Structured illumination microscopy using micro-pixellated light-emitting diodes

Structured illumination is a flexible and economical method of obtaining optical sectioning in wide-field microscopy [1]. In this technique the illumination system is modified to project a single-spatial frequency grid pattern onto the sample [2, 3]. The pattern can only be resolved in the focal plane and by recording images for different transverse grid positions (or phases) an image of the in-focus parts of the object can be calculated. Light emitting diodes (LEDs) are becoming increasingly popular for lighting and illumination systems due to their low cost, small dimensions, low coherence, uniform illumination, high efficiency and long lifetime. These properties, together with recent developments in high brightness, ultraviolet operation and microstructured emitter design offer great potential for LEDs as light sources for microscopy. In this paper we demonstrate a novel structured illumination microscope using a blue micro-structured light emitting diode as the illumination source. The system is potentially very compact and has no-moving-parts

Quantum Field Theory: Where We Are

We comment on the present status, the concepts and their limitations, and the successes and open problems of the various approaches to a relativistic quantum theory of elementary particles, with a hindsight to questions concerning quantum gravity and string theory.Comment: To appear in: An Assessment of Current Paradigms in the Physics of Fundamental Phenomena, to be published by Springer Verlag (2006

Whole Blood Profiling of T-cell-Derived microRNA Allows the Development of Prognostic models in Inflammatory Bowel Disease

Background: MicroRNAs [miRNAs] are cell-specific small non-coding RNAs that can regulate gene expression and have been implicated in inflammatory bowel disease [IBD] pathogenesis. Here we define the cell-specific miRNA profiles and investigate its biomarker potential in IBD. Methods: In a two-stage prospective multi-centre case control study, next generation sequencing was performed on a discovery cohort of immunomagnetically separated leukocytes from 32 patients (nine Crohn''s disease [CD], 14 ulcerative colitis [UC], eight healthy controls) and differentially expressed signals were validated in whole blood in 294 patients [97 UC, 98 CD, 98 non-IBD, 1 IBDU] using quantitative PCR. Correlations were analysed with phenotype, including need for early treatment escalation as a marker of progressive disease using Cox proportional hazards. Results: In stage 1, each leukocyte subset [CD4+ and CD8+ T-cells and CD14+ monocytes] was analysed in IBD and controls. Three specific miRNAs differentiated IBD from controls in CD4+ T-cells, including miR-1307-3p [p = 0.01], miR-3615 [p = 0.02] and miR-4792 [p = 0.01]. In the extension cohort, in stage 2, miR-1307-3p was able to predict disease progression in IBD (hazard ratio [HR] 1.98, interquartile range [IQR]: 1.20-3.27; logrank p = 1.80 × 10-3), in particular CD [HR 2.81; IQR: 1.11-3.53, p = 6.50 × 10-4]. Using blood-based multimarker miRNA models, the estimated chance of escalation in CD was 83% if two or more criteria were met and 90% for UC if three or more criteria are met. Interpretation: We have identified and validated unique CD4+ T-cell miRNAs that are differentially regulated in IBD. These miRNAs may be able to predict treatment escalation and have the potential for clinical translation; further prospective evaluation is now indicated

Quenched Lattice QCD with Domain Wall Fermions and the Chiral Limit

Quenched QCD simulations on three volumes, $8^3 \times$, $12^3 \times$ and $16^3 \times 32$ and three couplings, $\beta=5.7$, 5.85 and 6.0 using domain wall fermions provide a consistent picture of quenched QCD. We demonstrate that the small induced effects of chiral symmetry breaking inherent in this formulation can be described by a residual mass (\mres) whose size decreases as the separation between the domain walls ($L_s$) is increased. However, at stronger couplings much larger values of $L_s$ are required to achieve a given physical value of \mres. For $\beta=6.0$ and $L_s=16$, we find \mres/m_s=0.033(3), while for $\beta=5.7$, and $L_s=48$, \mres/m_s=0.074(5), where $m_s$ is the strange quark mass. These values are significantly smaller than those obtained from a more naive determination in our earlier studies. Important effects of topological near zero modes which should afflict an accurate quenched calculation are easily visible in both the chiral condensate and the pion propagator. These effects can be controlled by working at an appropriately large volume. A non-linear behavior of $m_\pi^2$ in the limit of small quark mass suggests the presence of additional infrared subtlety in the quenched approximation. Good scaling is seen both in masses and in $f_\pi$ over our entire range, with inverse lattice spacing varying between 1 and 2 GeV.Comment: 91 pages, 34 figure

