On-Site More Frequent Dialysis May Hasten Return Home for Nursing Home End-Stage Renal Disease Patients.

BACKGROUND: A direct outcome comparison between Skilled nursing facility (SNF) patients receiving on-site more frequent dialysis (MFD) targeting 14 hours of treatment over five sessions weekly compared to on-site conventional dialysis for death, hospitalization and speed of return home has not been reported. METHODS: From Jan 1, 2022, to July 1, 2023, in a retrospective prospective observational design, using an intent to treat and competing risk strategy, all new admissions to an on-site in SNF dialysis service admitted to nursing homes with on-site MFD dialysis were compared to admissions to nursing homes providing on-site conventional dialysis for the outcome goal of 90 day cumulative incidence of discharge to home, while monitoring safety issues represented by the competing risks of hospitalization and death. RESULTS: 10,246 MFD dialytic episodes and 3,451 conventional dialytic episodes were studied in 195 nursing homes in 12 states. At baseline the MFD population was consistently sicker than CONVENTIONAL dialysis population with a first systolic blood pressure in 23% vs 7.6% (p<.001), lower mean hemoglobin (9.3g/dl vs 10.4g/dl; p<.001), lower iron saturation (25.7% vs 26.6%; p=0.02), higher Charlson score (3.5 vs 3.0; p<.001), higher mean age (67.6 vs 66.7; p<.001). ), more complicated diabetes (31% vs 24%; P<.001), cerebrovascular disease ( 12.6% vs 6.8%:p<.001), and congestive heart failure (24% vs 18%). At 42 days, discharge to home was 25% greater in the MFD than conventional group (17.5% vs 14%) without worsened hospitalization or death. CONCLUSION: Despite a handicap of sicker patients at baseline, real-world application of MFD appears to hasten return home from SNFs compared to conventional dialysis. The findings suggests that MFD allows for SNF acceptance of sicker patients, presumably permitting earlier hospital discharge, without safety compromise as measured by death or rehospitalization benefitting hospitals, patients, and payers.

Feeding en route: Prey availability and traits influence prey selection by an avian predator on migration.

During animal migration, ephemeral communities of taxa at all trophic levels co-occur over space and time. The interactions between predators and prey along migration corridors are ecologically and evolutionarily significant. However, these interactions remain understudied in terrestrial systems and warrant further investigations using novel approaches. We investigated the predator-prey interactions between a migrating avivorous predator and ephemeral avian prey community in the fall migration season. We tested for associations between avian traits and prey selection and hypothesized that prey traits (i.e. relative size, flocking behaviour, habitat, migration tendency and availability) would influence prey selection by a sexually dimorphic raptor on migration. To document prey consumption, we sampled trace prey DNA from beaks and talons of migrating sharp-shinned hawks Accipiter striatus (n = 588). We determined prey availability in the ephemeral avian community by extracting weekly abundance indices from eBird Status and Trends data. We used discrete choice models to assess prey selection and visualized the frequency of prey in diet and availability on the landscape over the fall migration season. Using eDNA metabarcoding, we detected prey species on 94.1% of the hawks sampled (n = 525/588) comprising 1396 prey species detections from 65 prey species. Prey frequency in diet and eBird relative abundance of prey species were correlated over the migration season for top-selected prey species, suggesting prey availability is an important component of raptor-songbird interactions during fall. Prey size, flocking behaviour and non-breeding habitat association were prey traits that significantly influenced predator choice. We found differences between female and male hawk prey selection, suggesting that sexual size dimorphism has led to distinct foraging strategies on migration. This research integrated field data collected by a volunteer-powered raptor migration monitoring station and public-generated data from eBird to reveal elusive predator-prey dynamics occurring in an ephemeral raptor-songbird community during fall migration. Understanding dynamic raptor-songbird interactions along migration routes remains a relatively unexplored frontier in animal ecology and is necessary for the conservation and management efforts of migratory and resident communities.

