De novo missense variants in ZBTB47 are associated with developmental delays, hypotonia, seizures, gait abnormalities, and variable movement abnormalities.

The collection of known genetic etiologies of neurodevelopmental disorders continues to increase, including several syndromes associated with defects in zinc finger protein transcription factors (ZNFs) that vary in clinical severity from mild learning disabilities and developmental delay to refractory seizures and severe autism spectrum disorder. Here we describe a new neurodevelopmental disorder associated with variants in ZBTB47 (also known as ZNF651), which encodes zinc finger and BTB domain-containing protein 47. Exome sequencing (ES) was performed for five unrelated patients with neurodevelopmental disorders. All five patients are heterozygous for a de novo missense variant in ZBTB47, with p.(Glu680Gly) (c.2039A>G) detected in one patient and p.(Glu477Lys) (c.1429G>A) identified in the other four patients. Both variants impact conserved amino acid residues. Bioinformatic analysis of each variant is consistent with pathogenicity. We present five unrelated patients with de novo missense variants in ZBTB47 and a phenotype characterized by developmental delay with intellectual disability, seizures, hypotonia, gait abnormalities, and variable movement abnormalities. We propose that these variants in ZBTB47 are the basis of a new neurodevelopmental disorder.

Depressive symptoms anticipate behavioral and emotional factors among older adults: A prospective cross-lagged panel design.

This study examined the temporal relationship among depression, anxiety, insomnia, perceived stress, and physical activity in adults aged 60+ years with a history of major depressive disorder. We conducted a longitudinal study with 12 weeks of follow-up. Assessments consisted of phone or video interviews and included questionnaires evaluating depression, anxiety, insomnia, perceived stress, and physical activity. Our analytic approach consisted of a depression-focused cross-lagged panel model (CLPM) to examine week-to-week correlations among the five measures. The depression-focused CLPM identified statistically significant week-to-week self-predictive effects for each of the five measures. Higher depressive symptom burden was a strong predictor of increased stress, greater insomnia, and less physical activity the following week. No other cross-measure predictions were statistically significant. Our analytical approach clarifies the directional relationship among variables that typically co-occur with depression showing that higher depression symptom burden predisposes older adults to poor sleep, a reduced level of daytime activity, and a greater sense of stress. These findings support the need for longitudinal assessments and targeted interventions for reducing symptoms of depression in older adults.

Testing for complete spatial randomness on three dimensional bounded convex shapes.

There is currently a gap in theory for point patterns that lie on the surface of objects, with researchers focusing on patterns that lie in a Euclidean space, typically planar and spatial data. Methodology for planar and spatial data thus relies on Euclidean geometry and is therefore inappropriate for analysis of point patterns observed in non-Euclidean spaces. Recently, there has been extensions to the analysis of point patterns on a sphere, however, many other shapes are left unexplored. This is in part due to the challenge of defining the notion of stationarity for a point process existing on such a space due to the lack of rotational and translational isometries. Here, we construct functional summary statistics for Poisson processes defined on convex shapes in three dimensions. Using the Mapping Theorem, a Poisson process can be transformed from any convex shape to a Poisson process on the unit sphere which has rotational symmetries that allow for functional summary statistics to be constructed. We present the first and second order properties of such summary statistics and demonstrate how they can be used to construct a test statistics to determine whether an observed pattern exhibits complete spatial randomness or spatial preference on the original convex space. We compare this test statistic with one constructed from an analogue L -function for inhomogeneous point processes on the sphere. A study of the Type I and II errors of our test statistics are explored through simulations on ellipsoids of varying dimensions.

Therapeutic Benefit of Blood Transfusion in a Patient With Novel PGK1 Mutation (c.461T>C [p.L154P]).

Phosphoglycerate kinase (PGK) is glycolytic enzyme critical in the creation of adenosine triphosphate. Mutations in the gene for this enzyme, PGK1, are associated with PGK deficiency, which is characterized by neurologic symptoms, nonhereditary spherocytic hemolytic anemia, and myopathy. We present a 20-year-old male with a novel c.461T>C (p.L154P) PGK1 mutation and clinical disease complicated by anemia and neurological symptoms. There is no recommended treatment for PGK deficiency. Because of our patient's advanced disease progression, we initiated serial blood transfusions and report significant subjective improvement in the patient's physical condition before his passing from PGK deficiency-related complications.

