Eos Promotes TH2 Differentiation by Interacting with and Propagating the Activity of STAT5.

The Ikaros zinc-finger transcription factor Eos has largely been associated with sustaining the immunosuppressive functions of regulatory T cells. Paradoxically, Eos has more recently been implicated in promoting proinflammatory responses in the dysregulated setting of autoimmunity. However, the precise role of Eos in regulating the differentiation and function of effector CD4+ T cell subsets remains unclear. In this study, we find that Eos is a positive regulator of the differentiation of murine CD4+ TH2 cells, an effector population that has been implicated in both immunity against helminthic parasites and the induction of allergic asthma. Using murine in vitro TH2 polarization and an in vivo house dust mite asthma model, we find that EosKO T cells exhibit reduced expression of key TH2 transcription factors, effector cytokines, and cytokine receptors. Mechanistically, we find that the IL-2/STAT5 axis and its downstream TH2 gene targets are one of the most significantly downregulated pathways in Eos-deficient cells. Consistent with these observations, we find that Eos forms, to our knowledge, a novel complex with and supports the tyrosine phosphorylation of STAT5. Collectively, these data define a regulatory mechanism whereby Eos propagates STAT5 activity to facilitate TH2 cell differentiation.

HECT domain interaction with ubiquitin binding sites on Tsg101-UEV controls HIV-1 egress, maturation, and infectivity.

The HECT domain of HECT E3 ligases consists of flexibly linked N- and C-terminal lobes, with a ubiquitin (Ub) donor site on the C-lobe that is directly involved in substrate modification. HECT ligases also possess a secondary Ub binding site in the N-lobe, which is thought to play a role in processivity, specificity, or regulation. Here, we report the use of paramagnetic solution NMR to characterize a complex formed between the isolated HECT domain of neural precursor cell-expressed developmentally downregulated 4-1 and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). Both proteins are involved in endosomal trafficking, a process driven by Ub signaling, and are hijacked by viral pathogens for particle assembly; however, a direct interaction between them has not been described, and the mechanism by which the HECT E3 ligase contributes to pathogen formation has not been elucidated. We provide evidence for their association, consisting of multiple sites on the neural precursor cell-expressed developmentally downregulated 4-1 HECT domain and elements of the Tsg101 UEV domain involved in noncovalent ubiquitin binding. Furthermore, we show using an established reporter assay that HECT residues perturbed by UEV proximity define determinants of viral maturation and infectivity. These results suggest the UEV interaction is a determinant of HECT activity in Ub signaling. As the endosomal trafficking pathway is hijacked by several human pathogens for egress, the HECT-UEV interaction could represent a potential novel target for therapeutic intervention.

Anti-human immunodeficiency virus-1 activity of MoMo30 protein isolated from the traditional African medicinal plant Momordica balsamina.

BACKGROUND: Plants are used in traditional healing practices of many cultures worldwide. Momordica balsamina is a plant commonly used by traditional African healers as a part of a treatment for HIV/AIDS. It is typically given as a tea to patients with HIV/AIDS. Water-soluble extracts of this plant were found to contain anti-HIV activity. METHODS: We employed cell-based infectivity assays, surface plasmon resonance, and a molecular-cell model of the gp120-CD4 interaction to study the mechanism of action of the MoMo30-plant protein. Using Edman degradation results of the 15 N-terminal amino acids, we determined the gene sequence of the MoMo30-plant protein from an RNAseq library from total RNA extracted from Momordica balsamina. RESULTS: Here, we identify the active ingredient of water extracts of the leaves of Momordica balsamina as a 30 kDa protein we call MoMo30-plant. We have identified the gene for MoMo30 and found it is homologous to a group of plant lectins known as Hevamine A-like proteins. MoMo30-plant is distinct from other proteins previously reported agents from the Momordica species, such as ribosome-inactivating proteins such as MAP30 and Balsamin. MoMo30-plant binds to gp120 through its glycan groups and functions as a lectin or carbohydrate-binding agent (CBA). It inhibits HIV-1 at nanomolar levels and has minimal cellular toxicity at inhibitory levels. CONCLUSIONS: CBAs like MoMo30 can bind to glycans on the surface of the enveloped glycoprotein of HIV (gp120) and block entry. Exposure to CBAs has two effects on the virus. First, it blocks infection of susceptible cells. Secondly, MoMo30 drives the selection of viruses with altered glycosylation patterns, potentially altering their immunogenicity. Such an agent could represent a change in the treatment strategy for HIV/AIDS that allows a rapid reduction in viral loads while selecting for an underglycosylated virus, potentially facilitating the host immune response.