Durante la migración animal, las comunidades efímeras de taxones de todos los niveles tróficos coexisten en el espacio y el tiempo. Las interacciones entre depredadores y presas a lo largo de los corredores migratorios son significativas desde el punto de vista ecológica y evolutivo. Sin embargo, estas interacciones siguen siendo poco estudiadas en los sistemas terrestres y justifican más investigaciones utilizando enfoques novedosos. Investigamos las interacciones depredador­presa entre un depredador avívoro migratorio y una comunidad de presas aviares efímeras en la temporada migratoria otoñal. Probamos las asociaciones entre los rasgos de las aves y la selección de presas y planteamos la hipótesis de que los rasgos de las presas (tamaño relativo, comportamiento de bandada, hábitat, tendencia migratoria y disponibilidad) influirían en la selección de presas por parte de una rapaz sexualmente dimórfica durante la migración. Para documentar el consumo de presas, recogimos rastros de ADN de presas de picos y garras de Gavilán Americano Accipiter striatus (n = 588) migratorios. Determinamos la disponibilidad de presas en la comunidad de aves efímeras extrayendo índices de abundancia semanales de los datos de eBird Estado y Tendencias. Utilizamos modelos de elección discreta para evaluar la selección de presas y visualizamos la frecuencia de las presas en la dieta y la disponibilidad en el paisaje durante la temporada migratoria otoñal. Utilizando el metacódigo de barras del ADN ambiental, detectamos especies de presas en el 94,1% de los halcones muestreados (n = 525/588), comprendiendo 1396 detecciones de 65 especies de presas. La frecuencia de presas en la dieta y la abundancia relativa de especies de presas en eBird se correlacionaron a lo largo de la temporada de migración para las principales especies de presas seleccionadas, lo que sugiere que la disponibilidad de presas es un componente importante de las interacciones entre aves rapaces y aves canoras durante el otoño. El tamaño de las presas, el comportamiento de las bandadas y la asociación con el hábitat no reproductivo fueron rasgos de presa que influyeron significativamente en la elección de los depredadores. Encontramos diferencias entre la selección de presas de gavilán hembra y macho, lo que sugiere que el dimorfismo sexual de tamaño ha conducido a distintas estrategias de alimentación durante la migración. Esta investigación integró datos de campo recopilados por una estación de monitoreo de migración de rapaces impulsada por voluntarios y datos generados públicamente por eBird para revelar la esquiva dinámica depredador­presa que ocurre en una comunidad efímera de rapaces y aves canoras durante la migración otoñal. Comprender las interacciones dinámicas entre rapaces y aves canoras a lo largo de las rutas migratorias sigue siendo una frontera relativamente inexplorada en la ecología animal y es necesaria para los esfuerzos de conservación y gestión de las comunidades migratorias y residentes.

De-escalated radiation for human papillomavirus virus-related oropharyngeal cancer: Who, why, what, where, when, how, how much and what next?

The emergence of treatment de-escalation as a feasible option for patients with newly diagnosed human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma has generated considerable excitement among both providers and patients alike. Since HPV-positive oropharyngeal carcinoma has been shown to be a unique entity with distinct clinical and molecular characteristics, the rationale for customizing treatment for patients with this disease is compelling. Indeed, evidence has accumulated demonstrating that patients with HPV-positive oropharyngeal cancer have a significantly improved prognosis as a result of their exquisite radiosensitivity compared to their HPV-negative counterparts and thus might possibly be targeted with de-escalated approaches. The fundamental goal of de-escalation is to maintain the high cure and survival rates associated with traditional approaches while reducing the intensity of treatment and thus the incidence of both short- and long-term toxicity. Given the rapidly increasing incidence of this disease, particularly among younger patients who are generally healthy, the focus on quality of life seems germane. Although the exact reason for the improved sensitivity of HPV-positive oropharyngeal carcinoma to treatment is uncertain, prospective studies have now been published demonstrating that de-escalated radiation can successfully maintain the high rates of cure and preserve quality of life for appropriately selected patients with this disease. However, these studies have been fairly heterogeneous in design, and it remains questionable how to apply their findings to real-world practice. The potential of integrating translational approaches into clinical paradigms is also just starting to become recognized. Consequently, multiple uncertainties continue to exist with respect to de-escalation for HPV-positive oropharyngeal cancer, and these questions comprise the crux of this review.

Ray-tracing analytical absorption correction for X-ray crystallography based on tomographic reconstructions.

