Molecular and clinical presentation of UBA1-mutated myelodysplastic syndromes.

Mutations in UBA1, which are disease-defining for VEXAS syndrome, have been reported in patients diagnosed with myelodysplastic syndromes (MDS). Here, we define the prevalence and clinical associations of UBA1 mutations in a representative cohort of patients with MDS. Digital droplet PCR profiling of a selected cohort of 375 male patients lacking MDS disease-defining mutations or established WHO disease classification identified 28 patients (7%) with UBA1 p.M41T/V/L mutations. Using targeted sequencing of UBA1 in a representative MDS cohort (n=2,027), we identified an additional 27 variants in 26 patients (1%), which we classified as likely/pathogenic (n=12) and unknown significance (n=15). Among the total 40 patients with likely/pathogenic variants (2%), all were male and 63% were classified by WHO2016 as MDS-MLD/SLD. Patients had a median of one additional myeloid gene mutation, often in TET2 (n=12), DNMT3A (n=10), ASXL1 (n=3), or SF3B1 (n=3). Retrospective clinical review where possible showed that 83% (28/34) UBA1-mutant cases had VEXAS-associated diagnoses or inflammatory clinical presentation. The prevalence of UBA1-mutations in MDS patients argues for systematic screening for UBA1 in the management of MDS.

Learning rules of engagement for social exchange within and between groups.

Globalizing economies and long-distance trade rely on individuals from different cultural groups to negotiate agreement on what to give and take. In such settings, individuals often lack insight into what interaction partners deem fair and appropriate, potentially seeding misunderstandings, frustration, and conflict. Here, we examine how individuals decipher distinct rules of engagement and adapt their behavior to reach agreements with partners from other cultural groups. Modeling individuals as Bayesian learners with inequality aversion reveals that individuals, in repeated ultimatum bargaining with responders sampled from different groups, can be more generous than needed. While this allows them to reach agreements, it also gives rise to biased beliefs about what is required to reach agreement with members from distinct groups. Preregistered behavioral (N = 420) and neuroimaging experiments (N = 49) support model predictions: Seeking equitable agreements can lead to overly generous behavior toward partners from different groups alongside incorrect beliefs about prevailing norms of what is appropriate in groups and cultures other than one's own.

Application limits of the scaling relations for Monte Carlo simulations in diffuse optics. Part 1: theory.

Monte Carlo (MC) is a powerful tool to study photon migration in scattering media, yet quite time-consuming to solve inverse problems. To speed up MC-simulations, scaling relations can be applied to an existing initial MC-simulation to generate a new data-set with different optical properties. We named this approach trajectory-based since it uses the knowledge of the detected photon trajectories of the initial MC-simulation, in opposition to the slower photon-based approach, where a novel MC-simulation is rerun with new optical properties. We investigated the convergence and applicability limits of the scaling relations, both related to the likelihood that the sample of trajectories considered is representative also for the new optical properties. For absorption, the scaling relation contains smoothly converging Lambert-Beer factors, whereas for scattering it is the product of two quickly diverging factors, whose ratio, for NIRS cases, can easily reach ten orders of magnitude. We investigated such instability by studying the probability-distribution for the number of scattering events in trajectories of given length. We propose a convergence test of the scattering scaling relation based on the minimum-maximum number of scattering events in recorded trajectories. We also studied the dependence of MC-simulations on optical properties, most critical in inverse problems, finding that scattering derivatives are ascribed to small deviations in the distribution of scattering events from a Poisson distribution. This paper, which can also serve as a tutorial, helps to understand the physics of the scaling relations with the causes of their limitations and devise new strategies to deal with them.

Fast data fitting scheme for compressive multispectral fluorescence lifetime imaging.

A single-pixel camera combined with compressive sensing techniques is a promising fluorescence microscope scheme for acquiring a multidimensional dataset (space, spectrum, and lifetime) and for reducing the measurement time with respect to conventional microscope schemes. However, upon completing the acquisition, a computational step is necessary for image reconstruction and data analysis, which can be time-consuming, potentially canceling out the beneficial effect of compressive sensing. In this work, we propose and experimentally validate a fast-fit workflow based on global analysis and multiple linear fits, which significantly reduces the computation time from tens of minutes to less than 1 s. Moreover, as the method is interlaced with the measurement flow, it can be applied in parallel with the acquisitions.