Federated causal inference in heterogeneous observational data.

We are interested in estimating the effect of a treatment applied to individuals at multiple sites, where data is stored locally for each site. Due to privacy constraints, individual-level data cannot be shared across sites; the sites may also have heterogeneous populations and treatment assignment mechanisms. Motivated by these considerations, we develop federated methods to draw inferences on the average treatment effects of combined data across sites. Our methods first compute summary statistics locally using propensity scores and then aggregate these statistics across sites to obtain point and variance estimators of average treatment effects. We show that these estimators are consistent and asymptotically normal. To achieve these asymptotic properties, we find that the aggregation schemes need to account for the heterogeneity in treatment assignments and in outcomes across sites. We demonstrate the validity of our federated methods through a comparative study of two large medical claims databases.

Pericyte Progenitor Coupling to the Emerging Endothelium During Vasculogenesis via Connexin 43.

BACKGROUND: Vascular pericytes stabilize blood vessels and contribute to their maturation, while playing other key roles in microvascular function. Nevertheless, relatively little is known about involvement of their precursors in the earliest stages of vascular development, specifically during vasculogenesis. METHODS: We combined high-power, time-lapse imaging with transcriptional profiling of emerging pericytes and endothelial cells in reporter mouse and cell lines. We also analyzed conditional transgenic animals deficient in Cx43/Gja1 (connexin 43/gap junction alpha-1) expression within Ng2+ cells. RESULTS: A subset of Ng2-DsRed+ cells, likely pericyte/mural cell precursors, arose alongside endothelial cell differentiation and organization and physically engaged vasculogenic endothelium in vivo and in vitro. We found no overlap between this population of differentiating pericyte/mural progenitors and other lineages including hemangiogenic and neuronal/glial cell types. We also observed cell-cell coupling and identified Cx43-based gap junctions contributing to pericyte-endothelial cell precursor communication during vascular assembly. Genetic loss of Cx43/Gja1 in Ng2+ pericyte progenitors compromised embryonic blood vessel formation in a subset of animals, while surviving mutants displayed little-to-no vessel abnormalities, suggesting a resilience to Cx43/Gja1 loss in Ng2+ cells or potential compensation by additional connexin isoforms. CONCLUSIONS: Together, our data suggest that a distinct pericyte lineage emerges alongside vasculogenesis and directly communicates with the nascent endothelium via Cx43 during early vessel formation. Cx43/Gja1 loss in pericyte/mural cell progenitors can induce embryonic vessel dysmorphogenesis, but alternate connexin isoforms may be able to compensate. These data provide insight that may reshape the current framework of vascular development and may also inform tissue revascularization/vascularization strategies.

Cultured Skin Autograft in Subtotal Pediatric Scalp Avulsion.

ABSTRACT: Pediatric scalp avulsions represent a reconstructive challenge because of the unique features of scalp tissue. When microsurgical reimplantation is not feasible, alternative approaches such as skin grafting, free flap transfer with latissimus flap, or tissue expansion are considered. Generally, there is no consensus regarding management of this trauma, and, oftentimes, multiple reconstructive techniques may be needed for definitive coverage. This case study describes the reconstruction of a pediatric subtotal scalp avulsion using a dermal regeneration template and novel autologous homologous skin construct. This case was complicated by the absence of original tissue for reimplantation, excessive size of the defect relative to body habitus, and family concerns for future hair-bearing function. The reconstruction successfully provided definitive coverage and significantly reduced the size of the donor site and associated compilations. However, the hair-bearing potential of the tissue has yet to be determined.

Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets.

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.

Integrated STAT3 and Ikaros Zinc Finger Transcription Factor Activities Regulate Bcl-6 Expression in CD4+ Th Cells.

B cell lymphoma-6 (Bcl-6) is a transcriptional repressor that is required for the differentiation of T follicular helper (TFH) cell populations. Currently, the molecular mechanisms underlying the transcriptional regulation of Bcl-6 expression are unclear. In this study, we have identified the Ikaros zinc finger transcription factors Aiolos and Ikaros as novel regulators of Bcl-6. We found that increased expression of Bcl-6 in CD4+ Th cell populations correlated with enhanced enrichment of Aiolos and Ikaros at the Bcl6 promoter. Furthermore, overexpression of Aiolos or Ikaros, but not the related family member Eos, was sufficient to induce Bcl6 promoter activity. Intriguingly, STAT3, a known Bcl-6 transcriptional regulator, physically interacted with Aiolos to form a transcription factor complex capable of inducing the expression of Bcl6 and the TFH-associated cytokine receptor Il6ra Importantly, in vivo studies revealed that the expression of Aiolos was elevated in Ag-specific TFH cells compared with that observed in non-TFH effector Th cells generated in response to influenza infection. Collectively, these data describe a novel regulatory mechanism through which STAT3 and the Ikaros zinc finger transcription factors Aiolos and Ikaros cooperate to regulate Bcl-6 expression.