PEITH(Θ): perfecting experiments with information theory in Python with GPU support.

Motivation: Different experiments provide differing levels of information about a biological system. This makes it difficult, a priori, to select one of them beyond mere speculation and/or belief, especially when resources are limited. With the increasing diversity of experimental approaches and general advances in quantitative systems biology, methods that inform us about the information content that a given experiment carries about the question we want to answer, become crucial. Results: PEITH(Θ) is a general purpose, Python framework for experimental design in systems biology. PEITH(Θ) uses Bayesian inference and information theory in order to derive which experiments are most informative in order to estimate all model parameters and/or perform model predictions. Availability and implementation: https://github.com/MichaelPHStumpf/Peitho. Contact: m.stumpf@imperial.ac.uk or juliane.liepe@mpibpc.mpg.de.

Fusing multimodal microscopy data for improved cell boundary estimation and fluorophore localization of Pseudomonas aeruginosa.

With advances in experimental technologies, the use of biological imaging has grown rapidly and there is need for procedures to combine data arising from different modalities. We propose a procedure to combine yellow fluorescence protein excitation and differential interference contrast microscopy time lapse videos to better estimate the cellular boundary of Pseudomonas aeruginosa (P. aeruginosa) and localization of it's type VI secretion system (T6SS). By approximating the shape by an ellipse, we construct a penalized objective function which accounts for both sources; the minimum of which provides an elliptical approximation to their cellular boundaries. Our approach suggests improved localization of the T6SS on the estimated cell boundary of P. aeruginosa constructed using both sources of data compared to using each in isolation.

Direct ex vivo evaluation of long-lived protective antiviral memory B cell responses against hepatitis B virus.

BACKGROUND: The frequency of protective antiviral memory B cells after hepatitis B virus (HBV) vaccination is unknown. METHODS: A novel 2-step immunomagnetic protocol to assess the ex vivo frequency of protective HBV surface antigen (HBsAg)-specific memory B cells was used. RESULTS: HBsAg-specific memory B cells were detected in vaccinated individuals, although at very low frequency (median, 0.2% of CD19(+) cells [range, 0%-4% of CD19(+) cells]). No correlation existed between the frequency of HBsAg-specific memory B cells and the corresponding serum antibody titer or B cell enzyme-linked immunosorbent spot findings. CONCLUSION: Our results indicate sustained B cell-mediated protection against HBV despite waning antibody titers, which is consistent with clinical observations.

Defining the directionality and quality of influenza virus-specific CD8+ T cell cross-reactivity in individuals infected with hepatitis C virus.

Cross-reactivity of murine and recently human CD8(+) T cells between different viral peptides, i.e., heterologous immunity, has been well characterized. However, the directionality and quality of these cross-reactions is critical in determining their biological importance. Herein we analyzed the response of human CD8(+) T cells that recognize both a hepatitis C virus peptide (HCV-NS3) and a peptide derived from the influenza neuraminidase protein (Flu-NA). To detect the cross-reactive CD8(+) T cells, we used peptide-MHC class I complexes (pMHCs) containing a new mutant form of MHC class I able to bind CD8 more strongly than normal MHC class I complexes. T cell responses against HCV-NS3 and Flu-NA peptide were undetectable in normal donors. In contrast, some responses against the Flu-NA peptide were identified in HCV(+) donors who showed strong HCV-NS3-specific reactivity. The Flu-NA peptide was a weak agonist for CD8(+) T cells in HCV(+) individuals on the basis of novel pMHCs and functional assays. These data support the idea of cross-reactivity between the 2 peptides, but indicate that reactivity toward the Flu-NA peptide is highly CD8-dependent and occurs predominantly after priming during HCV infection. Our findings indicate the utility of the novel pMHCs in dissecting cross-reactivity and suggest that cross-reactivity between HCV and influenza is relatively weak. Further studies are needed to relate affinity and functionality of cross-reactive T cells.

Protection of hepatocytes from cytotoxic T cell mediated killing by interferon-alpha.

