Sequence specificity defines the effectiveness of PPMOs targeting Pseudomonas aeruginosa.

Development of new therapeutics against antibiotic resistant pathogenic bacteria is recognized as a priority across the globe. We have reported using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) as species-specific antibiotics. The oligo sequences, 11 bases are designed to be complementary to specific essential genes near the Shine-Dalgarno site and inhibit translation. Here, we analyzed target specificity and the impact of genetic mutations on lead PPMOs targeting the rpsJ or acpP gene of Pseudomonas aeruginosa. Mutants in P. aeruginosa PAO1 were generated with four, two, or one base-pair mutations within the 11-base target sequence of the rpsJ gene. All mutants exhibited increased MICs compared to wild-type PAO1 when treated with the RpsJ PPMO, and the increase in the MICs was proportional to the number of base-pair mutations. Among single base-pair mutants, mutations in the middle of the sequence were more impactful than mutations in 5' or 3' end of the sequence. The increased MICs shown by the rpsJ mutants could be reversed by PPMOs designed to target the mutated rpsJ sequence. BALB/c mice infected intratracheally with mutants demonstrated increased lung burden when treated with RpsJ PPMO compared to wild-type PAO1-infected mice treated with RpsJ PPMO. Treating mice with a PPMOs designed to specifically target the mutant sequence was more effective against these mutant strains. These experiments confirm target specificity of two lead P. aeruginosa PPMOs and illustrate one potential mechanism of resistance that could emerge from an antisense approach.

Refined diagnostic criteria for the bipolar disorders: phase two of the AREDOC project.

OBJECTIVE: As limitations exist across DSM criteria sets for defining and differentiating the bipolar disorders generally and their component bipolar I (BP-1) and bipolar II (BP-II) sub-types, we sought to generate empirically based criteria. METHOD: We formed an international Task Force (TF) comprising members with bipolar disorder expertise, and who recruited 74 patients with a TF-diagnosed bipolar I and 104 with a bipolar II condition (with patients responding to definitional queries and symptom questionnaires), while 33 unipolar depressed patients recruited by the first author also completed the symptom questionnaire. A factor analysis sought to determine granular hypo/manic constructs. RESULTS: The bipolar disorder subjects strongly affirmed a new general definition of a bipolar disorder (capturing both manic and hypomanic episodes). While DSM-5 requires impaired functioning, we established that a high percentage of individuals with a BP-I or a BP-II disorder reported improved functioning and therefore modified this criterion. Analyses identified syptoms with differential high rates in individuals with bipolar disorder and its sub-types (and thus not simply capturing happiness), while a factor analysis generated seven symptom constructs both linked with and differing from DSM-5 bipolar symptom criteria. CONCLUSION: This second-stage report details a new set of criteria for differentiating the bipolar disorders from unipolar depressive conditions, while arguing for BP-I and BP-II disorders being differentiated principally by the respective presence or absence of psychotic features. Future studies will evaluate whether further modifications are required and examine for differential treatment benefits for those with a BP-I versus a BP-II condition.

Final Results of the OPERA Experiment on ν_{τ} Appearance in the CNGS Neutrino Beam.

The OPERA experiment was designed to study ν_{µ}→ν_{τ} oscillations in the appearance mode in the CERN to Gran Sasso Neutrino beam (CNGS). In this Letter, we report the final analysis of the full data sample collected between 2008 and 2012, corresponding to 17.97×10^{19} protons on target. Selection criteria looser than in previous analyses have produced ten ν_{τ} candidate events, thus reducing the statistical uncertainty in the measurement of the oscillation parameters and of ν_{τ} properties. A multivariate approach for event identification has been applied to the candidate events and the discovery of ν_{τ} appearance is confirmed with an improved significance level of 6.1σ. |Δm_{32}^{2}| has been measured, in appearance mode, with an accuracy of 20%. The measurement of the ν_{τ} charged-current cross section, for the first time with a negligible contamination from ν[over ¯]_{τ}, and the first direct evidence for the ν_{τ} lepton number are also reported.

Banff Histopathological Consensus Criteria for Preimplantation Kidney Biopsies.

