpected viral rebound 4 years after HSCT. These 3 cases highlight the substantial changes to the HIV reservoir and the HIV immune response in patients undergoing allogeneic HSCT. The viral rebound observed in 1 patient indicates that replication competent HIV can re-emerge several years after HSCT despite these marked changes.


[Angelus Silesius--physician and mystic].
The priest as physician of the soul and the doctor as physician of the body, this ancient model of christian mentality was adapted by Johannes Scheffler-better known under his pseudonym Angelus Silesius-both in his biography and his literary works. Born 1624 in Breslau, he graduated to a Doctor philosophiae et medicinae and became physician in ordinary to an orthodox-Lutheran earl. Contacts to the esoteric Christianity, which was based on theosophy, mysticism and the physics of Johann Böhme, let Scheffler quit his profession. He converted to Catholicism in 1653 and was ordained as a catholic priest in 1661. His two main manuscripts, the Cherubinische Wandersmann and the Heilige Seelenlust, a poetical dyptichon, show the two possibilities of reaching the mystical recognition of god, either the capability of reason or the ability of emotion. The centre of all is the unio mystica, the union with god. Its a mysticism of the spontaneity of salvation, which occurs in a variety of prospects, metaphors and paradoxical language.


Does a p53 "Wild-type" Immunophenotype Exclude a Diagnosis of Endometrial Serous Carcinoma?
An aberrant p53 immunophenotype may be identified in several histotypes of endometrial carcinoma, and is accordingly recognized to lack diagnostic specificity in and of itself. However, based on the high frequency with which p53 aberrations have historically been identified in endometrial serous carcinoma, a mutation-type immunophenotype is considered to be highly sensitive for the histotype. Using an illustrative case study and a review of the literature, we explore a relatively routine diagnostic question: whether the negative predictive value of a wild-type p53 immunophenotype for serous carcinoma is absolute, that is, whether a p53-wild type immunophenotype is absolutely incompatible with a diagnosis of serous carcinoma. The case is an advanced stage endometrial carcinoma that was reproducibly classified by pathologists from 3 institutions as serous carcinoma based on its morphologic features. By immunohistochemistry, the tumor was p53-wild type (DO-7 clone), diffusely positive for p16 (block positivity), and showed retained expression of PTEN, MSH2, MSH6, MLH1, and PMS2. Next generation sequencing showed that there indeed was an underlying mutation in TP53 (D393fs*78, R213*). The tumor was microsatellite stable, had a low mutational burden (4 mutations per MB), and displayed no mutations in the exonuclease domain of DNA polymerase epsilon (POLE) gene. Other genomic alterations included RB1 mutation (R46fs*19), amplifications in MYST3 and CRKL, and ARID1A deletion (splice site 5125-94_5138del108). A review of the recent literature identified 5 studies in which a total of 259 cases of serous carcinoma were whole-exome sequenced. The average TP53 mutational rate in endometrial serous carcinoma was only 75% (range, 60 to 88). A total of 12 (33%) of 36 immunohistochemical studies reported a p53-aberrant rate of <80% in endometrial serous carcinoma. We discuss in detail several potential explanations that may underlie the scenario of serous carcinoma-like morphology combined with p53-wild-type immunophenotype, including analytic limitations, a nonserous histotype displaying morphologic mimicry of serous carcinoma, and true biological phenomena (including the possibility of a TP53-independent pathway of endometrial serous carcinogenesis). Ultimately, our central thematic question is provisionally answered in the negative. At present, the available data would not support a categorical conclusion that a p53 alteration is a necessary and obligate component in the genesis and/or diagnosis of endometrial serous carcinoma. On the basis of their collective experience, the authors proffer some recommendations on the use of p53 immunohistochemistry in the histotyping of endometrial carcinomas.


Goal setting and achievement for walking: A series of N-of-1 digital interventions.
Despite evidence that goal setting is valuable for physical activity promotion, recent studies highlighted a potential oversimplification in the application of this behavior change technique. While more difficult performance goals might trigger higher physical activity levels, higher performance goals might concurrently be more difficult to achieve, which could reduce long-term motivation. This study examined (a) the association between performance goal difficulty and physical activity and (b) the association between performance goal difficulty and goal achievement. This study used data from an e-Health intervention among inactive overweight adults (n = 20). The study duration included a 2-week baseline period and an intervention phase of 80 days. During the intervention, participants received a daily step goal experimentally manipulated by taking participants' baseline physical activity median (i.e., number of steps) multiplied by a pseudorandom factor ranging from 1 to 2.6. A continuous measure of goal achievement was inferred for each day by dividing the daily number of steps by the goal prescribed that day. Linear and generalized additive models were fit for each participant. The results confirm that, for a majority of the participants involved in the study, performance goal difficulty was positively and significantly associated with physical activity (n = 14), but, concurrently, negatively and significantly associated with goal achievement (n = 19). These associations were mainly linear. At the daily level, setting a higher physical activity goal leads to engaging in higher physical activity levels, but concurrently lower goal achievement. (PsycInfo Database Record (c) 2020 APA, all rights reserved).