Carcinoembryonic antigen-related cell adhesion molecule type 5 receptor-targeted fluorescent intraoperative molecular imaging tracer for lung cancer: a nonrandomized controlled trial

IMPORTANCE Localization of subcentimeter ground glass opacities during minimally invasive thoracoscopic lung cancer resections is a significant challenge in thoracic oncology. Intraoperative molecular imaging has emerged as a potential solution, but the availability of suitable fluorescence agents is a limiting factor.OBJECTIVE To evaluate the suitability of SGM-101, a carcinoembryonic antigen-related cell adhesion molecule type 5 (CEACAM5) receptor-targeted near-infrared fluorochrome, for molecular imagingguided lung cancer resections, because glycoprotein is expressed in more than 80% of adenocarcinomas.DESIGN, SETTING, AND PARTICIPANTS For this nonrandomized, proof-of-principal, phase 1 controlled trial, patients were divided into 2 groups between August 1, 2020, and January 31, 2022. Patients with known CEACAM5-positive gastrointestinal tumors suggestive of lung metastasis were selected as proof-of-principle positive controls. The investigative group included patients with lung nodules suggestive of primary lung malignant neoplasms. Patients 18 years or older without significant comorbidities that precluded surgical exploration with suspicious pulmonary nodules requiring surgical biopsy were included in the study.INTERVENTIONS SGM-101 (10mg) was infused up to 5 days before index operation, and pulmonary nodules were imaged using a near-infrared camera system with a dedicated thoracoscope.MAIN OUTCOMES AND MEASURES SGM-101 localization to pulmonary nodules and its correlation with CEACAM5 glycoprotein expression by the tumor as quantified by tumor and normal pulmonary parenchymal fluorescence.RESULTS Ten patients (5 per group; 5 male and 5 female; median [IQR] age, 66 [58-69] years) with 14 total lesions (median [range] lesion size, 0.91 [0.90-2.00] cm) were enrolled in the study. In the control group of 4 patients (1 patient did not undergo surgical resection because of abnormal preoperative cardiac clearance findings that were not deemed related to SGM-101 infusion), the mean (SD) lesion size was 1.33 (0.48) cm, 2 patients had elevated serum CEA markers, and 2 patients had normal serum CEA levels. Of the 4 patients who underwent surgical intervention, those with 2+ and 3+ tissue CEACAM5 expression had excellent tumor fluorescence, with a mean (SD) tumor to background ratio of 3.11 (0.45). In the patient cohort, the mean (SD) lesion size was 0.68 (0.22) cm, and no elevations in serum CEA levels were found. Lack of SGM-101 fluorescence was associated with benign lesions and with lack of CEACAM5 staining.CONCLUSIONS AND RELEVANCE This in-human proof-of-principle nonrandomized controlled trial demonstrated SGM-101 localization to CEACAM5-positive tumors with the detection of real-time near-infrared fluorescence in situ, ex vivo, and by immunofluorescence microscopy. These findings suggest that SGM-101 is a safe, receptor-specific, and feasible intraoperative molecular imaging fluorochrome that should be further evaluated in randomized clinical trials.Surgical oncolog

Regulation of phosphorylase kinase by low concentrations of Ca ions upon muscle contraction: the connection between metabolism and muscle contraction and the connection between muscle physiology and Ca-dependent signal transduction

It had long been one of the crucial questions in muscle physiology how glycogenolysis is regulated in connection with muscle contraction, when we found the answer to this question in the last half of the 1960s. By that time, the two principal currents of muscle physiology, namely, the metabolic flow starting from glycogen and the mechanisms of muscle contraction, had already been clarified at the molecular level thanks to our senior researchers. Thus, the final question we had to answer was how to connect these two currents. We found that low concentrations of Ca ions (10−7–10−4 M) released from the sarcoplasmic reticulum for the regulation of muscle contraction simultaneously reversibly activate phosphorylase kinase, the enzyme regulating glycogenolysis. Moreover, we found that adenosine 3′,5′-monophosphate (cyclic AMP), which is already known to activate muscle phosphorylase kinase, is not effective in the absence of such concentrations of Ca ions. Thus, cyclic AMP is not effective by itself alone and only modifies the activation process in the presence of Ca ions (at that time, cyclic AMP-dependent protein kinase had not yet been identified). After a while, it turned out that our works have not only provided the solution to the above problem on muscle physiology, but have also been considered as the first report of Ca-dependent protein phosphorylation, which is one of the central problems in current cell biology. Phosphorylase kinase is the first protein kinase to phosphorylate a protein resulting in the change in the function of the phosphorylated protein, as shown by Krebs and Fischer. Our works further showed that this protein kinase is regulated in a Ca-dependent manner. Accordingly, our works introduced the concept of low concentrations of Ca ions, which were first identified as the regulatory substance of muscle contraction, to the vast field of Ca biology including signal transduction