The Banff working group on preimplantation biopsy was established to develop consensus criteria (best practice guidelines) for the interpretation of preimplantation kidney biopsies. Digitally scanned slides were used (i) to evaluate interobserver variability of histopathologic findings, comparing frozen sections with formalin-fixed, paraffin-embedded tissue of wedge and needle core biopsies, and (ii) to correlate consensus histopathologic findings with graft outcome in a cohort of biopsies from international medical centers. Intraclass correlations (ICCs) and univariable and multivariable statistical analyses were performed. Good to fair reproducibility was observed in semiquantitative scores for percentage of glomerulosclerosis, arterial intimal fibrosis and interstitial fibrosis on frozen wedge biopsies. Evaluation of frozen wedge and core biopsies was comparable for number of glomeruli, but needle biopsies showed worse ICCs for glomerulosclerosis, interstitial fibrosis and tubular atrophy. A consensus evaluation form is provided to help standardize the reporting of histopathologic lesions in donor biopsies. It should be recognized that histologic parameters may not correlate with graft outcome in studies based on organs deemed to be acceptable after careful clinical assessment. Significant limitations remain in the assessment of implantation biopsies.

Staphylococcus aureus and Pseudomonas aeruginosa co-infection is associated with cystic fibrosis-related diabetes and poor clinical outcomes.

Cystic fibrosis-related diabetes (CFRD) patients suffer from accelerated rates of pulmonary decline compared to cystic fibrosis (CF) patients with normal glucose tolerance (NGT). However, the mechanisms underlying this difference are unknown. While CFRD is associated with increased respiratory infections, a link between infection and enhanced pulmonary dysfunction remains unclear. The development of glucose intolerance is spectral, resulting in impaired glucose tolerance (IGT) prior to the diagnosis of CFRD. Inclusion of IGT patients within the NGT group may diminish the ability to identify correlations with CFRD. With this in mind, this study aimed to determine if the association between CFRD and respiratory infections is correlated with pulmonary decline. Respiratory cultures from 234 CF patients with confirmed diagnosis of NGT or CFRD were analyzed to measure rates of infection, focusing on the two most prevalent bacteria in CF, Staphylococcus aureus and Pseudomonas aeruginosa. Infection status was correlated with pulmonary function and confounding clinical variables including age, gender, blood glucose levels, and CF transmembrane conductance regulator (CFTR) phenotype were considered in multivariate analyses. CFRD patients, particularly those with extremely high blood glucose levels, were more likely than NGT patients to be co-infected with S. aureus and P. aeruginosa, compared to infection with only one pathogen. Co-infection was associated with decreased lung function and increased frequency of pulmonary exacerbations, even after adjustment for confounding variables. Alterations in the microbial community composition, as opposed to the presence of a single pathogen, may account for greater pulmonary decline in CFRD patients.

Single-item migraine screening tests, self-reported bothersome headache or stripe pattern hypersensitivity?

BACKGROUND: A simple screening tool may potentially help the migraine diagnosis in a primary care setting. The use of single-item tests, such as stripe pattern hypersensitivity test and self-reported bothersome headache (HA) question, as migraine screening tools have not been fully explored. METHODS: Two hundred and fifty-four subjects (patients and companions) were randomly enrolled from an OB/GYN clinic (men 82, women 172; age 38 ± 14). They were instructed to rate the stripe sensitivity level (0-4) and to report any bothersome HA (yes/no). A brief structured HA interview was conducted to describe the HA characteristics and for migraine diagnosis based on the ICHD-IIIß criteria. RESULTS: In a multivariate model, bothersome HA question and stripe pattern hypersensitivity test were both significantly associated with EM+PM+CM (odds ratio: 24.0, P < 0.01 vs 2.6, P = 0.01) or EM (odds ratio: 16.2, P < 0.01 vs 3.0, P < 0.01). Bothersome HA question had a greater screening power than stripe pattern hypersensitivity for screening EM+PM+CM (area under the ROC curve: 0.84 [95% CI 0.78-0.89] vs 0.62 [95% CI 0.55-0.69]) or EM (area under the ROC curve: 0.80 [95% CI 0.73-0.86] vs 0.64 [95% CI 0.56-0.72]). CONCLUSION: When performed in an OB/GYN clinic, self-reported bothersome HA question seemed more powerful than visual stripe pattern test in screening migraine thus could potentially be used as a single-item screening test.