BACKGROUND: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV) infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL) to recognise and kill hepatocytes under cytokine stimulation. METHODS/PRINCIPLE FINDINGS: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-alpha treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-alpha stimulated hepatocyte lines were still able to present antigen and induce IFN-gamma expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-alpha, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor-proteinase inhibitor 9 (PI-9). PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection. CONCLUSION/SIGNIFICANCE: IFN-alpha induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.

Quantification and localisation of FOXP3+ T lymphocytes and relation to hepatic inflammation during chronic HCV infection.

BACKGROUND/AIMS: T lymphocyte-mediated immune reactions are closely involved in the pathogenesis of HCV-induced chronic liver disease. Regulatory T cells are able to suppress HCV-specific T lymphocyte proliferation and cytokine secretion during chronic HCV infection. We wished to address to what extent regulatory T cells exist in the livers of HCV+ individuals, and what the role of such cells might be in disease progression. METHODS: We analysed the frequency and distribution of FOXP3+ cells, along with CD4, CD8 and CD20+ cells, in liver biopsies of 28 patients with chronic HCV and 14 patients with PBC, and correlated these data with histological parameters. RESULTS: A striking number of FOXP3+ cells were present in the portal tract infiltrates of HCV-infected livers. FOXP3+ cells were largely CD4+ and there was a remarkably consistent ratio of total CD4+:FOXP3+ cells in liver across a wide range of disease states of around 2:1. This differed substantially from the ratio observed in PBC (10:1, P=0.001). CONCLUSIONS: An unexpectedly high proportion of the cellular infiltrate in persistent HCV infection comprises FOXP3+ cells. The relative proportion of FOXP3+ cells remains similar in both mild and severe fibrosis. These cells are likely to play a critical role in intrahepatic immune regulation, although their overall role in disease progression remains to be determined.

Donation after cardiac death has a minimal impact on thoracic organ recovery.

BACKGROUND: Our organ procurement organization recently developed an aggressive donation after cardiac death program. Thoracic organs are rarely recovered from non-heart-beating donors. Therefore, there is concern that donation after cardiac death may affect the recovery of thoracic organs from donors not allowed to progress to brain death. OBJECTIVE: To evaluate the potential impact of donation after cardiac death on the recovery of thoracic organs. METHODS: On the assumption that prolongation of care on all cases would result in a diagnosis of brain death. By retrospective chart review, all donations after cardiac death were evaluated for thoracic organ potential using the same standards that were used to evaluate brain-dead donors. RESULTS: During the study period there were 34 of 44 (77%) non-heart-beating donors qualified to donate abdominal organs only. Ten of 44 non-heart-beating donors (24%) qualified to potentially donate thoracic organs; the families of 4 of 10 of these donors insisted on the immediate withdrawal of life support, leaving only 6 donors with thoracic organ potential. All 6 of these donors qualified as potential heart donors and 3 as potential lung donors. CONCLUSIONS: A total of 97 organs were recovered and successfully transplanted from 44 non-heart-beating donors. If all the donors who qualified to donate thoracic organs progressed to brain death and if their thoracic organs were transplantable, then 6 additional hearts and 3 pairs of lungs may have been recovered. These data demonstrate that an aggressive donation after cardiac death program contributes significantly to the organ donor pool, with a minimal impact on potential thoracic organ recovery.

Mutationally altered signal output in the Nart (NarX-Tar) hybrid chemoreceptor.

Signal-transducing proteins that span the cytoplasmic membrane transmit information about the environment to the interior of the cell. In bacteria, these signal transducers include sensor kinases, which typically control gene expression via response regulators, and methyl-accepting chemoreceptor proteins, which control flagellar rotation via the CheA kinase and CheY response regulator. We previously reported that a chimeric protein (Nart) that joins the ligand-binding, transmembrane, and linker regions of the NarX sensor kinase to the signaling and adaptation domains of the Tar chemoreceptor elicits a repellent response to nitrate and nitrite. As with NarX, nitrate evokes a stronger response than nitrite. Here we show that mutations targeting a highly conserved sequence (the P box) in the periplasmic domain alter chemoreception by Nart and signaling by NarX similarly. In particular, the G51R substitution converts Nart from a repellent receptor into an attractant receptor for nitrate. Our results underscore the conclusion that the fundamental mechanism of transmembrane signaling is conserved between homodimeric sensor kinases and chemoreceptors. They also highlight the plasticity of the coupling between ligand binding and signal output in these systems.

