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ObjectivesImmunotherapy with immune checkpoint inhibitors has shown only limited success in the management of metastatic soft tissue sarcoma. Overall response rates (ORR) with single agent pembrolizumab were 18% and median PFS was 18 weeks on the clinical trial SARC028. One strategy to improve the responses to immunotherapy is with stereotactic body radiation therapy (SBRT), which can enhance the antitumor CD8 T cell response through the release of tumor-specific antigens, potentially priming a more diverse class of T cell receptors.MethodsThis is a phase 0, pilot prospective study taking place at a single center with 2 arms. In Arm A, patients are treated with pembrolizumab 400 mg IV infusion on day 1 of a 42-day cycle. Stereotactic body radiation therapy (SBRT) is delivered in 1-5 fractions starting on C1D15-28 and given every other day. In Arm B, patients who have started an immune checkpoint inhibitor within 60 days are treated with SBRT in addition to the current therapy.ResultsIn this study we outline testing the feasibility of adding SBRT to pembrolizumab.ConclusionThe ultimate goal of combination therapy is improved overall response, including tumors not treated with SBRT. This trial can be found registered online: NCT05488366.

The P450 enzyme CYP121 from Mycobacterium tuberculosis catalyzes a carbon-carbon (C-C) bond coupling cyclization of the dityrosine substrate containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in less than 5 ms in the rapid freeze-quenching study of CYP121 during the shunt reaction with peracetic acid or hydrogen peroxide in acetic acid solution. We show that this intermediate can also be observed in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing method for time-resolved serial femtosecond crystallography with X-ray free-electron laser (tr-SFX-XFEL) technology covering the millisecond time domain and without freezing, we structurally monitored the reaction in situ at room temperature. After a 200 ms peracetic acid reaction with the cocrystallized enzyme-substrate microcrystal slurry, a ferric-hydroperoxo intermediate is observed, and its structure is determined at 1.85 Å resolution. The structure shows a hydroperoxyl ligand between the heme and the native substrate, cYY. The oxygen atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion. The end-on binding ligand adopts a near-side-on geometry and is weakly associated with the iron ion, causing the unusual high-spin state. This compound 0 intermediate, spectroscopically and structurally observed during the catalytic shunt pathway, reveals a unique binding mode that deviates from the end-on compound 0 intermediates in other heme enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound cYY, suggesting an active oxidant role of the intermediate for direct substrate oxidation in the nonhydroxylation C-C bond coupling chemistry.

Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O-O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.

* Context.--Digital pathology using whole slide images has been recently approved to support primary diagnosis in clinical surgical pathology practices. Here we describe a novel imaging method, fluorescence-imitating brightfield imaging, that can capture the surface of fresh tissue without requiring prior fixation, paraffin embedding, tissue sectioning, or staining. Objective.--To compare the ability of pathologists to evaluate direct-to-digital images with standard pathology preparations. Design.--One hundred surgical pathology samples were obtained. Samples were first digitally imaged, then processed for standard histologic examination on 4-im hematoxylin-eosin-stained sections and digitally scanned. The resulting digital images from both digital and standard scan sets were viewed by each of 4 reading pathologists. The data set consisted of 100 reference diagnoses and 800 study pathologist reads. Each study read was compared to the reference diagnosis, and also compared to that reader's diagnosis across both modalities. Results.--The overall agreement rate, across 800 reads, was 97.9%. This consisted of 400 digital reads at 97.0% versus reference and 400 standard reads versus reference at 98.8%. Minor discordances (defined as alternative diagnoses without clinical treatment or outcome implications) were 6.1% overall, 7.2% for digital, and 5.0% for standard. Conclusions.--Pathologists can provide accurate diagnoses from fluorescence-imitating brightfield imaging slide-free images. Concordance and discordance rates are similar to published rates for comparisons of whole slide imaging to standard light microscopy of glass slides for primary diagnosis. It may be possible, therefore, to develop a slide-free, nondestructive approach for primary pathology diagnosis., While in vivo methods for disease detection and classification, including radiology and liquid biopsy, continue to improve with respect to spatial resolution (radiology) and molecular information, they have not, to [...]