Probing ultrafast shock-induced chemistry in liquids using broad-band mid-infrared absorption spectroscopy.

We probe shock-induced chemistry in two organic liquids by measuring broadband, midinfrared absorption in the 800-1400 cm-1 frequency range. To test this new method and understand the signatures of chemical reactions in time resolved vibrational spectra, we compared liquid benzene shocked to unreactive conditions (shocked to a pressure of 18 GPa for a duration of 300 ps) to nitromethane under reactive conditions (25 GPa). We see clear signatures of shock-induced chemistry that are distinguishable from the pressure- and temperature-induced changes in vibrational mode shapes. While shocked benzene shows primarily a broadening and shifting of the vibrational modes, the nitromethane vibrational modes vanish once the shock wave enters the liquid and simultaneously, a spectrally broad feature appears that we interpret as the infrared spectrum of the complex mixture of product and intermediate species. To further interpret these measurements, we compare them to reactive quantum molecular dynamics simulations, which gives qualitatively consistent results. This work demonstrates a promising method for time resolving shock-induced chemistry, illustrating that chemical reactions produce distinct changes in the vibrational spectra.

Innovative scheme for high-repetition-rate imaging of CN radical.

We have employed, to the best of our knowledge, a novel excitation scheme to perform the first high-repetition-rate planar laser-induced fluorescence (PLIF) measurements of a CN radical in combustion. The third harmonic of a Nd:YVO4 laser at 355 nm due to its relatively large linewidth overlaps with several R branch transitions in a CN ground electronic state. Therefore, the 355 nm beam was employed to directly excite the CN transitions with good efficiency. The CN measurements were performed in premixed CH4-N2O flames with varying equivalence ratios. A detailed characterization of the high-speed CN PLIF imaging system is presented via its ability to capture statistical and dynamical information in these premixed flames. Single-shot CN PLIF images obtained over a HMX pellet undergoing self-supported deflagration are presented as an example of the imaging system being applied towards characterizing the flame structure of energetic materials.

New Fpg probe chemistry for direct detection of recombinase polymerase amplification on lateral flow strips.

Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings.

Effectiveness of Prenatal Versus Postpartum Tetanus, Diphtheria, and Acellular Pertussis Vaccination in Preventing Infant Pertussis.

BACKGROUND: Most severe and fatal cases of pertussis occur in infants <8 weeks of age, before initiation of the primary pertussis vaccine series. Women are recommended to receive tetanus, diphtheria, and acellular pertussis (Tdap) vaccine at the start of the third trimester of each pregnancy to optimize transplacental transfer of antibodies to the fetus. This recommendation was made by the Advisory Committee for Immunization Practices based on immunogenicity data, and no studies in the United States have yet evaluated the effectiveness of this strategy in reducing pertussis incidence in infants. METHODS: We evaluated a cohort of mothers with documented Tdap vaccination histories in the California Immunization Registry to determine whether infants whose mothers received Tdap vaccine at 27-36 weeks gestation had a lower risk of pertussis at <8 weeks of age than infants born to women who received Tdap vaccine within 14 days post partum. RESULTS: Tdap vaccination received at 27-36 weeks gestation was found to be 85% (95% confidence interval, 33%-98%) more effective than postpartum Tdap vaccination at preventing pertussis in infants <8 weeks of age . Vaccination at 27-36 weeks gestation was more effective at preventing pertussis in infant than vaccination during the second trimester. CONCLUSIONS: Tdap vaccination at 27-36 weeks gestation was 85% more effective than postpartum vaccination at preventing pertussis in infants <8 weeks of age. Efforts should be made by prenatal care providers to provide Tdap vaccine to pregnant women during routine prenatal visits at the earliest opportunity between 27 and 36 weeks gestation.

Aerosols: A Sustainable Route to Functional Materials.

