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The notion that T cell insulitis increases as type 1 diabetes (T1D) develops is unsurprising, however, the quantitative analysis of CD4 + and CD8 + T cells within the islet mass is complex and limited with standard approaches. Optical microscopy is an important and widely used method to evaluate immune cell infiltration into pancreatic islets of Langerhans for the study of disease progression or therapeutic efficacy in murine T1D. However, the accuracy of this approach is often limited by subjective and potentially biased qualitative assessment of immune cell subsets. In addition, attempts at quantitative measurements require significant time for manual analysis and often involve sophisticated and expensive imaging software. In this study, we developed and illustrate here a streamlined analytical strategy for the rapid, automated and unbiased investigation of islet area and immune cell infiltration within (insulitis) and around (peri-insulitis) pancreatic islets. To this end, we demonstrate swift and accurate detection of islet borders by modeling cross-sectional islet areas with convex polygons (convex hulls) surrounding islet-associated insulin-producing β cell and glucagon-producing α cell fluorescent signals. To accomplish this, we used a macro produced with the freeware software ImageJ equipped with the Fiji Is Just ImageJ (FIJI) image processing package. Our image analysis procedure allows for direct quantification and statistical determination of islet area and infiltration in a reproducible manner, with location-specific data that more accurately reflect islet areas as insulitis proceeds throughout T1D. Using this approach, we quantified the islet area infiltrated with CD4 + and CD8 + T cells allowing statistical comparison between different age groups of non-obese diabetic (NOD) mice progressing towards T1D. We found significantly more CD4 + and CD8 + T cells infiltrating the convex hull-defined islet mass of 13-week-old non-diabetic and 17-week-old diabetic NOD mice compared to 4-week-old NOD mice. We also determined a significant and measurable loss of islet mass in mice that developed T1D. This approach will be helpful for the location-dependent quantitative calculation of islet mass and cellular infiltration during T1D pathogenesis and can be combined with other markers of inflammation or activation in future studies.

The rhodamine motif has been modified in myriad ways to produce probes with specific fluorescent and chemical properties optimal for a variety of microscopy experiments. Recently, far-red emitting silicon rhodamines have become popular in single-molecule localization microscopy (SMLM), since these dyes are membrane-permeable and can be used alongside red fluorophores for two-color imaging. While this has expanded multi-color SMLM imaging capabilities, we demonstrate that silicon rhodamines can create previously unreported photoproducts with significantly blueshifted emissions, which appear as bright single-molecule crosstalk in the red emission channel. We show that this fluorescence is caused by the replacement of the central silicon group with oxygen after 640 nm illumination, turning far-red silicon rhodamines (JFX650, JF669, etc.) into their red oxygen rhodamine counterparts (JFX554, JF571, etc.). While this blueshifted population can cause artifacts in two-color SMLM data, we demonstrate up to 16-fold reduction in crosstalk using oxygen-scavenging systems. We also leverage this far-red photoconversion to demonstrate UV-free photoactivated localization microscopy (PALM) without the need for additives, and with 5-fold higher efficiency than the Cy5 to Cy3 conversion. Finally, we demonstrate multiplexed pseudo two-color PALM in a single emission channel by separating localizations by their photo-activation wavelengths instead of their emission wavelengths.

An improved understanding of etiological mechanisms in Parkinson’s disease (PD) is urgently needed because the number of affected individuals is projected to increase rapidly as populations age. We present results from a blood-based methylome-wide association study of PD involving meta-analysis of 229 K CpG probes in 1,132 cases and 999 controls from two independent cohorts. We identify two previously unreported epigenome-wide significant associations with PD, including cg06690548 on chromosome 4. We demonstrate that cg06690548 hypermethylation in PD is associated with down-regulation of the SLC7A11 gene and show this is consistent with an environmental exposure, as opposed to medications or genetic factors with effects on DNA methylation or gene expression. These findings are notable because SLC7A11 codes for a cysteine-glutamate anti-porter regulating levels of the antioxidant glutathione, and it is a known target of the environmental neurotoxin β-methylamino-L-alanine (BMAA). Our study identifies the SLC7A11 gene as a plausible biological target in PD. Parkinson’s disease (PD) is a common neurodegenerative disorder with a complex etiology involving genetics and the environment. Here, Vallerga et al. identify two CpG probes associated with PD in a blood cell type-corrected epigenome-wide meta-analysis, implicating the SLC7A11 gene as a plausible biological target.