The newly constructed Bois d’Arc Lake Reservoir in Fannin County, Texas, USA, inevitably flooded a large ground surface area (67.34 km2) when the reservoir began impounding water in April 2021. Inside this (now) flooded area, land-based archaeological data recovery investigations discovered and documented several archaeological sites, now registered in the state of Texas; though, only two neighboring sites, namely, 41FN178 and 41FN244, are examined here. The first phase of archaeological testing at these sites included shovel testing, test unit excavations, and geoarchaeological trenching that yielded archaeological artifacts suggesting that Middle Caddo Indian peoples (AD 1200–1400) might have occupied this landscape. As the sites were recognized before the reservoir’s impoundment phase, this merited a non-invasive, non-destructive, high-resolution near-surface geophysical study to map strategic areas within sites 41FN178 and 41FN244 that might yield potential shallow targets of archaeological context. The adopted geophysical survey comprised 3D direct current electrical resistivity imaging (ERI) and land horizontal magnetic gradiometry (HMG), each mapping a total surface area of 2133 and 15,640 m2, respectively. The combination of 3D ERI and land HMG surveys was instrumental in rapidly mapping the horizontal and vertical extent of shallowly buried anomalies within a large area prior to the completion of the dam and the beginning of water impoundment. Based on the geophysical insights, the outline of several Caddo houses with functional internal and external features (e.g., burnt cooking surfaces, storage pits, refuse pits, fired soil, ditches, a dump site, and a compound fence) are thought to exist within the uppermost 2 m of the Quaternary stratigraphy at both sites. At site 41FN244, 3D ERI found numerous resistive anomalies surrounding a conductive anomaly, collectively interpreted as a group of post-holes surrounding the remains of a Caddo house’s inner clay floor. It also found a cluster of several resistive anomalies interpreted as midden or middens. The HMG survey carried across areas from which archaeological test units also yielded positive findings, at sites 41FN178 and 41FN244, identified numerous scattered monopolar and dipolar anomalies interpreted as post-molds of Caddo houses, compound enclosures or fences, and adjacent middens. Archaeological excavations guided by the geophysical results yielded significant cultural material and post-mold features at site 244, which validate the geophysical interpretation in a preliminary context. Additionally, several dispersed magnetic anomalies are thought to be shallowly buried hearths, burn cooking surfaces, storage pits, and ditches. The mapped magnetic anomalies agree with the location and distribution of previously found archaeological artifacts and the extent of resistive and conductive resistivity anomalies. Follow-up archaeological excavations of these geophysical anomalies have preliminarily confirmed interpretations.

Abstract Background Glaucoma patients with irreversible visual field loss often experience decreased quality of life, impaired mobility, and mental health challenges. Perceptual learning (PL) and transcranial electrical stimulation (tES) have emerged as promising interventions for vision rehabilitation, showing potential in restoring residual visual functions. The Glaucoma Rehabilitation using ElectricAI Transcranial stimulation (GREAT) project aims to investigate whether combining PL and tES is more effective than using either method alone in maximizing the visual function of glaucoma patients. Additionally, the study will assess the impact of these interventions on brain neural activity, blood biomarkers, mobility, mental health, quality of life, and fear of falling. Methods The study employs a three-arm, double-blind, randomized, superiority-controlled design. Participants are randomly allocated in a 1:1:1 ratio to one of three groups receiving: (1) real PL and real tES, (2) real PL and sham tES, and (3) placebo PL and sham tES. Each participant undergoes 10 sessions per block (~ 1 h each), with a total of three blocks. Assessments are conducted at six time points: baseline, interim 1, interim 2, post-intervention, 1-month post-intervention, and 2-month post-intervention. The primary outcome is the mean deviation of the 24-2 visual field measured by the Humphrey visual field analyzer. Secondary outcomes include detection rate in the suprathreshold visual field, balance and gait functions, and electrophysiological and biological responses. This study also investigates changes in neurotransmitter metabolism, biomarkers, self-perceived quality of life, and psychological status before and after the intervention. Discussion The GREAT project is the first study to assess the effectiveness of PL and tES in the rehabilitation of glaucoma. Our findings will offer comprehensive assessments of the impact of these treatments on a wide range of brain and vision-related metrics including visual field, neural activity, biomarkers, mobility, mental health, fear of falling, and quality of life. Trial registration ClinicalTrials.gov NCT05874258 . Registered on May 15, 2023.