Sustainability is an increasingly important topic in the design and manufacture of materials, with the need to reduce the environmental impact of producing materials being of paramount significance. A competing interest to this is the ability to produce functional materials in large volumes from a fast, on-line process, which can be integrated easily into existing industrial setups. Herein, we present aerosol-assisted chemical vapour deposition (AACVD) routes to advanced functional materials. We will show that by careful design of precursors and manipulation of deposition conditions, it is possible to achieve high sustainability whilst maintaining fast growth rates and large scale production of thin film functional materials.

TIPS pentacene loaded PEO-PDLLA core-shell nanoparticles have similar cellular uptake dynamics in M1 and M2 macrophages and in corresponding in vivo microenvironments.

Nanoparticle based drug delivery platforms have the potential to transform disease treatment paradigms and therapeutic strategies, especially in the context of pulmonary medicine. Once administered, nanoparticles disperse throughout the lung and many are phagocytosed by macrophages. However, there is a paucity of knowledge regarding cellular up-take dynamics of nanoparticles due largely to macrophage heterogeneity. To address this issue, we sought to better define nanoparticle up-take using polarized M1 and M2 macrophages and novel TIPS-pentacene loaded PEO-PDLLA nanoparticles. Our data reveal that primary macrophages polarized to either M1 or M2 phenotypes have similar levels of nanoparticle phagocytosis. Similarly, M1 and M2 polarized macrophages isolated from the lungs of mice following either acute (Th1) or allergic (Th2) airway inflammation also demonstrated equivalent levels of nanoparticle up-take. Together, these studies provide critical benchmark information pertaining to cellular up-take dynamics and biodistribution of nanoparticles in the context of clinically relevant inflammatory microenvironments.

T follicular helper cell programming by cytokine-mediated events.

CD4+ T cells, or T helper cells, are critical mediators and coordinators of adaptive immunity. Unique effector T helper cell populations have been identified that perform distinct functions in response to pathogenic infection. The T follicular helper (Tfh) cells are one such subset, which has been identified as the primary T-cell population responsible for interacting with B cells to promote effective antibody-mediated immune responses. Since their initial description at the turn of the century, and subsequent classification as a distinct T helper cell subset, there has been substantial interest in elucidating the regulatory mechanisms that govern Tfh cell formation. The collective insight from this body of work has demonstrated that Tfh cell differentiation is a complex and multistage process regulated by a litany of cell-intrinsic and cell-extrinsic factors. As with the development of the other recognized T helper cell subsets, specific cytokines exercise prominent roles in both the positive and negative regulation of Tfh cell development. However, the exact composition of, and stage-specific requirements for, these environmental factors in the governance of Tfh cell differentiation remain incompletely understood. In this review, we summarize what is known regarding the role of cytokines in both the promotion and inhibition of Tfh cell differentiation and function.

Loss of Sirt1 promotes prostatic intraepithelial neoplasia, reduces mitophagy, and delays PARK2 translocation to mitochondria.

Prostatic intraepithelial neoplasia is a precursor to prostate cancer. Herein, deletion of the NAD(+)-dependent histone deacetylase Sirt1 induced histological features of prostatic intraepithelial neoplasia at 7 months of age; these features were associated with increased cell proliferation and enhanced mitophagy. In human prostate cancer, lower Sirt1 expression in the luminal epithelium was associated with poor prognosis. Genetic deletion of Sirt1 increased mitochondrial superoxide dismutase 2 (Sod2) acetylation of lysine residue 68, thereby enhancing reactive oxygen species (ROS) production and reducing SOD2 activity. The PARK2 gene, which has several features of a tumor suppressor, encodes an E3 ubiquitin ligase that participates in removal of damaged mitochondria via mitophagy. Increased ROS in Sirt1(-/-) cells enhanced the recruitment of Park2 to the mitochondria, inducing mitophagy. Sirt1 restoration inhibited PARK2 translocation and ROS production requiring the Sirt1 catalytic domain. Thus, the NAD(+)-dependent inhibition of SOD2 activity and ROS by SIRT1 provides a gatekeeper function to reduce PARK2-mediated mitophagy and aberrant cell survival.

Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Aß(1-42) secretion in SH-SY5Y neural cells.