Processing of single-crystal X-ray diffraction data from area detectors can be separated into two steps. First, raw intensities are obtained by integration of the diffraction images, and then data correction and reduction are performed to determine structure-factor amplitudes and their uncertainties. The second step considers the diffraction geometry, sample illumination, decay, absorption and other effects. While absorption is only a minor effect in standard macromolecular crystallography (MX), it can become the largest source of uncertainty for experiments performed at long wavelengths. Current software packages for MX typically employ empirical models to correct for the effects of absorption, with the corrections determined through the procedure of minimizing the differences in intensities between symmetry-equivalent reflections; these models are well suited to capturing smoothly varying experimental effects. However, for very long wavelengths, empirical methods become an unreliable approach to model strong absorption effects with high fidelity. This problem is particularly acute when data multiplicity is low. This paper presents an analytical absorption correction strategy (implemented in new software AnACor) based on a volumetric model of the sample derived from X-ray tomography. Individual path lengths through the different sample materials for all reflections are determined by a ray-tracing method. Several approaches for absorption corrections (spherical harmonics correction, analytical absorption correction and a combination of the two) are compared for two samples, the membrane protein OmpK36 GD, measured at a wavelength of λ = 3.54 Å, and chlorite dismutase, measured at λ = 4.13 Å. Data set statistics, the peak heights in the anomalous difference Fourier maps and the success of experimental phasing are used to compare the results from the different absorption correction approaches. The strategies using the new analytical absorption correction are shown to be superior to the standard spherical harmonics corrections. While the improvements are modest in the 3.54 Šdata, the analytical absorption correction outperforms spherical harmonics in the longer-wavelength data (λ = 4.13 Å), which is also reflected in the reduced amount of data being required for successful experimental phasing.

Cancer Incidence After Diagnosis of Abdominal Aortic Aneurysm-Brief Report.

BACKGROUND: Epidemiological and mechanistic data support a potential causal link between cardiovascular disease (CVD) and cancer. Abdominal aortic aneurysms (AAAs) represent a common form of CVD with at least partially distinct genetic and biologic pathogenesis from other forms of CVD. The risk of cancer and how this risk differs compared with other forms of CVD, is unknown among AAA patients. We conducted a retrospective cohort study using the IBM MarketScan Research Database to test whether individuals with AAA have a higher cancer risk independent of traditional shared risk factors. METHODS: All individuals ≥18 years of age with ≥36 months of continuous coverage between 2008 and 2020 were enrolled. Those with potential Mendelian etiologies of AAA, aortic aneurysm with nonspecific anatomic location, or a cancer diagnosis before the start of follow-up were excluded. A subgroup analysis was performed of individuals having the Health Risk Assessment records including tobacco use and body mass index. The following groups of individuals were compared: (1) with AAA, (2) with non-AAA CVD, and (3) without any CVD. RESULTS: The propensity score-matched cohort included 58 993 individuals with AAA, 117 986 with non-AAA CVD, and 58 993 without CVD. The 5-year cumulative incidence of cancer was 13.1% (12.8%-13.5%) in participants with AAA, 10.1% (9.9%-10.3%) in participants with non-AAA CVD, and 9.6% (9.3%-9.9%) in participants without CVD. Multivariable-adjusted Cox proportional hazards regression models found that patients with AAA exhibited a higher cancer risk than either those with non-AAA CVD (hazard ratio, 1.28 [95% CI, 1.23-1.32]; P<0.001) or those without CVD (hazard ratio, 1.32 [95% CI, 1.26-1.38]; P<0.001). Results remained consistent after excluding common smoking-related cancers and when adjusting for tobacco use and body mass index. CONCLUSIONS: Patients with AAA may have a unique risk of cancer requiring further mechanistic study and investigation of the role of enhanced cancer screening.

Translational risk-adapted approaches to de-escalated radiation for human papillomavirus-positive oropharyngeal cancer: Past, present, and future.

Interest in the use of de-escalated radiation to treat patients with newly diagnosed human papillomavirus (HPV)-positive oropharyngeal cancer has grown dramatically with the publication of prospective trials demonstrating the efficacy of such an approach. While the rationale for de-escalation--- namely to decrease treatment-related toxicity while maintaining the excellent rates of disease control historically observed in patients with this disease-is inherently obvious, uncertainty exists regarding how to best select patients for de-escalation. Consequently, risk-adapted strategies using a variety of translational and clinical platforms have been increasingly popularized to better refine treatment. These have integrated contemporary methods of mid-treatment response assessment using advanced technologies and molecular assays to customize the radiation dose. By monitoring the response as patients actively proceed through treatment, risk-adapted protocols have the potential to provide insight into the biological behavior of tumors and make individualized therapy possible. The purpose of this review is to summarize the evidence to date on risk-adapted approaches to de-escalated radiation-- highlighting the clinical, radiological, and biological data which may ultimately help usher the principles of precision medicine into practice for patients with HPV-positive oropharyngeal cancer.