Multiple myeloma (MM) is a plasma-cell neoplasm that is treated with high-dose chemotherapy, autologous stem cell transplant (ASCT) and long-term immunomodulatory drug (IMiD) maintenance. The presence of somatic mutations in the peripheral blood is termed clonal hematopoiesis of indeterminate potential (CHIP) and is associated with adverse outcomes. Targeted sequencing of the stem cell product from 629 MM patients treated by ASCT at the Dana-Farber Cancer Institute (2003–2011) detects CHIP in 136/629 patients (21.6%). The most commonly mutated genes are DNMT3A, TET2, TP53, ASXL1 and PPM1D. Twenty-one from fifty-six patients (3.3%) receiving first-line IMiD maintenance develop a therapy-related myeloid neoplasm (TMN). However, regardless of CHIP status, the use of IMiD maintenance associates with improved PFS and OS. In those not receiving IMiD maintenance, CHIP is associated with decreased overall survival (OS) (HR:1.34, p  = 0.02) and progression free survival (PFS) (HR:1.45, p  < 0.001) due to an increase in MM progression. Multiple myeloma (MM) is treated with induction chemotherapy, autologous stem cell transplant (ASCT) and long-term immunomodulatory drug (IMiD) maintenance. Here, the authors show that the presence of clonal haematopoiesis of indeterminate potential (CHIP) at time of ASCT is associated with adverse outcomes in MM patients.

An emerging field of research indicates that physical activity can benefit cognitive functions and academic achievements in children. However, less is known about how academic achievements can benefit from specific types of motor activities (e.g., fine and gross) integrated into learning activities. Thus, the aim of this study was to investigate whether fine or gross motor activity integrated into math lessons (i.e., motor-enrichment) could improve children's mathematical performance. A 6-week within school cluster-randomized intervention study investigated the effects of motor-enriched mathematical teaching in Danish preadolescent children ( = 165, age = 7.5 ± 0.02 years). Three groups were included: a control group (CON), which received non-motor enriched conventional mathematical teaching, a fine motor math group (FMM) and a gross motor math group (GMM), which received mathematical teaching enriched with fine and gross motor activity, respectively. The children were tested before (T0), immediately after (T1) and 8 weeks after the intervention (T2). A standardized mathematical test (50 tasks) was used to evaluate mathematical performance. Furthermore, it was investigated whether motor-enriched math was accompanied by different effects in low and normal math performers. Additionally, the study investigated the potential contribution of cognitive functions and motor skills on mathematical performance. All groups improved their mathematical performance from T0 to T1. However, from T0 to T1, the improvement was significantly greater in GMM compared to FMM (1.87 ± 0.71 correct answers) ( = 0.02). At T2 no significant differences in mathematical performance were observed. A subgroup analysis revealed that normal math-performers benefitted from GMM compared to both CON 1.78 ± 0.73 correct answers ( = 0.04) and FMM 2.14 ± 0.72 correct answers ( = 0.008). These effects were not observed in low math-performers. The effects were partly accounted for by visuo-spatial short-term memory and gross motor skills. The study demonstrates that motor enriched learning activities can improve mathematical performance. In normal math performers GMM led to larger improvements than FMM and CON. This was not the case for the low math performers. Future studies should further elucidate the neurophysiological mechanisms underlying the observed behavioral effects.

Although circulating tumor DNA (ctDNA) assays are increasingly used to inform clinical decisions in cancer care, they have limited ability to identify the transcriptional programs that govern cancer phenotypes and their dynamic changes during the course of disease. To address these limitations, we developed a method for comprehensive epigenomic profiling of cancer from 1 ml of patient plasma. Using an immunoprecipitation-based approach targeting histone modifications and DNA methylation, we measured 1,268 epigenomic profiles in plasma from 433 individuals with one of 15 cancers. Our assay provided a robust proxy for transcriptional activity, allowing us to infer the expression levels of diagnostic markers and drug targets, measure the activity of therapeutically targetable transcription factors and detect epigenetic mechanisms of resistance. This proof-of-concept study in advanced cancers shows how plasma epigenomic profiling has the potential to unlock clinically actionable information that is currently accessible only via direct tissue sampling. In a large multi-cancer cohort, a single liquid biopsy assay enables the detection of four epigenomic modifications, allowing the monitoring of expression of potential drug targets and resistance genes.