Discovery of τ Neutrino Appearance in the CNGS Neutrino Beam with the OPERA Experiment.

The OPERA experiment was designed to search for ν_{µ}→ν_{τ} oscillations in appearance mode, i.e., by detecting the τ leptons produced in charged current ν_{τ} interactions. The experiment took data from 2008 to 2012 in the CERN Neutrinos to Gran Sasso beam. The observation of the ν_{µ}→ν_{τ} appearance, achieved with four candidate events in a subsample of the data, was previously reported. In this Letter, a fifth ν_{τ} candidate event, found in an enlarged data sample, is described. Together with a further reduction of the expected background, the candidate events detected so far allow us to assess the discovery of ν_{µ}→ν_{τ} oscillations in appearance mode with a significance larger than 5σ.

Nuchal Translucency Quality Review (NTQR) program: first one and half million results.

OBJECTIVE: To evaluate the performance of first-trimester nuchal translucency (NT) measurement by providers (physician-sonologists and sonographers) within the Nuchal Translucency Quality Review (NTQR) program. METHODS: After training and credentialing providers, the NTQR monitored performance of NT measurement by the extent to which an individual's median multiple of the normal median (MoM) for crown-rump length (CRL) was within the range 0.9-1.1 MoM of a published normal median curve. The SD of log10 MoM and regression slope of NT on CRL were also evaluated. We report the distribution between providers of these performance indicators and evaluate potential sources of variation. RESULTS: Among the first 1.5 million scans in the NTQR program, performed between 2005 and 2011, there were 1 485 944 with CRL in the range 41-84 mm, from 4710 providers at 2150 ultrasound units. Among the 3463 providers with at least 30 scans in total, the median of the providers' median NT-MoMs was 0.913. Only 1901 (55%) had a median NT-MoM within the expected range; there were 89 above 1.1 MoM, 1046 at 0.8-0.9 MoM, 344 at 0.7-0.8 MoM and 83 below 0.7 MoM. There was a small increase in the median NT-MoM according to providers' length of time in the NTQR program and number of scans entered annually. On average, physician-sonologists had a higher median NT-MoM than did sonographers, as did those already credentialed before joining the program. The median provider SD was 0.093 and the median slope was 13.5%. SD correlated negatively with the median NT-MoM (r = -0.34) and positively with the slope (r = 0.22). CONCLUSION: Even with extensive training, credentialing and monitoring, there remains considerable variability between NT providers. There was a general tendency towards under-measurement of NT compared with expected values, although more experienced providers had performance closer to that expected.

Graft-versus-host disease after double-unit cord blood transplantation has unique features and an association with engrafting unit-to-recipient HLA match.

Manifestations of and risk factors for graft-versus-host disease (GVHD) after double-unit cord blood transplantation (DCBT) are not firmly established. We evaluated 115 DCBT recipients (median age, 37 years) who underwent transplantation for hematologic malignancies with myeloablative or nonmyeloablative conditioning and calcineurin inhibitor/mycophenolate mofetil immunosuppression. Incidence of day 180 grades II to IV and III to IV acute GVHD (aGVHD) were 53% (95% confidence interval, 44 to 62) and 23% (95% confidence interval, 15 to 31), respectively, with a median onset of 40 days (range, 14 to 169). Eighty percent of patients with grades II to IV aGVHD had gut involvement, and 79% and 85% had day 28 treatment responses to systemic corticosteroids or budesonide, respectively. Of 89 engrafted patients cancer-free at day 100, 54% subsequently had active GVHD, with 79% of those affected having persistent or recurrent aGVHD or overlap syndrome. Late GVHD in the form of classic chronic GVHD was uncommon. Notably, grades III to IV aGVHD incidence was lower if the engrafting unit human leukocyte antigen (HLA)-A, -B, -DRB1 allele match was >4/6 to the recipient (hazard ratio, 0.385; P = .031), whereas engrafting unit infused nucleated cell dose and unit-to-unit HLA match were not significant. GVHD after DCBT was common in our study, predominantly affected the gut, and had a high therapy response, and late GVHD frequently had acute features. Our findings support the consideration of HLA- A,-B,-DRB1 allele donor-recipient (but not unit-unit) HLA match in unit selection, a practice change in the field. Moreover, new prophylaxis strategies that target the gastrointestinal tract are needed.