"I don't think of it as a shelter. I say I'm going home": a qualitative evaluation of a low-threshold shelter for women who use drugs.

BACKGROUND: In 2021-2022, encampments in a downtown Boston neighborhood reached record heights, increasing the visibility of drug use and homelessness in the city. In response, the city planned a "sweep" (i.e., eradication of encampments) and requested support from social services and medical providers to pilot low-threshold shelters. Low-threshold shelters reduce barriers to staying in traditional congregate shelters with more flexible regulations, longer-term bed assignments, and secured storage for contraband (e.g., drugs, weapons) instead of forced disposal. One homeless service provider opened a harm reduction-focused shelter for women who use drugs. This report describes the low-threshold shelter design and program evaluation. METHODS: This program evaluation had two primary aims: (1) to examine guests' beliefs about shelter policies and practices; and (2) to understand the staff's experiences working in a low-threshold model. We conducted semi-structured qualitative interviews with 16 guests and 12 staff members during the summer 2022. Interviews were thematically analyzed. RESULTS: Guests expressed overwhelming approval for the shelter's policies, which they stated supported their autonomy, dignity, and safety. They emphasized the staff's willingness to build relationships, thus demonstrating true commitment to the guests. Guests highlighted the value of daytime access to the shelter, as it granted them autonomy over their time, reduced their substance use, and helped them build relationships with staff and other guests. The co-directors and staff designed the shelter quickly and without US models for reference; they turned to international literature, local harm reduction health care providers, and women living in encampments for guidance on the shelter policies. The staff were passionate and committed to the health and stability of the guests. Most staff found value in the low-threshold model, though some were challenged by it, believing it enabled drug use and did not require the guests to "get better." CONCLUSIONS: This evaluation indicates the value of low-threshold, harm reduction shelters as alternatives to traditional models. While these shelters do not mitigate the need for overarching housing reform, they are important measures to meet the needs of women experiencing unsheltered homelessness who face intersectional oppression.

Two-layer reconstruction of Raman spectra in diffusive media based on an analytical model in the time domain.

We derive and validate an analytical model that describes the migration of Raman scattered photons in two-layer diffusive media, based on the diffusion equation in the time domain. The model is derived under a heuristic approximation that background optical properties are identical on the excitation and Raman emission wavelengths. Methods for the reconstruction of two-layer Raman spectra have been developed, tested in computer simulations and validated on tissue-mimicking phantom measurements data. Effects of different parameters were studied in simulations, showing that the thickness of the top layer and number of detected photon counts have the most significant impact on the reconstruction. The concept of quantitative, mathematically rigorous reconstruction using the proposed model was finally proven on experimental measurements, by successfully separating the spectra of silicone and calcium carbonate (calcite) layers, showing the potential for further development and eventual application in clinical diagnostics.

Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

Above pile-up fluorescence microscopy with a 32 Mc/s single-channel time-resolved SPAD system.

One of the major drawbacks of time-correlated single-photon counting (TCSPC) is generally represented by pile-up distortion, which strongly bounds the maximum acquisition speed to a few percent of the laser excitation rate. Based on a previous theoretical analysis, recently we presented the first, to the best of our knowledge, low-distortion and high-speed TCSPC system capable of overcoming the pile-up limitation by perfectly matching the single-photon avalanche diode (SPAD) dead time to the laser period. In this work, we validate the proposed system in a standard fluorescence measurement by comparing experimental data with the reference theoretical framework. As a result, a count rate of 32 Mc/s was achieved with a single-channel system still observing a negligible lifetime distortion.

Enlarged Field of View in Spatially Modulated Selective Volume Illumination Microscopy.

Three-dimensional fluorescence microscopy is a key technology for inspecting biological samples, ranging from single cells to entire organisms. We recently proposed a novel approach called spatially modulated Selective Volume Illumination Microscopy (smSVIM) to suppress illumination artifacts and to reduce the required number of measurements using an LED source. Here, we discuss a new strategy based on smSVIM for imaging large transparent specimens or voluminous chemically cleared tissues. The strategy permits steady mounting of the sample, achieving uniform resolution over a large field of view thanks to the synchronized motion of the illumination lens and the camera rolling shutter. Aided by a tailored deconvolution method for image reconstruction, we demonstrate significant improvement of the resolution at different magnification using samples of varying sizes and spatial features.

Multispectral compressive fluorescence lifetime imaging microscopy with a SPAD array detector.

