Operation model of a skew-symmetric split-crystal neutron interferometer.

The observation of neutron interference using a triple Laue interferometer formed by two separate crystals opens the way to the construction and operation of skew-symmetric interferometers with extended arm separation and length. The specifications necessary for their successful operation are investigated here: most importantly, how the manufacturing tolerance and crystal alignments impact the interference visibility. In contrast with previous studies, both incoherent sources and the three-dimensional operation of the interferometer are considered. It is found that, with a Gaussian Schell model of an incoherent source, the integrated density of the particles leaving the interferometer is the same as that yielded by a coherent Gaussian source having a radius equal to the coherence length.

Photocatalytic Activation of Aryl(trifluoromethyl) Diazos to Carbenes for High-Resolution Protein Labeling with Red Light.

State-of-the-art methods in photoproximity labeling center on the targeted generation and capture of short-lived reactive intermediates to provide a snapshot of local protein environments. Diazirines are the current gold standard for high-resolution proximity labeling, generating short-lived aryl(trifluoromethyl) carbenes. Here, we present a method to access aryl(trifluoromethyl) carbenes from a stable diazo source via tissue-penetrable, deep red to near-infrared light (600-800 nm). The operative mechanism of this activation involves Dexter energy transfer from photoexcited osmium(II) photocatalysts to the diazo, thus revealing an aryl(trifluoromethyl) carbene. The labeling preferences of the diazo probe with amino acids are studied, showing high reactivity toward heteroatom-H bonds. Upon the synthesis of a biotinylated diazo probe, labeling studies are conducted on native proteins as well as proteins conjugated to the Os photocatalyst. Finally, we demonstrate that the conjugation of a protein inhibitor to the photocatalyst also enables selective protein labeling in the presence of spectator proteins and achieves specific labeling of a membrane protein on the surface of mammalian cells via a two-antibody photocatalytic system.

Extracellular vesicles released by cancer-associated fibroblast-induced myeloid-derived suppressor cells inhibit T-cell function.

Myeloid cells are known to play a crucial role in creating a tumor-promoting and immune suppressive microenvironment. Our previous study demonstrated that primary human monocytes can be polarized into immunosuppressive myeloid-derived suppressor cells (MDSCs) by cancer-associated fibroblasts (CAFs) in a 3D co-culture system. However, the molecular mechanisms underlying the immunosuppressive function of MDSCs, especially CAF-induced MDSCs, remain poorly understood. Using mass spectrometry-based proteomics, we compared cell surface protein changes among monocytes, in vitro differentiated CAF-induced MDSCs, M1/M2 macrophages, and dendritic cells, and identified an extracellular vesicle (EV)-mediated secretory phenotype of MDSCs. Functional assays using an MDSC/T-cell co-culture system revealed that blocking EV generation in CAF-induced MDSCs reversed their ability to suppress T-cell proliferation, while EVs isolated from CAF-induced MDSCs directly inhibited T-cell function. Furthermore, we identified fructose bisphosphatase 1 (FBP1) as a cargo protein that is highly enriched in EVs isolated from CAF-induced MDSCs, and pharmacological inhibition of FBP1 partially reversed the suppressive phenotype of MDSCs. Our findings provide valuable insights into the cell surface proteome of different monocyte-derived myeloid subsets and uncover a novel mechanism underlying the interplay between CAFs and myeloid cells in shaping a tumor-permissive microenvironment.

Ozone effect on the inflammatory and proteomic profile of human macrophages and airway epithelial cells.

Ozone (O3) is one of the most harmful urban pollutants, but its biological mechanisms have not been fully elucidated yet. Human bronchial epithelial cells (HBEpC) and human macrophage cells (differentiated human monocytic cell line) were exposed to O3 at the concentration of 240 µg/m3 (120 ppb), corresponding to the European Union alert threshold. Cell viability, reactive oxygen species (ROS) production, and pro-inflammatory cytokines release (IL-8 and TNF-α) were evaluated. Results indicated that O3 exposure increases ROS production in both cell types and enhances cytokines release in macrophages. O3 stimulated IL-8 and TNF-α in HBEpC when the cells were pretreated with Lipopolysaccharide, used to mimic a pre-existing inflammatory condition. Proteomics analysis revealed that, in HBEpC, O3 caused the up-regulation of aldo-keto reductase family 1 member B10, a recognized critical protein in lung carcinogenesis. In conclusion, our results show that 120 ppb O3 can lead to potential damage to human health suggesting the need for a revision of the actual alert levels.