Pediatric tuberculosis (TB) often goes undiagnosed because of the lack of reliable diagnostic methods. With the aim of assessing biomarker(s) that can aid in the diagnosis of TB infection and disease, we investigated 746 Indian children with suspected TB. Whole-blood mRNA from 210 children was examined by dual-color Reverse-Transcriptase Multiple Ligation-dependent Probe-Amplification for the expression of 45 genes and a Bio-Plex assay for the expression of cytokines/chemokines in QuantiFERON supernatants. The study shows that transcription of SEC14L1, GUSB, BPI, CCR7 and TGFβ-1 (all P ⩽0.05) was downregulated in TB disease compared with uninfected controls, while transcription of RAB33A was downregulated in TB disease compared with both latent TB ( P <0.05) and controls ( P <0.01). The transcription of CD4, TGFβ-1 ( P <0.01) and the expression of IL-2 ( P <0.01) and IL-13 ( P <0.05) was upregulated in latent TB compared with that in controls. Using the Least Absolute Shrinkage and Selection Operator (lasso) model, RAB33A alone discriminated between TB disease and latent TB (area under the curve (AUC) 77.5%), whereas a combination of RAB33A, CXCL10, SEC14L1, FOXP3 and TNFRSF1A was effective in discriminating between TB disease and controls (AUC 91.7%). A combination of 11 biomarkers predicted latent TB with moderate discriminatory power (AUC 72.2%). In conclusion, RAB33A is a potential biomarker for TB disease, whereas CD4 , TGFβ-1 and IL-2, IL-13 may identify latent TB in children.

The tuberculin skin test (TST) and QuantiFERON-TB-Gold-In-tube (QFTGIT) are adjunctive tests used in the diagnosis of pediatric tuberculosis (TB). Neither test can rule out TB; however, a positive test usually triggers preventive treatment in TB contacts aged <5 years. TST and QFTGIT can give divergent results and it is unclear how discordant results should be interpreted in terms of TB risk and preventive treatment. To understand the immune processes underlying concordant or discordant TST and QFTGIT results, we analyzed immune responses in children from Palamaner Taluk in India (a TB-endemic region with routine neonatal BCG vaccination) who were referred to a TB case verification ward on suspicion of TB. Two hundred and ten children aged <3 years were classified according to their TST and QFTGIT results, and their immune responses analyzed by dual-colour-Reverse-Transcriptase-Multiple-Ligation-dependent-Probe-Amplification, using a panel of 45 genes and a 10-plex antigen-specific enzyme-linked immunosorbent assay. We show that immune biomarkers FPR1, TNFRSF1A and interferon (IFN)-γ are upregulated (all P <0.05) in concordant test-positive children, whereas BPI is downregulated ( P <0.05). In contrast, SEC14L1 ( P =0.034) and Interferon gamma-induced protein 10 (IP-10) ( P =0.001) are differentially expressed between the TST+QFTGIT− /TST−QFTGIT+ groups. Known TB exposure was more frequent in concordant positive children and results were consistent with elevated expression of genes associated with inflammatory responses. Children with discordant test results displayed a mixed profile with activation of both pro- and anti-inflammatory markers. TST and/or QFTGIT positivity appears to reflect distinct but overlapping aspects of host immunity.

IntroductionAccurate tuberculosis (TB) incidence and optimal surveillance strategies are pertinent to TB vaccine trial design. Infants are a targeted population for new TB vaccines, but data from India, with the highest global burden of TB cases, is limited.MethodsIn a population-based prospective trial conducted between November 2006 and July 2008, BCG-vaccinated neonates in South India were enrolled and cluster-randomised to active or passive surveillance. We assessed the influence of surveillance strategy on TB incidence, case-finding rates and all-cause mortality. Predefined criteria were used to diagnose TB. All deaths were evaluated using a verbal autopsy.Results4382 children contributed to 8164 person-years (py) of follow-up (loss to follow-up 6.9%); 749 children were admitted for TB evaluation (active surveillance: 641; passive surveillance: 108). The TB incidence was 159.2/100 000 py and the overall case-finding rate was 3.19 per 100 py (95% CI 0.82 to 18.1). Whereas, the case-finding rate for definite TB was similar using active or passive case finding, the case-finding rate for probable TB was 1.92/100 py (95% CI 0.83 to 3.78) with active surveillance, significantly higher than 0.3/100 py (95% CI 0.01 to 1.39, p=0.02) with passive surveillance. Compared to passive surveillance, children with active surveillance had decreased risk of dying (OR 0.68, 95%CI 0.47 to 0.98) which was mostly attributable to reduction of death from pneumonia/respiratory infections (OR 0.34, 95%CI 0.14 to 0.80).ConclusionWe provide reliable estimates of TB incidence in South Indian children <2 years of age. Active surveillance increased the case-finding rates for probable TB and was associated with reduced all-cause mortality.