Promoter methylation of serotonin transporter gene is associated with obesity measures: a monozygotic twin study.

OBJECTIVE: Epigenetic mechanisms are increasingly being recognized as an important factor for obesity. The serotonin transporter gene (SLC6A4) has a critical role in regulating food intake, body weight and energy balance. This study examines the potential association between SLC6A4 promoter methylation and obesity measures in a monozygotic (MZ) twin sample. METHODS: We studied 84 MZ twin pairs drawn from the Vietnam Era Twin Registry. Obesity measures include body mass index (BMI), body weight, waist circumference (WC) and waist-hip ratio (WHR). The SLC6A4 promoter methylation profile in peripheral blood leukocytes was quantified by bisulfite pyrosequencing. The association between methylation variation and obesity parameters was examined by mixed-model regression and matched pair analysis, adjusting for age, smoking, alcohol consumption, physical activity and total daily energy intake. Multiple testing was controlled using the adjusted false discovery rate (q-value). RESULTS: Mean methylation level was positively correlated with BMI (r=0.29; P=0.0002), body weight (r=0.31; P<0.0001) and WC (r=0.20; P=0.009), but not WHR. Intra-pair differences in mean methylation were significantly correlated with intra-pair differences in BMI, body weight and WC, but not WHR. On average, a 1% increase in mean methylation was associated with 0.33 kg m(-2) increase in BMI (95% CI: 0.02-0.65; P=0.03), 1.16 kg increase in body weight (95% CI, 0.16-2.16; P=0.02) and 0.78 cm increase in WC (95% CI, 0.05-1.50; P=0.03) after controlling for potential confounders. CONCLUSIONS: SLC6A4 promoter hypermethylation is significantly associated with an increased prevalence of obesity within a MZ twin study.

Antibodies to phosphatidylserine/prothrombin complex in suspected antiphospholipid syndrome in the absence of antibodies to cardiolipin or Beta-2-glycoprotein I.

Antibodies to phosphatidylserine/prothrombin (aPS/PT) complex were measured in 728 serum specimens from patients suspected of having antiphospholipid syndrome (APS), but without diagnostic elevations in the levels of antibodies to cardiolipin or Beta-2 Glycoprotein 1 (ß2-GP1). Of the 728 specimens, 41 had elevated levels of aPS/PT. Thrombotic events occurred in 11 of the 22 patients with accessible medical histories. Six of the patients with accessible medical records also had laboratory evidence of the lupus anticoagulant. The identification of aPS/PT in patients without evidence of antibodies to cardiolipin, ß2-GP1, or the lupus anticoagulant can contribute to the identification of APS in patients that may go undetected with current testing methods.

Prenatal diagnosis of sickle cell anaemia and thalassaemia by analysis of fetal cells in maternal blood.

Currently, amniocentesis, chorionic villus sampling (CVS) and fetal blood sampling are used to obtain fetal cells for genetic diagnosis. These invasive procedures pose a small but not negligible risk for the fetus. Efforts have been directed towards the enrichment of fetal cells, such as erythroblasts, from maternal blood and progress has been made in the diagnosis of some chromosomal disorders and in sex determinations. We now report the detection of point mutations in single gene disorders using this method of prenatal diagnosis by enriching fetal cells from maternal blood by magnetic cell sorting followed by isolation of pure fetal cells by microdissection. In two pregnancies at risk for sickle cell anaemia and beta-thalassaemia, we successfully identified the fetal genotypes. Thus, prenatal diagnosis of single gene disorders by recovering fetal cells from maternal circulation appears to be a feasible approach.

Behavioral dynamics and neural grounding of a dynamic field theory of multi-object tracking.

The ability to dynamically track moving objects in the environment is crucial for efficient interaction with the local surrounds. Here, we examined this ability in the context of the multi-object tracking (MOT) task. Several theories have been proposed to explain how people track moving objects; however, only one of these previous theories is implemented in a real-time process model, and there has been no direct contact between theories of object tracking and the growing neural literature using ERPs and fMRI. Here, we present a neural process model of object tracking that builds from a Dynamic Field Theory of spatial cognition. Simulations reveal that our dynamic field model captures recent behavioral data examining the impact of speed and tracking duration on MOT performance. Moreover, we show that the same model with the same trajectories and parameters can shed light on recent ERP results probing how people distribute attentional resources to targets vs. distractors. We conclude by comparing this new theory of object tracking to other recent accounts, and discuss how the neural grounding of the theory might be effectively explored in future work.