Multispectral/hyperspectral fluorescence lifetime imaging microscopy (λFLIM) is a promising tool for studying functional and structural biological processes. The rich information content provided by a multidimensional dataset is often in contrast with the acquisition speed. In this work, we develop and experimentally demonstrate a wide-field λFLIM setup, based on a novel time-resolved 18×1 single-photon avalanche diode array detector working in a single-pixel camera scheme, which parallelizes the spectral detection, reducing measurement time. The proposed system, which implements a single-pixel camera with a compressive sensing scheme, represents an optimal microscopy framework towards the design of λFLIM setups.

Non-alcoholic beriberi, Wernicke encephalopathy and long-term eating disorder: case report and a mini-review.

INTRODUCTION: Nowadays, reports of beriberi are rare in developed countries. Wernicke encephalopathy may be present in about 25% of patients with beriberi. CASE REPORT: We report the case of a woman with history of depression and chronic eating disorder, who complained Wernicke encephalopathy and beriberi. Sural nerve and muscular biopsy were performed, showing severe axonal neuropathy. Thiamine supplementation was started with rapid improvement of the pulmonary and cardiac affections; improvement of peripheral neuropathy was incomplete. CONCLUSIONS: Thiamine deficiency can be misdiagnosed. Beriberi is an important cause of acute flaccid paralysis; hence, clinicians should consider this diagnosis and prompt start thiamine treatment to avoid permanent neurological sequelae.

Visible-Light Photoredox-Catalyzed Amidation of Benzylic Alcohols.

A new photocatalyzed route to amides from alcohols and amines mediated by visible light is presented. The reaction is carried out in ethyl acetate as a solvent. Ethyl acetate can be defined a green and bio-based solvent. The starting materials such as the energy source are easily available, stable, and inexpensive. The reaction has shown to be general and high yielding.

Time-resolved multispectral imaging based on an adaptive single-pixel camera.

Time-resolved multispectral imaging has many applications in different fields, which range from characterization of biological tissues to environmental monitoring. In particular, optical techniques, such as lidar and fluorescence lifetime imaging, require imaging at the subnanosecond scales over an extended area. In this paper, we demonstrate experimentally a time-resolved multispectral acquisition scheme based on single-pixel imaging. Single-pixel imaging is an emerging paradigm that provides low-cost high-quality images. Here, we use an adaptive strategy that allows acquisition and image reconstruction times to be reduced drastically or full basis scans. Adaptive time-resolved multispectral imaging scheme can have significant applications in biological imaging, at scales from macroscopic to microscopic.

Time- and frequency-resolved fluorescence with a single TCSPC detector via a Fourier-transform approach.

We introduce a broadband single-pixel spectro-temporal fluorescence detector, combining time-correlated single photon counting (TCSPC) with Fourier transform (FT) spectroscopy. A birefringent common-path interferometer (CPI) generates two time-delayed replicas of the sample's fluorescence. Via FT of their interference signal at the detector, we obtain a two-dimensional map of the fluorescence as a function of detection wavelength and emission time, with high temporal and spectral resolution. Our instrument is remarkably simple, as it only requires the addition of a CPI to a standard single-pixel TCSPC system, and it shows a readily adjustable spectral resolution with inherently broad bandwidth coverage.

Frequency offset Raman spectroscopy (FORS) for depth probing of diffusive media.

We present a new technique, frequency offset Raman spectroscopy (FORS), to probe Raman spectra of diffusive media in depth. The proposed methodology obtains depth sensitivity exploiting changes in optical properties (absorption and scattering) with excitation wavelengths. The approach was demonstrated experimentally on a two-layer tissue phantom and compared with the already consolidated spatially offset Raman spectroscopy (SORS) technique. FORS attains a similar enhancement of signal from deep layers as SORS, namely 2.81 against 2.62, while the combined hybrid FORS-SORS approach leads to a markedly higher 6.0 enhancement. Differences and analogies between FORS and SORS are discussed, suggesting FORS as an additional or complementary approach for probing heterogeneous media such as biological tissues in depth.

Multiple-view diffuse optical tomography system based on time-domain compressive measurements.