Dominant influence of an HLA-B27 restricted CD8+ T cell response in mediating HCV clearance and evolution.

Virus-specific CD8+ T cell responses play an important role in the natural course of infection; however, the impact of certain CD8+ T cell responses in determining clinical outcome has not been fully defined. A well-defined cohort of women inoculated with HCV from a single source showed that HLA-B27 has a strong association with spontaneous clearance. The immunological basis for this association is unknown. However, the finding is especially significant because HLA-B27 has also been shown to have a protective role in HIV infection. We report the identification of an HLA-B27 restricted hepatitis C virus (HCV)-specific CD8+ T cell epitope that is recognized in the majority of recovered HLA-B27 positive women. In chronically HCV-infected individuals, analysis of the corresponding viral sequence showed a strong association between sequence variations within this epitope and expression of HLA-B27, indicating allele-specific selection pressure at the population level. Functional analysis in 3 chronically HCV-infected patients showed that the emerging variant viral epitopes represent escape mutations. In conclusion, our results suggest a dominant role of HLA-B27 in mediating spontaneous viral clearance as well as viral evolution in HCV infection and mechanistically link both associations to a dominant novel CD8+ T cell epitope. These results support the central role of virus-specific CD8+ T cells and the genetically determined restriction of the virus-specific T cell repertoire in HCV infection.

Nitrate-dependent activation of the Dif signaling pathway of Myxococcus xanthus mediated by a NarX-DifA interspecies chimera.

Myxococcus xanthus fibril exopolysaccharide (EPS), essential for the social gliding motility and development of this bacterium, is regulated by the Dif chemotaxis-like pathway. DifA, an MCP homolog, is proposed to mediate signal input to the Dif pathway. However, DifA lacks a prominent periplasmic domain, which in classical chemoreceptors is responsible for signal perception and for initiating transmembrane signaling. To investigate the signaling properties of DifA, we constructed a NarX-DifA (NafA) chimera from the sensory module of Escherichia coli NarX and the signaling module of M. xanthus DifA. We report here the first functional chimeric signal transducer constructed using genes from organisms in two different phylogenetic subdivisions. When expressed in M. xanthus, NafA restored fruiting body formation, EPS production, and S-motility to difA mutants in the presence of nitrate. Studies with various double mutants indicate that NafA requires the downstream Dif proteins to function. We propose that signal inputs to the Dif pathway and transmembrane signaling by DifA are essential for the regulation of EPS production in M. xanthus. Despite the apparent structural differences, DifA appears to share similar transmembrane signaling mechanisms with enteric sensor kinases and chemoreceptors.

Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection.

The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8(+) T cells. The role of CD4(+)CD25(+) T regulatory (T(reg)) cells in priming and expanding virus-specific CD8(+) T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8(+) T-cell proliferation and gamma interferon (IFN-gamma) frequency were analyzed with/without depletion of T(reg) cells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4(+)CD25(+) T(reg) cells inhibited anti-CD3/CD28 CD8(+) T-cell proliferation and perforin expression. Depletion of CD4(+)CD25(+) T(reg) cells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P = 0.0005 and P = 0.002, respectively) and also the number of HCV- and EBV-specific IFN-gamma-expressing CD8(+) T cells. Although stimulated CD8(+) T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4(+)CD25(+) regulatory T cells on CD8(+) T-cell proliferation. In conclusion, marked CD4(+)CD25(+) regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8(+) T-cell responses and viral persistence.

Preferential loss of IL-2-secreting CD4+ T helper cells in chronic HCV infection.

Hepatitis C virus (HCV) becomes persistent in the majority of infected individuals. In doing so, the virus evades host adaptive immune responses, although the mechanisms responsible in this evasion are not clear. Several groups have demonstrated weak or absent HCV-specific CD4+ T cell responses during chronic HCV infection using proliferation assays and, more recently, class II tetramers. However, the functional status of HCV-specific CD4+ T cells in resolved and persistent infection is poorly understood. Using interferon gamma (IFN-gamma) and interleukin 2 (IL-2) enzyme-linked immunospot assays, we analyzed cytokine secretion patterns in chronically infected patients and compared them with those with resolved infection. In the spontaneous resolver group, strong IL-2 secretion in relation to IFN-gamma secretion was observed. However, in the persistently infected group, a consistent and significant loss of IL-2-secreting cells, compared with IFN-gamma-secreting cells, was identified. In vitro addition of IL-2 had a substantial effect in restoring CD4+ T cell activity. In conclusion, failure of IL-2 secretion, as opposed to physical deletion or complete functional unresponsiveness, appears to be an important determinant of the status of CD4+ T cell populations in chronic HCV infection. Loss of IL-2 secretory capacity may lead to disruption of IFN-gamma and proliferative function in vivo-a status that characterizes the cellular immune response in both CD4+ and CD8+ compartments in chronic disease.