Wild populations of aquatic turtles are imperiled because of anthropogenic activities (e.g., consumption, collection, fisheries bycatch) (1); more than half (186/357) of the recognized species of aquatic turtles in the [...]

Context.—Digital pathology using whole slide images has been recently approved for primary diagnosis in clinical surgical pathology practices. Here we describe a novel imaging method, fluorescence-imitating brightfield imaging, that can capture the surface of fresh tissue without requiring prior fixation, paraffin embedding, tissue sectioning, or staining.Objective.—To compare the ability of pathologists to evaluate direct-to-digital images with standard pathology preparations.Design.—One hundred surgical pathology samples were obtained. Samples were first digitally imaged, then processed for standard histologic examination on 4-μm hematoxylin-eosin-stained sections and digitally scanned. The resulting digital images from both digital and standard scan sets were viewed by each of 4 reading pathologists. The data set consisted of 100 reference diagnoses and 800 study pathologist reads. Each study read was compared to the reference diagnosis, and also compared to that reader's diagnosis across both modalities.Results.—The overall agreement rate, across 800 reads, was 97.9%. This consisted of 400 digital reads at 97.0% versus reference and 400 standard reads versus reference at 98.8%. Minor discordances (defined as alternative diagnoses without clinical treatment or outcome implications) were 6.1% overall, 7.2% for digital, and 5.0% for standard.Conclusions.—Pathologists can provide accurate diagnoses from fluorescence-imitating brightfield imaging slide-free images. Concordance and discordance rates are similar to published rates for comparisons of whole slide imaging to standard light microscopy of glass slides for primary diagnosis. It may be possible, therefore, to develop a slide-free, nondestructive approach for primary pathology diagnosis.

Abstract In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life-stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. We demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.

Abstract Background While research on substance using youth experiencing homelessness (YEH) is increasing, there is a dearth of information regarding effective prevention interventions for these youth. Suicide is the leading cause of death among YEH and most youth do not access services that may be available to them. Therefore, this study seeks to address this gap in the research literature with the goal to identify an effective suicide prevention intervention that can be readily adopted by communities that serve these youth. Methods Three hundred (N = 300) YEH with recent substance use and suicidal ideation or a recent suicide attempt will be recruited from the streets as well as a drop-in center serving YEH. After the baseline assessment, all youth will be randomly assigned to Cognitive Therapy for Suicide Prevention (CTSP) + Services as Usual (SAU) (N = 150) or to SAU alone (N = 150). SAU includes outreach, advocacy, and service linkage whereas YEH who receive CTSP will also receive ten CTSP sessions and an optional nine booster sessions. Follow-up assessments will be conducted at 3, 6, 9, and 12 months post-baseline. Theoretically derived mediators (e.g., cognitive distortions) will be tested to shed light on mechanisms associated with change, and the moderating effects of sex, race, sexual orientation, and baseline service connection will be examined. In order to ease future dissemination of the intervention to agencies serving YEH, we will rigorously assess acceptability, feasibility, fidelity, and cost associated with the delivery of our intervention approach using a mixed-methods approach. Discussion This study adds to a very small number of clinical trials seeking to prevent lethal suicide among a very high-risk group by addressing suicidal ideation directly rather than underlying conditions. It is hypothesized that youth receiving CTSP + SAU will show greater reductions in suicidal ideation (primary outcome), substance use, and depressive symptoms (secondary outcomes) over time compared to SAU alone, as well as improved risk and protective factors. Trial registration NCT05994612. Date of Registration: August 16, 2023.