Dual fluorescent labeling of GLP-1R in live cells via enzymatic tagging and bioorthogonal chemistry.

To study GPCR conformational dynamics in live cells, here we report an integrated approach combining enzymatic SNAP-tagging with bioorthogonal chemistry for dual fluorescent labeling of GLP-1R. The resulting GLP-1R conformational biosensors permit a FRET-based analysis of the receptor subdomain movement in response to ligand stimulation in live cells.

Anti-HIV antibody development up to 1 year after antiretroviral therapy initiation in acute HIV infection.

Early initiation of antiretroviral therapy (ART) in acute HIV infection (AHI) is effective at limiting seeding of the HIV viral reservoir, but little is known about how the resultant decreased antigen load affects long-term Ab development after ART. We report here that Env-specific plasma antibody (Ab) levels and Ab-dependent cellular cytotoxicity (ADCC) increased during the first 24 weeks of ART and correlated with Ab levels persisting after 48 weeks of ART. Participants treated in AHI stage 1 had lower Env-specific Ab levels and ADCC activity on ART than did those treated later. Importantly, participants who initiated ART after peak viremia in AHI developed elevated cross-clade ADCC responses that were detectable 1 year after ART initiation, even though clinically undetectable viremia was reached by 24 weeks. These data suggest that there is more germinal center (GC) activity in the later stages of AHI and that Ab development continues in the absence of detectable viremia during the first year of suppressive ART. The development of therapeutic interventions that can enhance earlier development of GCs in AHI and Abs after ART initiation could provide important protection against the viral reservoir that is seeded in individuals treated early in the disease.

Multidisciplinary approach to a septic COVID-19 patient undergoing veno-venous extracorporeal membrane oxygenation and receiving thoracic surgery.

A multidisciplinary approach appears to be fundamental for the treatment of critically ill patients with COVID-19, improving clinical outcomes, even in the most severe cases. Such severe cases are advisable to be collegially discussed between intensivists, surgeons, infectious disease, and other physicians potentially involved.

Mechanism of the small ATP-independent chaperone Spy is substrate specific.

ATP-independent chaperones are usually considered to be holdases that rapidly bind to non-native states of substrate proteins and prevent their aggregation. These chaperones are thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy's action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action.

The applicability of forensic time since death estimation methods for buried bodies in advanced decomposition stages.

Estimation of the postmortem interval in advanced postmortem stages is a challenging task. Although there are several approaches available for addressing postmortem changes of a (human) body or its environment (ecologically and/or biochemically), most are restricted to specific timeframes and/or individual and environmental conditions. It is well known, for instance, that buried bodies decompose in a remarkably different manner than on the ground surface. However, data on how established methods for PMI estimation perform under these conditions are scarce. It is important to understand whether and how postmortem changes are affected under burial conditions, if corrective factors could be conceived, or if methods have to be excluded for respective cases. We present the first multi-methodological assessment of human postmortem decomposition carried out on buried body donors in Europe, at the Amsterdam Research Initiative for Sub-surface Taphonomy and Anthropology (ARISTA) in the Netherlands. We used a multidisciplinary approach to investigate postmortem changes of morphology, skeletal muscle protein decomposition, presence of insects and other necrophilous animals as well as microbial communities (i.e., microbiomes) from August to November 2018 associated with two complete body exhumations and eight partial exhumations. Our results clearly display the current possibilities and limitations of methods for PMI estimation in buried remains and provide a baseline for future research and application.

Pupillary effects in habitual cannabis consumers quantified with pupillography.

Driving under the influence of alcohol (DUIA) and drugs (DUID) is considered an elevated risk for traffic safety. When assessing a driver's fitness to drive, standardized and objective measurement methods are still required, in order to clarify the question whether an individual is under the influence of substances acting on the central nervous system (CNS). We exposed healthy test subjects (n=41) as well as persons who were under the influence of cannabis after repeated inhalation to multiple light stimuli using infrared technology and measured the pupillary light reflex (PLR). Toxicological tests of blood samples taken from every subject followed. The aims of this study were to assess the differences in pupillography response between cannabis consumers after a washout period and no cannabis consumers as well as the dose related effects on pupillography parameters of cannabis in cannabis consumers. All four pupillary parameters changed according to a weakened pupil function after acute administration of cannabis in all test subjects. Furthermore, it could be observed that habitual cannabis consumers showed an altered pupillary function just before the first dose was taken, suggesting that the long-term effects and addiction also have to be taken into account, when effects of the CNS are discussed. The results of the present study show that almost all pupil parameters could be reliable indicators for the detection of subjects under the acute effect of cannabis.