Incidence and prevalence of lymphedema in patients following burn injury: a five-year retrospective and three-month prospective study.

BACKGROUND: Burn injuries and the disease process of lymphedema are complex medical conditions involving the integument. The possibility of a relationship between burn injury and the development of lymphedema was studied. METHODS AND RESULTS: The incidence of lymphedema was explored by conducting a 5-year retrospective chart review on edema related ICD-9 codes. The prevalence of lymphedema was assessed in a prospective study of patients in a burn clinic. The techniques used to diagnose lymphedema were utilized on all patients presenting to clinic with extremity edema. The prospective analysis reviewed the lymphedema tests and measures and burn characteristics to determine those that were appropriate to diagnose or predict the risk of lymphedema in a burn patient. A 6-month follow-up study was also conducted to re-examine subjects initially diagnosed with lymphedema. CONCLUSIONS: The retrospective review did not reveal significant findings to determine incidence of lymphedema following burn injury. The prospective study identified eight patients with a clinical diagnosis of lymphedema. Two of the eight subjects had a previous diagnosis of lymphedema prior to the burn injury. The six remaining subjects represent a prevalence of 1.0%. Specific tests and measures, Stemmer Sign, deepening of skin folds, and the lack of venous alterations, were found to be appropriate measures to diagnose lymphedema in the burn patient population. Specific burn characteristics, circumferential extremity involvement, and fascial excision, also identified burn patients who may be at risk for developing lymphedema. Lastly, the results of the 6-month re-evaluation confirmed the original diagnoses.

Virus-specific CD8+ T lymphocytes within the normal human liver.

The frequency and phenotype of human antiviral memory CD8(+) T cells in blood are well studied, yet little is known about their distribution within tissues. Analysis of antiviral CD8(+) T cell populations derived from a unique set of normal liver and blood samples identified a consistent population of virus-specific cells within the liver. In comparison to the circulating T cells, the liver-derived T cells were present at frequencies which were variably enriched compared to that in the blood, and showed significant differences with regard to the expression of CD45RA, CD45RO, CD95, CCR7, CD27 and CD28. The differences in these cell surface markers are consistent with a mature 'effector memory' phenotype of antigen-specific CD8(+) T cells within the liver. An enrichment of an activated subset of NKT cells (V alpha 24/V beta 11) was also observed, a finding which may be relevant to the regulation of the antiviral populations.

Ultra-sensitive class I tetramer analysis reveals previously undetectable populations of antiviral CD8+ T cells.

A major breakthrough in cellular immunology has been the development of HLA class I tetramers to analyze CD8(+) T cell responses. However, in many situations, including persistent virus infection, specific T cell responses are rarely detected using this technology. This raises the question of whether such responses are 'deleted' (or 'exhausted') or present below the conventional detection limit for class I tetramer staining. In particular, persistent hepatitis C virus (HCV) infection is characterized by very weak or apparently absent specific CD8(+) T cell responses, even though they are readily detectable in acute disease. Therefore, we assessed the use of anti-PE-labeled magnetic beads to enrich tetramer-positive HCV-specific T cells and identify previously undetectable populations. Using the enrichment technique, HCV-specific T cells could be detected in the majority of infected individuals, whereas these responses were not detected using conventional tetramer staining (8/15 vs. 1/15; p=0.01). Magnetic enrichment could reliably detect very rare HCV-specific responses at frequencies of >0.0011% of CD8(+) T cells (approximately 1/million PBMC), and phenotypic analysis of these rare populations was possible. Therefore, this direct ex vivo technique revealed the persistence of very low frequencies of virus-specific CD8(+) T cells during chronic virus infection and is readily transferable to the study of other viral, self- or tumor-specific T cells.