Classics in Chemical Neuroscience: Xylazine.

Xylazine (also known as "tranq") is a potent nonopioid veterinary sedative that has recently experienced a surge in use as a drug adulterant, most often combined with illicitly manufactured fentanyl. This combination may heighten the risk of fatal overdose. Xylazine has no known antidote approved for use in humans, and age-adjusted overdose deaths involving xylazine were 35 times higher in 2021 than 2018. In April 2023, the Biden Administration declared xylazine-laced fentanyl an emerging drug threat in the United States. In 2022, the Drug Enforcement Agency (DEA) reported nearly a quarter of seized fentanyl powder contained xylazine. This dramatic increase in prevalence has solidified the status of xylazine as an emerging drug of abuse and an evolving threat to public health. The following narrative review outlines the synthesis, pharmacokinetics, pharmacodynamics, and adverse effects of xylazine, as well as the role it may play in the ongoing opioid epidemic.

Visual cortex anodal transcranial direct current stimulation does not alter reading performance for Chinese presented character-by-character to normal peripheral vision in older adults.

Visual cortex anodal transcranial direct current stimulation (a-tDCS) has been shown to reduce crowding in normal peripheral vision and may improve the reading of English words in patients with macular degeneration. Given the different visual requirements of reading English words and Chinese characters, the effect of a-tDCS on peripheral reading performance in English might differ from Chinese. This study recruited 20 participants (59-73 years of age) with normal vision and tested the hypothesis that a-tDCS would improve the reading of Chinese characters presented at 10° eccentricity compared with sham stimulation. Chinese sentences of different print sizes and exposure durations were presented one character at a time, 10° below or to the left of fixation. The individual critical print size (CPS) - the smallest print size eliciting the maximum reading speed (MRS) - was determined. Reading accuracies for characters presented 0.2 logMAR smaller than the individually fitted CPS were measured at four time points: before, during, 5 min after, and 30 min after receiving active or sham visual cortex a-tDCS. Participants completed both the active and sham sessions in a random order following a double-blind, within-subject design. No effect of active a-tDCS on reading accuracy was observed, implying that a single session of a-tDCS did not improve Chinese character reading in normal peripheral vision. This may suggest that a-tDCS does not significantly reduce the crowding elicited within a single Chinese character. However, the effect of a-tDCS on between-character crowding is yet to be determined.

The DEAD-box RNA helicase PfDOZI imposes opposing actions on RNA metabolism in Plasmodium falciparum.

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.

Re-irradiation versus systemic therapy for the management of local-regionally recurrent head and neck cancer.

PURPOSE: The optimal management of local-regionally recurrent head and neck cancer that is not amenable to surgical resection is uncertain. We sought to compare outcomes among patients treated with and without re-irradiation in this setting. METHODS AND MATERIALS: A review of institutional registries identified 65 patients with local-regionally recurrent squamous cell carcinoma of the head and neck who were ineligible for surgery. Forty patients (62 %) opted for re-irradiation with the remaining 25 patients (38 %) undergoing initial systemic therapy alone. All patients had measurable disease. Forty-three patients (66 %) were male and twenty-two (33 %) were female. The median age at the time of recurrence was 59 years (range, 39-84 years). The most common primary sites of disease were the oropharynx, (n = 25), oral cavity (N = 19), and nasopharynx (n = 11). The median interval from completion of prior radiation to the diagnosis of recurrent disease was 35 months (range, 2-102 months). RESULTS: Re-irradiation improved 2-year overall survival, (32 % versus 11 %), progression-free survival (31 % versus 7 %), and local-regional control (39 % versus 3 %) compared to systemic therapy alone (p < 0.05, for both). The likelihood of developing any new grade 3+ toxicity was significantly higher among patients treated by re-irradiation compared to those treated by systemic therapy (53 % vs. 28 %, p < 0.001). There were 3 treatment-related fatalities, all of which occurred in the re-irradiation group. The incidence of grade 3+ late toxicity was 48 % and 12 % for patients in the re-irradiation and systemic therapy cohorts, respectively (p < 0.001). CONCLUSION: Although re-irradiation improved overall survival compared to systemic therapy for appropriately selected patients with local-regionally recurrent head and neck cancer, the relatively high risk of toxicity must be considered.