Clustered mutations in the GRIK2 kainate receptor subunit gene underlie diverse neurodevelopmental disorders

Kainate receptors (KARs) are glutamate-gated cation channels with diverse roles in the central nervous system. Bi-allelic loss of function of the KAR-encoding gene GRIK2 causes a nonsyndromic neurodevelopmental disorder (NDD) with intellectual disability and developmental delay as core features. The extent to which mono-allelic variants in GRIK2 also underlie NDDs is less understood because only a single individual has been reported previously. Here, we describe an additional eleven individuals with heterozygous de novo variants in GRIK2 causative for neurodevelopmental deficits that include intellectual disability. Five children harbored recurrent de novo variants (three encoding p.Thr660Lys and two p.Thr660Arg), and four children and one adult were homozygous for a previously reported variant (c.1969G>A [p.Ala657Thr]). Individuals with shared variants had some overlapping behavioral and neurological dysfunction, suggesting that the GRIK2 variants are likely pathogenic. Analogous mutations introduced into recombinant GluK2 KAR subunits at sites within the M3 transmembrane domain (encoding p.Ala657Thr, p.Thr660Lys, and p.Thr660Arg) and the M3-S2 linker domain (encoding p.Ile668Thr) had complex effects on functional properties and membrane localization of homomeric and heteromeric KARs. Both p.Thr660Lys and p.Thr660Arg mutant KARs exhibited markedly slowed gating kinetics, similar to p.Ala657Thr-containing receptors. Moreover, we observed emerging genotype-phenotype correlations, including the presence of severe epilepsy in individuals with the p.Thr660Lys variant and hypomyelination in individuals with either the p.Thr660Lys or p.Thr660Arg variant. Collectively, these results demonstrate that human GRIK2 variants predicted to alter channel function are causative for early childhood development disorders and further emphasize the importance of clarifying the role of KARs in early nervous system development.Genetics of disease, diagnosis and treatmen

Clustered mutations in the GRIK2 kainate receptor subunit gene underlie diverse neurodevelopmental disorders (vol 108, pg 1692, 2021)

Neurolog

The prevalence and transcriptional activity of the mucosal microbiota of ulcerative colitis patients

Active microbes likely have larger impact on gut health status compared to inactive or dormant microbes. We investigate the composition of active and total mucosal microbiota of treatment-naïve ulcerative colitis (UC) patients to determine the microbial picture at the start-up phase of disease, using both a 16S rRNA transcript and gene amplicon sequencing. DNA and RNA were isolated from the same mucosal colonic biopsies. Our aim was to identify active microbial members of the microbiota in early stages of disease and reveal which members are present, but do not act as major players. We demonstrated differences in active and total microbiota of UC patients when comparing inflamed to non-inflamed tissue. Several taxa, among them the Proteobacteria phyla and families therein, revealed lower transcriptional activity despite a high presence. The Bifidobacteriaceae family of the Actinobacteria phylum showed lower abundance in the active microbiota, although no difference in presence was detected. The most abundant microbiota members of the inflamed tissue in UC patients were not the most active. Knowledge of active members of microbiota in UC patients could enhance our understanding of disease etiology. The active microbial community composition did not deviate from the total when comparing UC patients to non-IBD controls

Search for single vector-like B quark production and decay via B → bH(b¯b) in pp collisions at √s = 13 TeV with the ATLAS detector

A search is presented for single production of a vector-like B quark decaying into a Standard Model b-quark and a Standard Model Higgs boson, which decays into a b¯b pair. The search is carried out in 139 fb−1 of √s = 13 TeV proton-proton collision data collected by the ATLAS detector at the LHC between 2015 and 2018. No significant deviation from the Standard Model background prediction is observed, and mass-dependent exclusion limits at the 95% confidence level are set on the resonance production cross-section in several theoretical scenarios determined by the couplings cW, cZ and cH between the B quark and the Standard Model W, Z and Higgs bosons, respectively. For a vector-like B occurring as an isospin singlet, the search excludes values of cW greater than 0.45 for a B resonance mass (mB) between 1.0 and 1.2 TeV. For 1.2 TeV < mB < 2.0 TeV, cW values larger than 0.50–0.65 are excluded. If the B occurs as part of a (B, Y) doublet, the smallest excluded cZ coupling values range between 0.3 and 0.5 across the investigated resonance mass range 1.0 TeV < mB < 2.0 TeV