Compressive sensing is a powerful tool to efficiently acquire and reconstruct an image even in diffuse optical tomography (DOT) applications. In this work, a time-resolved DOT system based on structured light illumination, compressive detection, and multiple view acquisition has been proposed and experimentally validated on a biological tissue-mimicking phantom. The experimental scheme is based on two digital micromirror devices for illumination and detection modulation, in combination with a time-resolved single element detector. We fully validated the method and demonstrated both the imaging and tomographic capabilities of the system, providing state-of-the-art reconstruction quality.

Time-domain Raman analytical forward solvers.

A set of time-domain analytical forward solvers for Raman signals detected from homogeneous diffusive media is presented. The time-domain solvers have been developed for two geometries: the parallelepiped and the finite cylinder. The potential presence of a background fluorescence emission, contaminating the Raman signal, has also been taken into account. All the solvers have been obtained as solutions of the time dependent diffusion equation. The validation of the solvers has been performed by means of comparisons with the results of "gold standard" Monte Carlo simulations. These forward solvers provide an accurate tool to explore the information content encoded in the time-resolved Raman measurements.

Interaction between human Ctf4 and the Cdc45/Mcm2-7/GINS (CMG) replicative helicase.

Chromosome transmission fidelity 4 (Ctf4) is a conserved protein required for DNA replication. In this report, interactions between human Ctf4 (hCtf4) and the replicative helicase containing the cell division cycle 45 (Cdc45)/minichromosome maintenance 2-7 (Mcm2-7)/Go, Ichi, Nii, and San (GINS) (CMG) proteins [human CMG (hCMG) complex] were examined. The hCtf4-CMG complex was isolated following in vitro interaction of purified proteins (hCtf4 plus the hCMG complex), coinfection of Spodoptera frugiperda (Sf9) insect cells with viruses expressing the hCMG complex and hCtf4, and from HeLa cell chromatin after benzonase and immunoprecipitation steps. The stability of the hCtf4-CMG complex depends upon interactions between hCtf4 and multiple components of the hCMG complex. The hCtf4-CMG complex, like the hCMG complex, contains DNA helicase activity that is more salt-resistant than the helicase activity of the hCMG complex. We demonstrate that the hCtf4-CMG complex contains a homodimeric hCtf4 and a monomeric hCMG complex and suggest that the homodimeric hCtf4 acts as a platform linking polymerase α to the hCMG complex. The role of the hCMG complex as the core of the replisome is also discussed.

Fast silicon photomultiplier improves signal harvesting and reduces complexity in time-domain diffuse optics.

We present a proof of concept prototype of a time-domain diffuse optics probe exploiting a fast Silicon PhotoMultiplier (SiPM), featuring a timing resolution better than 80 ps, a fast tail with just 90 ps decay time-constant and a wide active area of 1 mm2. The detector is hosted into the probe and used in direct contact with the sample under investigation, thus providing high harvesting efficiency by exploiting the whole SiPM numerical aperture and also reducing complexity by avoiding the use of cumbersome fiber bundles. Our tests also demonstrate high accuracy and linearity in retrieving the optical properties and suitable contrast and depth sensitivity for detecting localized inhomogeneities. In addition to a strong improvement in both instrumentation cost and size with respect to legacy solutions, the setup performances are comparable to those of state-of-the-art time-domain instrumentation, thus opening a new way to compact, low-cost and high-performance time-resolved devices for diffuse optical imaging and spectroscopy.

Properties of the human Cdc45/Mcm2-7/GINS helicase complex and its action with DNA polymerase epsilon in rolling circle DNA synthesis.

In eukaryotes, although the Mcm2-7 complex is a key component of the replicative DNA helicase, its association with Cdc45 and GINS (the CMG complex) is required for the activation of the DNA helicase. Here, we show that the CMG complex is localized to chromatin in human cells and describe the biochemical properties of the human CMG complex purified from baculovirus-infected Sf9 cells. The isolated complex binds to ssDNA regions in the presence of magnesium and ATP (or a nonhydrolyzable ATP analog), contains maximal DNA helicase in the presence of forked DNA structures, and translocates along the leading strand (3' to 5' direction). The complex hydrolyses ATP in the absence of DNA; unwinds duplex regions up to 500 bp; and either replication protein A or Escherichia coli single stranded binding protein increases the efficiency of displacement of long duplex regions. Using a 200-nt primed circular DNA substrate, the combined action of human DNA polymerase ε and the human CMG complex leads to the formation of products >10 kb in length. These findings suggest that the coordinated action of these replication complexes supports leading strand synthesis.