Integrating Overall Survival and Tumor Control Probability Models to Predict Local Progression After Brain Metastasis Radiosurgery.

Purpose: Stereotactic radiosurgery (SRS) for brain metastases is frequently prescribed to the maximum tolerated dose to minimize the probability of local progression. However, many patients die from extracranial disease prior to local progression and may not require maximally aggressive treatment. Recently, improvements in models of SRS tumor control probability (TCP) and overall survival (OS) have been made. We predicted that by combining models of OS and TCP, we could better predict the true risk of local progression after SRS than by using TCP modeling alone. Methods and Materials: Records of patients undergoing SRS at a single institution were reviewed retrospectively. Using established TCP and OS models, for each patient, the probability of 1-year survival [p(OS)] was calculated, as was the probability of 1-year local progression [p(LP)]) for each treated lesion. Joint-probability was used to combine the models [p(LP,OS)=p(LP)*p(OS)]. Analyses were conducted at the individual metastasis and whole-patient levels. Fine-Gray regression was used to model p(LP) or p(LP,OS) on the risk of local progression after SRS, with death as a competing risk. Results: At the patient level, 1-year local progression was 0.08 (95% CI, 0.03-0.15), median p(LP,OS) was 0.13 (95% CI, 0.07-0.2), and median p(LP) was 0.29 (95% CI, 0.22-0.38). At the metastasis level, 1-year local progression was 0.02 (95% CI, 0.01-0.04), median p(LP,OS) was 0.05 (95% CI, 0.02-0.07), and median p(LP) was 0.10 (95% CI, 0.07-0.13). p(LP,OS) was found to be significantly associated with the risk of local progression at the patient level (P = .048) and metastasis level (P = .007); however, p(LP) was not (P = .16 and P = .28, respectively). Conclusions: Simultaneous modeling of OS and TCP more accurately predicted local progression than TCP modeling alone. Better understanding which patients with brain metastases are at risk of local progression after SRS may help personalize treatment to minimize risk without sacrificing efficacy.

Micro-extraction method for the analysis of flame retardants in dust collected from air filters from HVAC systems.

Dust is a sink for many semi-volatile compounds including flame retardants of the organophosphate ester (OPE) and brominated flame-retardant (BFR) classes. Given the large amount of time that we spend indoors, our exposure to these compounds via dust is of significant interest. Here, we present a novel microextraction approach to determine quantitative levels of selected OPEs and BFRs sampled from residential air filters from HVAC systems using a small volume of solvent. Dust samples (25 mg) is extracted with 1 mL of hexane/acetone (50/50, v/v). Upon solvent extraction of these HVAC dust samples, the analytes (TCPP, TDCPP, TPHP, T24DtBPP, TBBPA, and TriBBPA) were quantified via gas chromatography-mass spectrometry (GC/MS) or liquid chromatography-mass spectrometry (LC/MS). The methods for extracting these compounds from HVAC dust samples are detailed here with extensive method validation data to demonstrate accuracy and precision of these methods. •Dust is a sink for many semi-volatile compounds, including novel or emerging indoor pollutants like the organophosphate ester flame retardant T24DtBPP.•Here, a small amount of dust (25 mg) is extracted with a small volume of solvent (1 mL hexane and acetone) prior to analysis via chromatographic separation and mass spectrometric detection.

CD4 T Cell-Derived IL-21 Is Critical for Sustaining Plasmodium Infection-Induced Germinal Center Responses and Promoting the Selection of Memory B Cells with Recall Potential.

Development of Plasmodium-specific humoral immunity is critically dependent on CD4 Th cell responses and germinal center (GC) reactions during blood-stage Plasmodium infection. IL-21, a cytokine primarily produced by CD4 T cells, is an essential regulator of affinity maturation, isotype class-switching, B cell differentiation, and maintenance of GC reactions in response to many infection and immunization models. In models of experimental malaria, mice deficient in IL-21 or its receptor IL-21R fail to develop memory B cell populations and are not protected against secondary infection. However, whether sustained IL-21 signaling in ongoing GCs is required for maintaining GC magnitude, organization, and output is unclear. In this study, we report that CD4+ Th cells maintain IL-21 expression after resolution of primary Plasmodium yoelii infection. We generated an inducible knockout mouse model that enabled cell type-specific and timed deletion of IL-21 in peripheral, mature CD4 T cells. We found that persistence of IL-21 signaling in active GCs had no impact on the magnitude of GC reactions or their capacity to produce memory B cell populations. However, the memory B cells generated in the absence of IL-21 exhibited reduced recall function upon challenge. Our data support that IL-21 prevents premature cellular dissolution within the GC and promotes stringency of selective pressures during B cell fate determination required to produce high-quality Plasmodium-specific memory B cells. These data are additionally consistent with a temporal requirement for IL-21 in fine-tuning humoral immune memory responses during experimental malaria.