In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infection have diminished. One exception is HIV-associated neurocognitive disorder (HAND). HAND is a spectrum of disorders in cognitive function that ranges from asymptomatic disease to severe dementia (HAD). The milder form of HAND has actually remained the same or slightly increased in prevalence in the CART era. Even in individuals who have maintained undetectable HIV RNA loads, viral proteins such as Nef and Tat can continue to be expressed. In this report, we show that Nef protein and nef messenger RNA (mRNA) are packaged into exosomes that remain in circulation in patients with HAD. Plasma-derived Nef exosomes from patients with HAD have the ability to interact with the neuroblastoma cell line SH-SY5Y and deliver nef mRNA. The mRNA can induce expression of Nef in target cells and subsequently increase expression and secretion of beta-amyloid (Aß) and Aß peptides. Increase secretion of amyloid peptide could contribute to cognitive impairment seen in HAND.

Quality indicators in pituitary surgery: a need for reliable and valid assessments. What should be measured?

Engagement with improving the quality of clinical care is a key component of medical professionalism. Central to Quality Improvement (QI) agenda are the development of valid, reliable and accurate quality metrics. We cannot improve what we do not measure. Pituitary surgery, which in the 21st century usually means trans-sphenoidal surgery (TSS), is unusual; it is a neurosurgical procedure in which complex outcomes can be measured precisely. We have clear guidelines for establishing remission/cure in functional endocrine disease and precise diagnostic tools with which to investigate our patients. Visual recovery can be equally precisely measured with standardised equipment. Moreover, TSS is one of the commonest major surgical procedures carried out in the 34 UK individual neurosurgical units. Most will carry out about 30-40 procedures each year, with four or five units notably higher with numbers in excess of one hundred cases. There are, potentially, plenty of data out there. Given this background, how best should we measure quality in this important area of clinical practice?

Effect of Stereotactic Body Radiotherapy on the Growth Kinetics and Enhancement Pattern of Primary Renal Tumors.

OBJECTIVE: The objective of our study was to assess the growth rate and enhancement of renal masses before and after treatment with stereotactic body radiotherapy (SBRT). MATERIALS AND METHODS: This retrospective study included all patients with renal masses who underwent SBRT during a 5-year period. Orthogonal measurements of renal masses were obtained on pre- and posttreatment CT or MRI. Pre- and posttreatment growth rates were compared for renal mass diameter and volume using the t test. Pre- and posttreatment tumor enhancement values were compared for tumors that underwent multiphasic contrast-enhanced MRI. RESULTS: Forty patients underwent SBRT for the treatment of 41 renal tumors: clear cell renal cell carcinomas (RCCs) (n = 16), papillary RCCs (n = 6), oncocytic neoplasms (n = 8), unclassified RCCs (n = 2), urothelial carcinoma (n = 1), and no pathologic diagnosis (n = 8). The mean maximum tumor diameter before treatment was 3.9 cm (range, 1.6-8.3 cm). Three hundred thirty-eight pre- and posttreatment imaging studies were analyzed: 214 MRI studies and 124 CT studies. The mean pre- and posttreatment lengths of observation were 416 days (range, 2-1800 days) and 561 days (83-1366 days), respectively. The mean pretreatment tumor growth rate of 0.68 cm/y decreased to -0.37 cm/y post treatment (p < 0.0001), and the mean tumor volume growth rate of 21.2 cm(3)/y before treatment decreased to -5.35 cm(3)/y after treatment (p = 0.002). Local control-defined as less than 5 mm of growth-was achieved in 38 of 41 (92.7%) tumors. The Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 showed progression in one tumor (2.4%), stability in 31 tumors (75.6%), partial response in eight tumors (19.5%), and complete response in one tumor (2.4%). No statistically significant change in tumor enhancement was shown (mean follow-up, 142 days; range, 7-581 days). CONCLUSION: Renal tumors treated with SBRT show statistically significant reductions in growth rate and tumor size after treatment but do not show statistically significant differences in enhancement in the initial (mean, 142 days) posttreatment period.

Association of Cytokines With Exosomes in the Plasma of HIV-1-Seropositive Individuals.

Human immunodeficiency virus (HIV)-infected and viremic individuals exhibit elevated levels of plasma cytokines. Here we show that most cytokines are not in free form but appear associated with exosomes that are distinct from virions. Purified exosomes were analyzed to determine the levels of 21 cytokines and chemokines and compared with exosome-depleted plasma. Most cytokines were markedly enriched in exosomes from HIV-positive individuals relative to negative controls and to plasma. Moreover, exposure of naive peripheral blood mononuclear cells to exosomes purified from HIV-positive patients induced CD38 expression on naive and central memory CD4(+) and CD8(+) T cells, probably contributing to inflammation and viral propagation via bystander cell activation.