Candesartan in migraine prevention: results from a retrospective real-world study.

Randomized studies have reported a positive effect of candesartan, an angiotensin II receptor antagonist, in migraine prevention. The aim of our study was to explore patient subjective efficacy of candesartan in a real-world sample of migraine patients and try to identify predictors of candesartan response. We audited the clinical records of 253 patients who attended the King's College Hospital, London, from February 2015 to December 2017, looking specifically at their response to candesartan. Univariate and multivariate logistic regression models were used to identify predictors of headache benefit. Odds ratios (OR) with confidence intervals (CI) 95% were calculated. Eighty-one patients (chronic migraine, n = 68) were included in the final analysis. Thirty-eight patients reported a positive response to candesartan, while 43 patients did not have a meaningful therapeutic effect. The median dose of candesartan was 8 mg and the median treatment period was 6 months. In a univariate logistic regression model, the presence of daily headache was associated with reduced odds of headache benefit (OR 0.39, 95% CI 0.16-0.96, p = 0.04). In multivariate logistic regression model, younger age (OR 0.92, 95% CI 0.87-0.98, p = 0.006) and longer disease duration (OR 1.06, 95% CI 1.01-1.12, p = 0.03) were associated with a good response to candesartan, while the presence of daily headache was associated with reduced odds of headache benefit (OR 0.16, 95% CI 0.04-0.71, p = 0.01). Having failed up to nine preventives in patients did not predict a treatment failure with candesartan as well. Candesartan yields clinical benefits in difficult-to-treat migraine patients, irrespective of previous failed preventives.

Brief Report: Group Sex and Methamphetamine Use Fuel an Explosive Epidemic of Hepatitis C Among HIV-Infected Men Who Have Sex With Men in Bangkok, Thailand.

BACKGROUND: Increased rates of hepatitis C virus (HCV) infection among HIV-infected men who have sex with men (MSM) and who deny injecting drugs have been reported in resource-rich settings. SETTING: We measured HCV prevalence and incidence in a predominantly MSM cohort with acute HIV infection in Bangkok, Thailand. METHODS: In 2009-2018, participants with acute HIV infection were enrolled into the SEARCH010/RV254 cohort. HCV antibody was measured at enrollment and at least once annually. Infection was confirmed with HCV RNA. Risk factors for HCV were analyzed by proportional hazards regression, with hazard ratios (HRs) calculated in a multivariable model. RESULTS: Of 573 participants, 94% were MSM, with a median age of 26 years (range 18-70 years). The prevalence of HCV antibody was 9 of the 573, or 1.6% [95% confidence interval (CI): 0.7% to 3.0%]. In 1883 person-years (PY) of follow-up, 39 incident cases were identified (20.7 per 1000 PY, 95% CI: 15.1 to 28.3). All incident cases were identified from 2014 onward, and incidence rose from a range of 7.5-11.4 per 1000 PY between 2014 and 2016 to 44.8 per 1000 PY in 2018 (P = 0.001). Most cases (97.4%) were MSM and denied injecting drugs (37 of the 39, 94.5%). In multivariate analysis, methamphetamine use [adjusted HR 2.33 (95% CI: 1.13 to 4.8), P = 0.022], group sex [adjusted HR 2.54 (95% CI: 1.26 to 5.12), P = 0.009], and a history of positive Treponema pallidum hemagglutination or rapid plasma reagin [adjusted HR 2.43 (95% CI: 1.22 to 4.85), P = 0.012] were significantly associated with incident HCV. CONCLUSION: We report an HCV epidemic among this cohort of HIV-infected Bangkok-based MSM. Access to timely HCV diagnosis and treatment is needed to prevent morbidity and to decrease onward transmission.

A field study to evaluate PMI estimation methods for advanced decomposition stages.

Estimating the postmortem interval (PMI) is one of the major tasks and a continuous challenge in forensic pathology. It is often an exclusion process of available methods, which ultimately can lead to an unsatisfactory outcome due to poor reliability. This problem is most acute in the late PMI, when decomposition proceeds and some methods (such as rigor, livor, and algor mortis) are no longer applicable. Several methods, such as forensic entomology, skeletal muscle protein degradation, and the study of body decomposition by application of a morphological scoring, are expected to provide further information; however, all have certain limitations and weaknesses. Availability of a tool-box of methods allows a case-specific selection of the most appropriate one(s), or eventually provides improvements in the overall accuracy and precision of the PMI estimation by merging and combining methods. To investigate practical (field) application, eventual interferences, and/or synergetic effects, as well as the robustness of these methods towards specific influencing factors, a field study was conducted, using eight pig cadavers of different body weights and physical coverage, left to decompose under natural conditions for 16 days. Morphological changes during decomposition were assessed using the total body score (TBS), muscle samples were collected to analyze protein degradation, and insect colonization was evaluated. The results reveal strengths and current limitations of all tested methods, as well as promising synergistic effects, and thus, provide a baseline for targeted future research.