Guidelines for the diagnosis of antibody-mediated rejection in pancreas allografts-updated Banff grading schema.

The first Banff proposal for the diagnosis of pancreas rejection (Am J Transplant 2008; 8: 237) dealt primarily with the diagnosis of acute T-cell-mediated rejection (ACMR), while only tentatively addressing issues pertaining to antibody-mediated rejection (AMR). This document presents comprehensive guidelines for the diagnosis of AMR, first proposed at the 10th Banff Conference on Allograft Pathology and refined by a broad-based multidisciplinary panel. Pancreatic AMR is best identified by a combination of serological and immunohistopathological findings consisting of (i) identification of circulating donor-specific antibodies, and histopathological data including (ii) morphological evidence of microvascular tissue injury and (iii) C4d staining in interacinar capillaries. Acute AMR is diagnosed conclusively if these three elements are present, whereas a diagnosis of suspicious for AMR is rendered if only two elements are identified. The identification of only one diagnostic element is not sufficient for the diagnosis of AMR but should prompt heightened clinical vigilance. AMR and ACMR may coexist, and should be recognized and graded independently. This proposal is based on our current knowledge of the pathogenesis of pancreas rejection and currently available tools for diagnosis. A systematized clinicopathological approach to AMR is essential for the development and assessment of much needed therapeutic interventions.

Intravoxel incoherent motion imaging of tumor microenvironment in locally advanced breast cancer.

Diffusion-weighted imaging plays important roles in cancer diagnosis, monitoring, and treatment. Although most applications measure restricted diffusion by tumor cellularity, diffusion-weighted imaging is also sensitive to vascularity through the intravoxel incoherent motion effect. Hypervascularity can confound apparent diffusion coefficient measurements in breast cancer. We acquired multiple b-value diffusion-weighted imaging at 3 T in a cohort of breast cancer patients and performed biexponential intravoxel incoherent motion analysis to extract tissue diffusivity (D(t)), perfusion fraction (f(p)), and pseudodiffusivity (D(p)). Results indicated significant differences between normal fibroglandular tissue and malignant lesions in apparent diffusion coefficient mean (±standard deviation) values (2.44 ± 0.30 vs. 1.34 ± 0.39 µm(2)/msec, P < 0.01) and D(t) (2.36 ± 0.38 vs. 1.15 ± 0.35 µm(2)/msec, P < 0.01). Lesion diffusion-weighted imaging signals demonstrated biexponential character in comparison to monoexponential normal tissue. There is some differentiation of lesion subtypes (invasive ductal carcinoma vs. other malignant lesions) with f(p) (10.5 ± 5.0% vs. 6.9 ± 2.9%, P = 0.06), but less so with D(t) (1.14 ± 0.32 µm(2)/msec vs. 1.18 ± 0.52 µm(2)/msec, P = 0.88) and D(p) (14.9 ± 11.4 µm(2)/msec vs. 16.1 ± 5.7 µm(2)/msec, P = 0.75). Comparison of intravoxel incoherent motion biomarkers with contrast enhancement suggests moderate correlations. These results suggest the potential of intravoxel incoherent motion vascular and cellular biomarkers for initial grading, progression monitoring, or treatment assessment of breast tumors.

Education, Healthy Ageing and Vaccine Literacy.

Importance and Objective: The Covid pandemic is a timely opportunity to try to broaden our understanding of the links between education and health literacy and explore the vaccine-decision process with a view to identifying interventions that will positively influence vaccine uptake. EVIDENCE: Health and vaccine literacy encompass people's knowledge, motivation, and competence to access, understand, appraise and apply health information in order to make judgements and take decisions in everyday life concerning health care, disease prevention and health promotion. FINDINGS: Appropriate vaccine communication, which depends greatly on personal and contextual determinants, as well as on societal and environmental circumstances, is essential to reassure people about vaccine efficacy, safety, and possible side effects. However, vaccine confidence is not solely a question of trust in the vaccine's efficacy, safety. and individual protective benefit of vaccination. It also encompasses the mechanism(s) of vaccine activity, immunization schedules, organization and trust in the healthcare system that promotes and delivers the vaccines, and at what costs. When healthcare professionals as science brokers of vaccine knowledge attempt to increase vaccine knowledge and confidence, they must adjust their communication to the educational or health literacy level of their intended audience. Even if their messages are apparently clear and simple, they absolutely need to verify that they are properly understood. RELEVANCE: Specific vaccine communication training appears essential to increase vaccine communication skills among healthcare providers. Moreover, further randomized controlled studies are warranted to improve vaccine empowerment among different populations, from a variety of educational backgrounds.