Suicide prevention for substance using youth experiencing homelessness: study protocol for a randomized controlled trial.

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.

Droplet microfluidics for time-resolved serial crystallography.

Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here, we report the use of droplet miniaturization for the controlled production of uniform crystals, providing an avenue for controlled substrate addition and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3 µm crystals of lysozyme and 2 µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 ms were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be utilized to advance time-resolved serial crystallography.

A Pilot Study of Pembrolizumab Combined With Stereotactic Ablative Radiotherapy for Patients With Advanced or Metastatic Sarcoma.

OBJECTIVES: Immunotherapy 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. METHODS: This 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. RESULTS: In this study we outline testing the feasibility of adding SBRT to pembrolizumab. CONCLUSION: The ultimate goal of combination therapy is improved overall response, including tumors not treated with SBRT. This trial can be found registered online: NCT05488366.

A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions.

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease-serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease-serpin in the framework of heparin that we review includes the following: thrombin-antithrombin III, plasmin-anti-plasmin, C1 esterase/kallikrein-C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)-alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer.

Intranasal SARS-CoV-2 RBD decorated nanoparticle vaccine enhances viral clearance in the Syrian hamster model.

Multiple vaccines have been developed and licensed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). While these vaccines reduce disease severity, they do not prevent infection. To prevent infection and limit transmission, vaccines must be developed that induce immunity in the respiratory tract. Therefore, we performed proof-of-principle studies with an intranasal nanoparticle vaccine against SARS-CoV-2. The vaccine candidate consisted of the self-assembling 60-subunit I3-01 protein scaffold covalently decorated with the SARS-CoV-2 receptor-binding domain (RBD) using the SpyCatcher-SpyTag system. We verified the intended antigen display features by reconstructing the I3-01 scaffold to 3.4 A using cryogenicelectron microscopy. Using this RBD-grafted SpyCage scaffold (RBD + SpyCage), we performed two intranasal vaccination studies in the "gold-standard" pre-clinical Syrian hamster model. The initial study focused on assessing the immunogenicity of RBD + SpyCage combined with the LTA1 intranasal adjuvant. These studies showed RBD + SpyCage vaccination induced an antibody response that promoted viral clearance but did not prevent infection. Inclusion of the LTA1 adjuvant enhanced the magnitude of the antibody response but did not enhance protection. Thus, in an expanded study, in the absence of an intranasal adjuvant, we evaluated if covalent bonding of RBD to the scaffold was required to induce an antibody response. Covalent grafting of RBD was required for the vaccine to be immunogenic, and animals vaccinated with RBD + SpyCage more rapidly cleared SARS-CoV-2 from both the upper and lower respiratory tract. These findings demonstrate the intranasal SpyCage vaccine platform can induce protection against SARS-CoV-2 and, with additional modifications to improve immunogenicity, is a versatile platform for the development of intranasal vaccines targeting respiratory pathogens.IMPORTANCEDespite the availability of efficacious COVID vaccines that reduce disease severity, SARS-CoV-2 continues to spread. To limit SARS-CoV-2 transmission, the next generation of vaccines must induce immunity in the mucosa of the upper respiratory tract. Therefore, we performed proof-of-principle, intranasal vaccination studies with a recombinant protein nanoparticle scaffold, SpyCage, decorated with the RBD of the S protein (SpyCage + RBD). We show that SpyCage + RBD was immunogenic and enhanced SARS-CoV-2 clearance from the nose and lungs of Syrian hamsters. Moreover, covalent grafting of the RBD to the scaffold was required to induce an immune response when given via the intranasal route. These proof-of-concept findings indicate that with further enhancements to immunogenicity (e.g., adjuvant incorporation and antigen optimization), the SpyCage scaffold has potential as a versatile, intranasal vaccine platform for respiratory pathogens.