Cancer-Associated Fibroblasts Promote Immunosuppression by Inducing ROS-Generating Monocytic MDSCs in Lung Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8+ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.

Patient-Centered Risk Assessment in Implant Treatment Planning.

PURPOSE: To evaluate the current scientific evidence on estimating cumulative risk for biologic complications relating to dental implants and to develop a patient-centered risk assessment tool for establishing aggregate risk. MATERIALS AND METHODS: A review of the scientific literature on risk indicators relating to dental implants was completed with the goal of identifying and weighting individual risk indicators so aggregate biologic risk could be estimated. Three authors completed independent reviews of the literature, identifying 31 systematic reviews on risk indicators for biologic complications with dental implants, from which 24 potential risk indicators were considered. Due to inconclusive scientific data on risk indicators, a Delphi process was used to gather structured expert opinion to supplement findings from the literature. Eleven Delphi participants with expertise in prosthodontics or periodontics participated in two email exchanges and one face-to-face meeting to comment and debate on the initial identification and weighting of risk indicators, propose the addition or removal of risk indicators, and provide recommended clinical management for each risk indicator. RESULTS: After three rounds of debate, literature review, and additions and removals of various risk indicators, consensus (defined as 95% or more in agreement) was achieved on 20 risk indicators. The Delphi group concluded that the risk indicators of smoking, diabetes, antiresorptive agents, and cemented restorations should include subcategories to more accurately identify and represent patient-specific risk. Clinical recommendations based on individual and aggregate risk were established by consensus. CONCLUSION: The literature on risk indicators for biologic complications was conflicting and inconclusive. The Delphi method was used to identify and establish the weighting of individual risk indicators, resulting in a risk assessment tool for estimating aggregate risk.

Aortic aneurysm endovascular treatment with the parallel graft technique from the aortic arch to the iliac axis.

BACKGROUND: The chimney technique has been developed for the treatment of complex aortic aneurysms. We analyzed the midterm to long-term outcomes of this approach from a single-center experience. METHODS: From October 2008 to July 2016, 58 patients underwent endovascular aortic aneurysm repair using the chimney technique. Indications for treatment were thoracic aortic aneurysm (TAA) (N.=11), thoracoabdominal aortic aneurysm (TAAA) (N.=2), pararenal aortic aneurysm (PAAA) (N.=15), aortoiliac/isolated hypogastric artery aneurysm (N.=25), type I endoleak after previous TEVAR/EVAR (N.=4), proximal pseudoaneurysm after AAA open repair (N.=1). Elective (82.8%) and emergent (17.2%) procedures were included. RESULTS: The immediate technical success was 100%. Single, double and triple chimneys were performed in 46, 10, and two patients, respectively. Overall, 61 target vessels (three left common carotid arteries, eight left subclavian arteries, three celiac trunks, three superior mesenteric arteries, 19 renal arteries and 25 hypogastric arteries) were involved. Postoperative mortality was 0. No neurologic complications were registered. Primary patency rate of the chimney stent/stent graft was 98.3%. Low-flow type I endoleak was observed in four patients (6.9%). Postoperative chimney graft re-intervention rate was 1.7%. The median follow-up was 32±20 months (range 3-96 months). Overall estimated survival at 12, 50, and 80 months was 100%, 89% and 44%, respectively. Estimated freedom from endoleak at 1, 12, 24, and 36 months was 96.5%, 95%, 95%, and 93%, respectively. One hypogastric artery stent-graft occluded at the 3rd month of follow-up. No reintervention was performed. CONCLUSIONS: Our experience with the chimney technique for aortic aneurysms from the aortic arch to the iliac axis shows promising and durable mid- and long-term results. Endograft oversizing, associated with the chimney graft diameter and length choice remain fundamental to reduce the risk of the most frequent procedure complications: type I endoleak and CG occlusion. The wider use of this technique should be justified in patients considered at high risk for open repair and/or not suitable for the custom-made branched/fenestrated endografts.