Geographic variation in shape and size of anuran tadpoles: Interpopulation comparisons in Scinax fuscovarius (Anura, Hylidae).

Among anuran species with biphasic life cycle, the occurrence of intraspecific larval morphotypes has been related to variations in developmental time, diet, geographical variation, or response to predators. Here, we evaluated the external morphological variation of larvae among three populations, located more than 270 km apart, of the anuran hylid Scinax fuscovarius by linear and geometric methods, to elucidate the presence of geographically different morphs. Comparisons targeted development, growth, and external morphology. Studied populations exhibited differences in reproductive seasonality, growth rate, timing of development, shape, and size. Shape and size comparisons revealed two well-differentiated morphs, one of them shared by the two closest populations. Morphological differences evidenced a smaller and depressed form of the entire body plan in the most distant population, which showed continuous reproduction throughout the rainy season and under more unpredictable conditions. We interpret the occurrence of the two different larval morphs in S. fuscovarius as a by-product of local geographical conditions, and discuss on possible associations with biotic and abiotic factors cues.

OPERA tau neutrino charged current interactions.

The OPERA experiment was designed to discover the vτ appearance in a vµ beam, due to neutrino oscillations. The detector, located in the underground Gran Sasso Laboratory, consisted of a nuclear photographic emulsion/lead target with a mass of about 1.25 kt, complemented by electronic detectors. It was exposed from 2008 to 2012 to the CNGS beam: an almost pure vµ beam with a baseline of 730 km, collecting a total of 1.8·1020 protons on target. The OPERA Collaboration eventually assessed the discovery of vµâ†’vτ oscillations with a statistical significance of 6.1 σ by observing ten vτ CC interaction candidates. These events have been published on the Open Data Portal at CERN. This paper provides a detailed description of the vτ data sample to make it usable by the whole community.

HLA-derived peptides which inhibit T cell function bind to members of the heat-shock protein 70 family.

Synthetic peptides corresponding to sequences of HLA class I molecules have inhibitory effects on T cell function. The peptides investigated in this study have sequences corresponding to the relatively conserved region of the alpha 1 helix of HLA class I molecules that overlaps the "public epitope" Bw4/Bw6. These HLA-derived peptides exhibit inhibitory effects on T lymphocytes and have beneficial effects on the survival of allogenic organ transplants in mice and rats. Peptides corresponding to the Bw4a epitope appear most potent as they inhibit the differentiation of T cell precursors into mature cytotoxic T lymphocytes (CTL) and target cell lysis by established CTL lines and clones. To elucidate the mechanism through which these peptides mediate their inhibitory effect on T lymphocytes, peptide binding proteins were isolated from T cell lysates. We show that the inhibitory Bw4a peptide binds two members of the heat-shock protein (HSP) 70 family, constitutively expressed HSC70 and heat-inducible HSP70. Peptide binding to HSC/HSP70 is sequence specific and follows the rules defined by the HSC70 binding motif. Most intriguing, however, is the strict correlation of peptide binding to HSC/HSP70 and the functional effects such that only inhibitory peptides bind to HSC70 and HSP70 whereas noninhibitory peptides do not bind. This correlation suggests that small molecular weight HLA-derived peptides may modulate T cell responses by directly interacting with HSPs. In contrast to numerous reports of HSP70 expression at the surface of antigen-presenting cells and some tumor cells, we find no evidence that HSC/HSP70 are expressed at the surface of the affected T cells. Therefore, we believe that the peptides' immunodulatory effects are not mediated through a signaling event initiated by interaction of peptide with surface HSP, but favor a model similar to the action of other immunomodulatory compounds, FK506 and cyclosporin A, with a role for HSC/HSP70 similar to that for immunophilins, FKBPs and CyP40.